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https://openalex.org/W4376131901
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https://www.research.ed.ac.uk/files/353261617/s00412_023_00797_4.pdf
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English
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Replisome-cohesin interactions provided by the Tof1-Csm3 and Mrc1 cohesion establishment factors
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Chromosoma
| 2,023
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cc-by
| 17,142
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Replisome-cohesin interactions provided by the Tof1-Csm3 and
Mrc1 cohesion establishment factors Citation for published version:
Shrestha, S, Minamino, M, Chen, ZA, Bouchoux, C, Rappsilber, J & Uhlmann, F 2023, 'Replisome-cohesin
interactions provided by the Tof1-Csm3 and Mrc1 cohesion establishment factors', Chromosoma, vol. 132,
no. 2, pp. 117-135. https://doi.org/10.1007/s00412-023-00797-4 Citation for published version:
Shrestha, S, Minamino, M, Chen, ZA, Bouchoux, C, Rappsilber, J & Uhlmann, F 2023, 'Replisome-cohesin
interactions provided by the Tof1-Csm3 and Mrc1 cohesion establishment factors', Chromosoma, vol. 132,
no. 2, pp. 117-135. https://doi.org/10.1007/s00412-023-00797-4 General rights
C
i h f
h General rights
Copyright for the publications made accessible via the Edinburgh Research Explorer is retained by the author(s)
and / or other copyright owners and it is a condition of accessing these publications that users recognise and
abide by the legal requirements associated with these rights. Edinburgh Research Explorer Abstract The chromosomal cohesin complex establishes sister chromatid cohesion during S phase, which forms the basis for faithful
segregation of DNA replication products during cell divisions. Cohesion establishment is defective in the absence of either
of three non-essential Saccharomyces cerevisiae replication fork components Tof1-Csm3 and Mrc1. Here, we investigate
how these conserved factors contribute to cohesion establishment. Tof1-Csm3 and Mrc1 serve known roles during DNA
replication, including replication checkpoint signaling, securing replication fork speed, as well as recruiting topoisomerase
I and the histone chaperone FACT. By modulating each of these functions independently, we rule out that one of these
known replication roles explains the contribution of Tof1-Csm3 and Mrc1 to cohesion establishment. Instead, using purified
components, we reveal direct and multipronged protein interactions of Tof1-Csm3 and Mrc1 with the cohesin complex. Our
findings open the possibility that a series of physical interactions between replication fork components and cohesin facilitate
successful establishment of sister chromatid cohesion during DNA replication. Keywords Sister chromatid cohesion · DNA replication · Cohesin · Tof1-Csm3 · Mrc1 · S. cerevisiae Replisome‑cohesin interactions provided by the Tof1‑Csm3 and Mrc1
cohesion establishment factors Sudikchya Shrestha1 · Masashi Minamino1 · Zhuo A. Chen2 · Céline Bouchoux1 · Juri Rappsil Received: 17 March 2023 / Revised: 25 April 2023 / Accepted: 26 April 2023 / Published online: 11 May 2023
© The Author(s) 2023 * Frank Uhlmann
frank.uhlmann@crick.ac.uk Take down policy Take down policy
The University of Edinburgh has made every reasonable effort to ensure that Edinburgh Research Explorer
content complies with UK legislation. If you believe that the public display of this file breaches copyright please
contact openaccess@ed.ac.uk providing details, and we will remove access to the work immediately and
investigate your claim. Download date: 24. Oct. 2024 Chromosoma (2023) 132:117–135
https://doi.org/10.1007/s00412-023-00797-4 RESEARCH RESEARCH * Frank Uhlmann
frank.uhlmann@crick.ac.uk
1
Chromosome Segregation Laboratory, The Francis Crick
Institute, London NW1 1AT, UK
2
Bioanalytics Unit, Institute of Biotechnology, Technische
Universität Berlin, 13355 Berlin, Germany
3
Wellcome Centre for Cell Biology, University of Edinburgh,
Edinburgh EH9 3BF, UK 2
Bioanalytics Unit, Institute of Biotechnology, Technische
Universität Berlin, 13355 Berlin, Germany 1
Chromosome Segregation Laboratory, The Francis Crick
Institute, London NW1 1AT, UK 3
Wellcome Centre for Cell Biology, University of Edinburgh,
Edinburgh EH9 3BF, UK The replication checkpoint and sister chromatid
cohesion establishment The replication checkpoint is crucial in moments of replica-
tion fork stalling, e.g., at sites of DNA damage or follow-
ing nucleotide depletion (Pardo et al. 2017). A low level
of replication checkpoint activation is detectable even dur-
ing unperturbed S phase (Forey et al. 2020). It is therefore
conceivable that replisome-cohesin encounters lead to tem-
porary checkpoint activation, which might facilitate sister
chromatid cohesion establishment. In support of this hypoth-
esis, single molecule observations in Xenopus egg extracts
revealed frequent fork stalling upon replisome-cohesin
encounters (Kanke et al. 2016). Tof1-Csm3 and Mrc1 have been studied for their diverse
roles during DNA replication. Mrc1, originally identified
as mediator of the replication checkpoint, promotes Rad53
checkpoint kinase activation in response to replication stress
(Osborn and Elledge 2003; Pardo et al. 2017). Tof1-Csm3
and Mrc1 couple helicase and polymerase progression when
forks slow down, and for this role have become known as
the “fork protection complex” (Katou et al. 2003). Tof1-
Csm3 and Mrc1 also act to secure full replisome speed under
undisturbed conditions, leading to their alternative denomi-
nation as “replisome progression complex” (Tourriere et al. 2005; Yeeles et al. 2017). Tof1, first known as topoisomer-
ase I interacting factor (Park and Sternglanz 1999), further-
more recruits topoisomerase I to the replisome, a function
that may relate to the role of Tof1 in imposing fork paus-
ing at genetically encoded replication fork barriers (Shyian
et al. 2020). Tof1 also interacts with the nucleosome chap-
erone FACT to enhance nucleosome disassembly (Safaric
et al. 2022). Additional reported roles for Tof1-Csm3 and To determine whether the replication checkpoint con-
tributes to cohesion establishment, we measured sister
chromatid cohesion in rad53-11 cells harboring a defec-
tive replication checkpoint effector kinase Rad53 (Shimada
et al. 2002; Forey et al. 2020). Rad53-11 protein is sparsely
expressed and fails to become phosphorylated in response
to exposure to the replication inhibitor hydroxyurea (HU)
(Fig. S1A). To assess the state of sister chromatid cohe-
sion, we synchronized rad53-11 cells in G1 using α-factor,
followed by release to progression through S phase and
arrest in mitosis using the spindle poison nocodazole. Cells
expressed a tet repressor-GFP fusion protein that marks a
tet operator array integrated at the URA3 locus, close to
the centromere of chromosome 5. Premature splitting of
the resulting GFP dot in a mitotic arrest indicates defec-
tive sister chromatid cohesion (Fig. 1A). Introduction inevitably encounters cohesins. However, details of what
happens during these encounters remain elusive. Following
DNA replication, cohesion between sister chromatids has
been established—cohesin now topologically encircles two
sister DNAs (Haering et al. 2008). Furthermore, cohesin is
acetylated at two conserved lysine residues of its Smc3 subu-
nit by the Eco1 acetyl transferase, stabilizing cohesin’s asso-
ciation with DNA (Ben-Shahar et al. 2008; Ünal et al. 2008;
Zhang et al. 2008). Chromosome segregation then occurs
after sister chromatid alignment on the mitotic apparatus,
when cohesins are proteolytically cleaved by the enzyme
separase to trigger anaphase (Uhlmann et al. 2000).f The monumental effort of DNA replication would be wasted
without sister chromatid cohesion, the process that keeps rep-
licated chromosomes together until their timely separation
in anaphase. Concomitantly with DNA replication, cohesion
between sister chromatids is established by the ring-shaped
cohesin complex, which topologically entraps DNA (Nas-
myth and Haering 2009; Peters and Nishiyama 2012; Uhl-
mann 2016). Cohesin molecules are loaded onto DNA during
G1 phase of the cell cycle with the help of a cohesin loader
complex. The turnover of cohesin on DNA at this point is
high, as cohesin’s DNA association is dynamic and subject
to the unloading action of Wapl (Gerlich et al. 2006; Lopez-
Serra et al. 2013). During S phase, the DNA replication fork Different scenarios have been contemplated to explain
cohesion establishment during replication fork-cohesin
encounters. Firstly, G1-loaded cohesin might be converted
into cohesive cohesin during replication fork passage. This
could be achieved either by replication fork passage through
cohesin rings, or through transfer of cohesin behind the fork
(Lengronne et al. 2006). In a second model, cohesin is de
novo loaded behind the replication fork, sequentially entrap-
ping the leading and lagging strands (Murayama et al. 2018). Thirdly, cohesin might be pushed by replication forks to rep-
lication termination sites where sister chromatid cohesion
is established (Cameron et al. 2022). These models are not (0121 3456789)
3 118 Chromosoma (2023) 132:117–135 Mrc1 include replication through secondary structures and
repetitive DNA (Razidlo and Lahue 2008; Gellon et al. 2019; Lerner et al. 2020), as well as replisome disassembly
(Deegan et al. 2020). mutually exclusive, as genetic experiments have found evi-
dence that more than one parallel cohesion establishment
pathway operates in cells (Xu et al. 2007; Borges et al. 2013;
Srinivasan et al. 2020). Introduction Several non-essential components of the replisome have
been identified as “cohesion establishment factors,” i.e., pro-
teins that enable the establishment of sister chromatid cohe-
sion but are not part of the cohesin complex. These include
Ctf4, Chl1, Ctf18-RFC, Tof1-Csm3, and Mrc1 (Hanna et al. 2001; Mayer et al. 2001; Mayer et al. 2004; Skibbens 2004;
Xu et al. 2004). Ctf4 serves as a replisome interaction hub
that, among other proteins, recruits the Chl1 helicase to con-
tact cohesin during cohesion establishment (Samora et al. 2016). The Ctf18-RFC complex increases PCNA levels at
the replication fork, on top of the PCNA that is required
for DNA synthesis (Liu et al. 2020). PCNA in turn recruits
Eco1 that, depending on transient DNA structures that arise
during DNA replication, acetylates Smc3 (Moldovan et al. 2006; Minamino et al. 2023). Tof1-Csm3 and Mrc1 form a
heterotrimeric complex that associates with the CMG heli-
case (Bando et al. 2009; Eickhoff et al. 2019; Baretić et al. 2020). Tof1 and Csm3 intimately interact to form a func-
tional unit, where deletion of either component has the same
consequence on sister chromatid cohesion as deleting both. While Tof1-Csm3 further associate with Mrc1, removing
Mrc1 results in sister chromatid cohesion defects additional
to those due to absence of Tof1-Csm3, suggesting that Mrc1
acts in a parallel cohesion establishment pathway (Xu et al. 2007). It has been suggested that Tof1-Csm3 contributes to
converting G1-loaded cohesin into a cohesive form, while
Mrc1 supports a de novo cohesin loading pathway (Srini-
vasan et al. 2020). The mechanisms by which Tof1-Csm3 or
Mrc1 perform any of these possible roles are yet unknown
and are the subject of our present study. Here, we investigate whether Tof1-Csm3 and Mrc1 con-
tribute to cohesion establishment through one of their repli-
cation functions. By systematically disrupting the replication
checkpoint, fork speed control, as well as topoisomerase I
and FACT recruitment, we find that the role of Tof1-Csm3
and Mrc1 in sister chromatid cohesion establishment is dis-
tinct from these known replication functions. Instead, we
find that Tof1-Csm3 and Mrc1 engage in a series of direct
physical interactions with the cohesin complex. We arrive
at a model in which replisome components not only secure
faithful DNA replication, but directly engage with the
cohesin complex to link DNA replication with sister chro-
matid cohesion establishment. The replication checkpoint and sister chromatid
cohesion establishment While cells lack-
ing Mrc1 (mrc1Δ) displayed a substantial cohesion defect,
compared to wild-type control cells, rad53-11 cells showed 1 3 3 119 Chromosoma (2023) 132:117–135 WT
rad53-11
mrc1
0
10
20
30
40
URA3 -GFP separation
(% of cells)
B
A
C
N
C
mrc1- C
Rad53 phosphosites
1
1096
843
*
N
C
1
1096
mrc1AQ
* *
* *
*
*
*
*
*
*
*
*
*
* Mec1 phosphosites
** *
WT
mrc1
MRC1-myc
mrc1AQ-myc
mrc1- C-myc
0
10
20
30
40
URA3 -GFP separation
(% of cells)
ac-Smc3
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
WT
rad53-11
mrc1
Rad53
Tubulin
G1 → M
(min)
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0.0
0.5
1.0
1.5
Time after G1 release into
metaphase arrest (min)
ac-Smc3 / Tubulin
D
ac-Smc3
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
MRC1
-myc
mrc1AQ
-myc
mrc1
mrc1- C
-myc
Mrc1-Myc
Tubulin
WT
G1 → M
(min)
*
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0.0
0.5
1.0
1.5
2.0
Time after G1 release into metaphase arrest (min)
ac-Smc3 / Tubulin
Fig. 1 The replication checkpoint and cohesion establishment. A Representative example of cells imaged in the GFP dot assay. One
GFP dot (bottom left cell) shows intact cohesion, while separated
GFP dots (top right cell) indicate a cohesion defect. One hundred to
200 cells were scored for each indicated genotype. Three independent
repeats of the experiment were performed. The means and the
individual values are shown, revealing a significant cohesion defect
in mrc1Δ cells (unpaired t test p < 0.0001) as well as a smaller yet
significant defect in rad53-11 cells (p = 0.018). B Immunoblotting
of synchronized cells from A at the indicated time points to analyze
Smc3 acetylation and Rad53. Tubulin served as a loading control. The ac-Smc3/tubulin ratios were normalized to the highest ratio
observed in wild-type cells. Means and individual values from three
independent repeat experiments are shown. Acetylation levels
significantly lower in mrc1Δ cells and higher in rad53-11 (two
ANOVA tests, p = 0.048 and p = 0.0081, respectively). The replication checkpoint and sister chromatid
cohesion establishment The means and the
individual values are shown, revealing a significant cohesion defect
in mrc1Δ cells (unpaired t test p < 0.0001) as well as a smaller yet
significant defect in rad53-11 cells (p = 0.018). B Immunoblotting
of synchronized cells from A at the indicated time points to analyze
Smc3 acetylation and Rad53. Tubulin served as a loading control. The ac-Smc3/tubulin ratios were normalized to the highest ratio
observed in wild-type cells. Means and individual values from three Fig. 1 The replication checkpoint and cohesion establishment. A Representative example of cells imaged in the GFP dot assay. One
GFP dot (bottom left cell) shows intact cohesion, while separated
GFP dots (top right cell) indicate a cohesion defect. One hundred to
200 cells were scored for each indicated genotype. Three independent
repeats of the experiment were performed. The means and the
individual values are shown, revealing a significant cohesion defect
in mrc1Δ cells (unpaired t test p < 0.0001) as well as a smaller yet
significant defect in rad53-11 cells (p = 0.018). B Immunoblotting
of synchronized cells from A at the indicated time points to analyze
Smc3 acetylation and Rad53. Tubulin served as a loading control. The ac-Smc3/tubulin ratios were normalized to the highest ratio
observed in wild-type cells. Means and individual values from three independent repeat experiments are shown. Acetylation levels were
significantly lower in mrc1Δ cells and higher in rad53-11 (two-way
ANOVA tests, p = 0.048 and p = 0.0081, respectively). C Schematic
of the Mrc1 mutants analyzed, as well as result from the GFP dot
assay to assess sister chromatid cohesion. Compared to increased
GFP dot separation in mrc1Δ cells vs. wild type (unpaired t test,
p = 0.0002), mrc1AQ-myc and mrc1-DC-myc cells showed a smaller
but also significant cohesion defect (p = 0.0064 and p = 0.048,
respectively). D Smc3 acetylation was assessed by immunoblotting;
Mrc1 was detected via its myc epitope tag. Acetylation differences,
assessed using a two-way ANVOA test, remained insignificant at
p < 0.05. An antibody background band is marked with an asterisk independent repeat experiments are shown. Acetylation levels were
significantly lower in mrc1Δ cells and higher in rad53-11 (two-way
ANOVA tests, p = 0.048 and p = 0.0081, respectively). C Schematic
of the Mrc1 mutants analyzed, as well as result from the GFP dot
assay to assess sister chromatid cohesion. Compared to increased
GFP dot separation in mrc1Δ cells vs. The replication checkpoint and sister chromatid
cohesion establishment C Sche
of the Mrc1 mutants analyzed, as well as result from the GF
assay to assess sister chromatid cohesion. Compared to incr
GFP dot separation in mrc1Δ cells vs. wild type (unpaired t
p = 0.0002), mrc1AQ-myc and mrc1-DC-myc cells showed a sm
but also significant cohesion defect (p = 0.0064 and p = 0
respectively). D Smc3 acetylation was assessed by immunoblo
Mrc1 was detected via its myc epitope tag. Acetylation differe
assessed using a two-way ANVOA test, remained insignifica
p < 0.05. An antibody background band is marked with an asteri WT
rad53-11
mrc1
0
10
20
30
40
URA3 -GFP separation
(% of cells)
A B
ac-Smc3
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
WT
rad53-11
mrc1
Rad53
Tubulin
G1 → M
(min)
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0.0
0.5
1.0
1.5
ac-Smc3 / Tubulin WT
rad53-11
mrc1
0
10
20
30
40
URA3 -GFP separation
(% of cells) B A A Time after G1 release into
metaphase arrest (min)
D
ac-Smc3
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
MRC1
-myc
mrc1AQ
-myc
mrc1
mrc1- C
-myc
Mrc1-Myc
Tubulin
WT
G1 → M
(min)
*
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0.0
0.5
1.0
1.5
2.0
Time after G1 release into metaphase arrest (min)
ac-Smc3 / Tubulin C
N
C
mrc1- C
Rad53 phosphosites
1
1096
843
*
N
C
1
1096
mrc1AQ
* *
* *
*
*
*
*
*
*
*
*
*
* Mec1 phosphosites
** *
WT
mrc1
MRC1-myc
mrc1AQ-myc
mrc1- C-myc
0
10
20
30
40
URA3 -GFP separation
(% of cells) D C Time after G1 release into metaphase arrest (min) Fig. 1 The replication checkpoint and cohesion establishment. A Representative example of cells imaged in the GFP dot assay. One
GFP dot (bottom left cell) shows intact cohesion, while separated
GFP dots (top right cell) indicate a cohesion defect. One hundred to
200 cells were scored for each indicated genotype. Three independent
repeats of the experiment were performed. The replication checkpoint and sister chromatid
cohesion establishment wild type (unpaired t test,
p = 0.0002), mrc1AQ-myc and mrc1-DC-myc cells showed a smaller
but also significant cohesion defect (p = 0.0064 and p = 0.048,
respectively). D Smc3 acetylation was assessed by immunoblotting;
Mrc1 was detected via its myc epitope tag. Acetylation differences,
assessed using a two-way ANVOA test, remained insignificant at
p < 0.05. An antibody background band is marked with an asterisk reduced in mrc1Δ cells compared to wild type, but instead
appeared slightly elevated in rad53-11 cells (Fig. 1B). These observations reveal largely successful sister chro-
matid cohesion establishment in the absence of a functional
replication checkpoint. a much smaller increase in GFP dot separation (Fig. 1A). As a complementary approach to assess successful sister
chromatid cohesion establishment, we monitored Smc3
acetylation levels during S phase by immunoblotting. As
previously seen (Borges et al. 2013), Smc3 acetylation was 1 3 120 Chromosoma (2023) 132:117–135 The Rad53 checkpoint kinase, as well as the main
upstream sensor of the replication checkpoint, Mec1, are
essential proteins for their role in regulating dNTP synthesis. They can be deleted in the absence of a cellular inhibitor of
dNTP synthesis, Sml1 (Zhao et al. 1998). rad53Δ sml1Δ
and mec1Δ sml1Δ cells showed a small increase in the fre-
quency of GFP dot separation when compared to wild-type
cells, well below the cohesion defects seen in mrc1Δ cells
(Fig. S1B). replication speed of ca. 2100 bp/min) (Theulot et al. 2022). We therefore examined sister chromatid cohesion in wild-
type cultures, supplemented with 20 mM HU during their
progression through S phase. Flow cytometric analysis of
DNA content at 5-min intervals confirmed slower DNA
synthesis, comparable to that in mrc1Δ cells (Fig. 2A). At later times, mrc1Δ cells completed S phase more effi-
ciently than wild-type cells exposed to 20 mM HU, which
could be due to activation of late replication origins in
mrc1Δ but not wild-type cells (Koren et al. 2010). The Rad53 checkpoint kinase, as well as the main
upstream sensor of the replication checkpoint, Mec1, are
essential proteins for their role in regulating dNTP synthesis. They can be deleted in the absence of a cellular inhibitor of
dNTP synthesis, Sml1 (Zhao et al. 1998). rad53Δ sml1Δ
and mec1Δ sml1Δ cells showed a small increase in the fre-
quency of GFP dot separation when compared to wild-type
cells, well below the cohesion defects seen in mrc1Δ cells
(Fig. S1B). The replication checkpoint and sister chromatid
cohesion establishment As an additional way to probe a possible replication
checkpoint contribution to sister chromatid cohesion, we
utilized two checkpoint defective Mrc1 alleles (Fig. 1C). mrc1AQ lacks 17 SQ and TQ motifs that are recognized by
the Mec1 upstream kinase to mediate the checkpoint signal
(Osborn and Elledge 2003). mrc1-ΔC in turn is missing the
Mrc1 C-terminus that harbors a cluster of Rad53 kinase rec-
ognition sites (McClure and Diffley 2021). Previous studies
reported conflicting observations with mrc1AQ cells, report-
ing wild-type levels of sister chromatid cohesion (Xu et al. 2004) or cohesion defects similar to mrc1Δ cells (Tsai et al. 2015). We observed an intermediate frequency of GFP dot
separation in both mrc1AQ and mrc1-ΔC cells, above wild
type but below what is observed in mrc1Δ cells (Fig. 1C). Meanwhile, Smc3 acetylation in mrc1AQ and mrc1-ΔC cells
remained at wild-type levels, unlike the acetylation defect
seen in mrc1Δ cells (Fig. 1D). It is possible that small sister
chromatid cohesion defects, revealed in the GFP dot assay,
arose because the mrc1AQ and mrc1-ΔC alleles also com-
promise Mrc1 function outside of the replication checkpoint. While the exact relationship between the replication check-
point and cohesion establishment merits further exploration,
taken together, our results suggest that cohesion establish-
ment is achieved largely independently of the replication
checkpoint. Unlike mrc1Δ cells, wild-type cells treated with 20 mM
HU did not show increased GFP dot separation—they had
therefore successfully established sister chromatid cohe-
sion (Fig. 2B). Measuring Smc3 acetylation levels in cells
treated with 20 mM HU revealed no reduction, compared
to that seen in mrc1Δ cells (Fig. 2C). On the contrary,
acetylation levels were slightly higher than in wild-type
control cells, an observation that we will return to below. Together, these observations suggest that a slowdown of
replication fork progression is not by itself a cause of sister
chromatid cohesion defects. As an alternative way to probe the relationship between
fork speed and sister chromatid cohesion establishment,
we aimed to restore fork progression in mrc1Δ cells to
wild-type rates. Removing the ribonucleotide reductase
inhibitor Sml1 results in augmented cellular dNTP pools
and an increased fork progression rate (Poli et al. 2012;
Theulot et al. 2022). Faster replication fork progression
in sml1Δ single mutant cells, compared to wild type, did
not cause sister chromatid cohesion defects (Fig. 2A–C),
strengthening the impression that cohesion establishment
occurs independently of a specific fork speed. Replisome speed and cohesion establishment Mrc1 and Tof1/Csm3 ensure that the replisome progresses at
full speed (Tourriere et al. 2005; Yeeles et al. 2017), raising
the question of whether successful cohesion establishment
requires replication fork encounters at a certain pace. Per-
haps cohesion establishment reactions occur at a rate that
must be coordinated with that of fork progression. We began
to investigate this possibility by manipulating replisome
speed independently of Tof1-Csm3 and Mrc1, by altering
the dNTP pools available to DNA polymerases (Fig. 2A). HU inhibits ribonucleotide reductase, the enzyme com-
plex that synthesizes dNTPs. While high HU concentra-
tions (100–200 mM) block replication progression, lower
concentrations lead to gradual replisome slowdown. Wild-
type cells supplemented with 20 mM HU show replica-
tion rates similar to those observed in mrc1Δ cells with-
out HU (both ca. 1100 bp/min, compared to a wild-type Mrc1 and Tof1/Csm3 ensure that the replisome progresses at
full speed (Tourriere et al. 2005; Yeeles et al. 2017), raising
the question of whether successful cohesion establishment
requires replication fork encounters at a certain pace. Per-
haps cohesion establishment reactions occur at a rate that
must be coordinated with that of fork progression. We began
to investigate this possibility by manipulating replisome
speed independently of Tof1-Csm3 and Mrc1, by altering
the dNTP pools available to DNA polymerases (Fig. 2A). Tof1-Csm3 act in a cohesion establishment path-
way parallel to Mrc1 (Xu et al. 2007). We therefore also
explored the effect of restoring replication speed to tof1Δ
and csm3Δ cells. However, neither sister chromatid cohe-
sion nor Smc3 acetylation were improved in tof1Δ or
csm3Δ cells by removing Sml1 (Fig. 2D, E). Replication
speed in the absence of Tof1 or Csm3 decreases more
modestly when compared to mrc1Δ cells, akin to what
is observed in wild-type cells treated with 5–10 mM HU
(Yeeles et al. 2017; Theulot et al. 2022). However, treat-
ing wild-type cells with 5 mM or 10 mM HU also did
not result in noticeable sister chromatid cohesion or Smc3
acetylation defects (Fig. S2A). Together, we conclude that
successful sister chromatid cohesion establishment is pos-
sible at a range of DNA polymerase speeds. HU inhibits ribonucleotide reductase, the enzyme com-
plex that synthesizes dNTPs. While high HU concentra-
tions (100–200 mM) block replication progression, lower
concentrations lead to gradual replisome slowdown. The replication checkpoint and sister chromatid
cohesion establishment Sml1 dele-
tion in mrc1Δ cells returned DNA synthesis rates in the
double mutant cells close to those observed in wild-type
controls (Fig. 2A). However, sister chromatid cohesion or
Smc3 acetylation did not improve (Fig. 2B, C). This sug-
gests that the cohesion defect in mrc1Δ cells did not arise
due to slow replication fork progression. 3 Replisome speed and cohesion establishment Wild-
type cells supplemented with 20 mM HU show replica-
tion rates similar to those observed in mrc1Δ cells with-
out HU (both ca. 1100 bp/min, compared to a wild-type 1 3 3 1
Chromosoma (2023) 132:117–135
A
B
D
WT
tof1
csm3
sml1
tof1 sml1
csm3 sml1
0
10
20
30
40
URA3 -GFP separation
(% of cells)
WT
tof1
tof1
csm3
csm3
sml1
sml1
sml1
ac-Smc3
Tubulin
Sml1
E
35
30
25
0
G1 release into
metaphase arrest (min)
45
60
120
40
1c 2c
1c 2c
1c 2c
1c 2c
1c 2c
WT
mrc1
sml1
mrc1
sml1
20 mM HU
polymerase speed
HU
slow
sml1
fast
pol ε
pol δ
C
ac-Smc3
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
Sml1
Tubulin
WT
mrc1
sml1
mrc1
sml1
20 mM HU
G1 → M
(min)
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0.0
0.5
1.0
1.5
Time after G1 release into metaphase arrest (min)
ac-Smc3/Tubulin
WT
sml1
tof1
csm3
tof1
sml1
csm3
sml1
0.0
0.5
1.0
1.5
ac-Smc3/Tubulin
WT
20mM HU
mrc1
sml1
sml1
mrc1
0
10
20
30
40
URA3 -GFP separation
(% of cells)
Fig. 2 Replisome speed and cohesion establishment. A Schematic
illustrating modulated DNA polymerase speeds. FACS analysis of
DNA content of cells of the indicated genotypes following G1 release
into medium containing nocodazole (and HU as indicated). The wild-
type profile is overlaid onto the others as a dark grey outline. B Sister
chromatid cohesion in the indicated strains, as judged by the GFP dot
assay. Mean and individual values of three independent experiments
are shown. Unpaired t tests revealed a significant cohesion defect in
mrc1Δ cells, compared to wild type (p = 0.0005), but no significant
defect following 20 mM HU treatment, and no significant difference
between mrcl1Δ and sml1Δ mrc1Δ cells. C Immunoblotting at
the indicated time points to analyze Smc3 acetylation and Sml1. Tubulin served as a loading control. The ac-Smc3/tubulin ratios were
normalized to the highest ratio observed in wild-type cells. Mea
and individual values from three independent repeat experimen
are shown. Replisome speed and cohesion establishment Two-way ANOVA tests revealed significantly increas
Smc3 acetylation following 20 mM HU treatment (p = 0.029
while other effect sizes did not reach significance at p < 0.05. As B, using strains of the indicated genotypes. Unpaired t tes
confirmed significant cohesion defects in tof1Δ and csm3Δ cel
compared to wild type (p = 0.0001 and p = 0.0002, respectively
but no significant differences due to sml1 deletion. E As C, but on
samples taken at 120 min were analyzed. Unpaired t tests confirm
significant acetylation defects in tof1Δ and csm3Δ cells, compar
to wild type (p = 0.0055 and p = 0.0007, respectively), but n
significant differences due to sml1 deletion 121 Chromosoma (2023) 132:117–135 35
30
25
0
G1 release into
metaphase arrest (min)
45
60
120
40
1c 2c
1c 2c
1c 2c
1c 2c
1c 2c
WT
mrc1
sml1
mrc1
sml1
20 mM HU A
polymerase speed
HU
slow
sml1
fast
pol ε
pol δ A 1
2
mrc1
sml1 G1 release into B
WT
20mM HU
mrc1
sml1
sml1
mrc1
0
10
20
30
40
URA3 -GFP separation
(% of cells) C
ac-Smc3
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
Sml1
Tubulin
WT
mrc1
sml1
mrc1
sml1
20 mM HU
G1 → M
(min)
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0.0
0.5
1.0
1.5
Time after G1 release into metaphase arrest (min)
ac-Smc3/Tubulin C B D
WT
tof1
csm3
sml1
tof1 sml1
csm3 sml1
0
10
20
30
40
URA3 -GFP separation
(% of cells) WT
tof1
tof1
csm3
csm3
sml1
sml1
sml1
ac-Smc3
Tubulin
Sml1
E
WT
sml1
tof1
csm3
tof1
sml1
csm3
sml1
0.0
0.5
1.0
1.5
ac-Smc3/Tubulin D E Fig. 2 Replisome speed and cohesion establishment. A Schematic
illustrating modulated DNA polymerase speeds. FACS analysis of
DNA content of cells of the indicated genotypes following G1 release
into medium containing nocodazole (and HU as indicated). The wild-
type profile is overlaid onto the others as a dark grey outline. B Sister
chromatid cohesion in the indicated strains, as judged by the GFP dot
assay. Mean and individual values of three independent experiments
are shown. HU augments Smc3 acetylation, independent
of cohesion establishment monitored by GFP dot separation and Smc3 acetylation
(Fig. 3B, C). This suggests that sister chromatid cohesion
establishment is proficient even at slower helicase progres-
sion rates. We had noticed that 20 mM HU treatment led to increased
Smc3 acetylation in wild-type cells (Fig. 2C). Titrating HU
concentrations between 5 and 20 mM (Fig. S2B) revealed
a dose-dependent increase in Smc3 acetylation. While HU-
mediated replication slowdown did not interfere with cohe-
sion establishment, we wondered whether, on the contrary,
increased Smc3 acetylation due to HU treatment could
improve cohesion establishment. We therefore supplemented
cultures of tof1Δ, csm3Δ, and mrc1Δ cells with 20 mM HU
and monitored Smc3 acetylation and sister chromatid cohe-
sion establishment. Indeed, HU exposure restored Smc3
acetylation in tof1Δ, csm3Δ, and mrc1Δ cells to levels seen
in wild-type cells. However, increased Smc3 acetylation was
not accompanied by improved sister chromatid cohesion
(Fig. S2C). This reveals an Smc3 acetylation reaction, in
response to HU treatment, that is independent of successful
sister chromatid cohesion establishment. The nature of this
reaction, as well as its relationship with Smc3 acetylation
during undisturbed replication fork progression, remains to
be investigated. As a complementary approach to address whether the
availability of ssDNA is limiting the efficiency of sister
chromatid cohesion establishment in tof1Δ, csm3Δ, and
mrc1Δ cells, we utilized a mutation in the large subunit of
the single-stranded DNA binding protein rfa1(G77E), which
shows reduced affinity for single-stranded DNA. This allele
improved the cohesion defect seen in the absence of Ctf18-
RFC, a cohesion establishment factor that loads PCNA to
serve as a potential adaptor for cohesin loading (Muray-
ama et al. 2018; Liu et al. 2020; Minamino et al. 2023). Unlike in the case of Ctf18-RFC, the cohesion defects in
the absence of Tof1-Csm3 or Mrc1 remained unaltered by
the rfa1(G77E) allele (Fig. S3A). This observation supports
the idea that Tof1-Csm3 and Mrc1 act in sister chromatid
cohesion establishment independently of affecting ssDNA
accessibility.i As a final approach to modulating the rate of replica-
tion fork progression, we utilized a yeast strain lacking the
catalytic domain of DNA polymerase ε (polε-Δcat). In these
cells, polymerase delta takes over leading strand synthesis,
albeit at markedly reduced DNA unwinding and synthesis
rates (Kesti et al. 1999; Yeeles et al. 2017) (Fig. S3B). polε-
Δcat cells showed a marked increase of GFP dot separation,
as well as slightly reduced Smc3 acetylation. Replisome speed and cohesion establishment Unpaired t tests revealed a significant cohesion defect in
mrc1Δ cells, compared to wild type (p = 0.0005), but no significant
defect following 20 mM HU treatment, and no significant difference
between mrcl1Δ and sml1Δ mrc1Δ cells. C Immunoblotting at
the indicated time points to analyze Smc3 acetylation and Sml1. Tubulin served as a loading control. The ac-Smc3/tubulin ratios were normalized to the highest ratio observed in wild-type cells. Means
and individual values from three independent repeat experiments
are shown. Two-way ANOVA tests revealed significantly increased
Smc3 acetylation following 20 mM HU treatment (p = 0.029),
while other effect sizes did not reach significance at p < 0.05. D
As B, using strains of the indicated genotypes. Unpaired t tests
confirmed significant cohesion defects in tof1Δ and csm3Δ cells,
compared to wild type (p = 0.0001 and p = 0.0002, respectively),
but no significant differences due to sml1 deletion. E As C, but only
samples taken at 120 min were analyzed. Unpaired t tests confirmed
significant acetylation defects in tof1Δ and csm3Δ cells, compared
to wild type (p = 0.0055 and p = 0.0007, respectively), but no
significant differences due to sml1 deletion normalized to the highest ratio observed in wild-type cells. Means
and individual values from three independent repeat experiments
are shown. Two-way ANOVA tests revealed significantly increased
Smc3 acetylation following 20 mM HU treatment (p = 0.029),
while other effect sizes did not reach significance at p < 0.05. D
As B, using strains of the indicated genotypes. Unpaired t tests
confirmed significant cohesion defects in tof1Δ and csm3Δ cells,
compared to wild type (p = 0.0001 and p = 0.0002, respectively),
but no significant differences due to sml1 deletion. E As C, but only
samples taken at 120 min were analyzed. Unpaired t tests confirmed
significant acetylation defects in tof1Δ and csm3Δ cells, compared
to wild type (p = 0.0055 and p = 0.0007, respectively), but no
significant differences due to sml1 deletion 1 3 1 3 122 Chromosoma (2023) 132:117–135 DNA helicase speed and cohesion establishment Above, we altered replisome progression by modulating
DNA polymerase speed via cellular dNTP pools. In com-
parison, Tof1-Csm3 and Mrc1 associate with the CMG
helicase, where they are thought to affect replisome pro-
gression by regulating the speed of DNA unwinding (Eick-
hoff et al. 2019; Baretić et al. 2020; McClure and Diffley
2021). The effects on replisomes of altering helicase or
polymerase speed might differ. For example, slowing down
polymerases while DNA unwinding continues at a constant
rate might result in increased availability of single-stranded
DNA (ssDNA). In contrast, slower DNA unwinding at a
constant rate of DNA synthesis might reduce the amount
of accessible ssDNA. As ssDNA is a substrate for cohesion
establishment (Murayama et al. 2018), we sought a way to
emulate the Tof1-Csm3 and Mrc1 effects more accurately
by modulating the rate of DNA unwinding. HU augments Smc3 acetylation, independent
of cohesion establishment This result can
be interpreted in two ways. It could be that sister chromatid
cohesion establishment is compromised at very low replica-
tion speeds in the polε-Δcat strain, which are substantially
lower than in tof1Δ, csm3Δ, or mrc1Δ cells. Alternatively,
the cohesion defect in polε-Δcat cells could stem from a
role of DNA polymerase ε that is independent of its role in
DNA replication. In support of the latter possibility, a small
C-terminal deletion in DNA polymerase ε elicits a cohesion
defect without apparent effect on DNA replication (Edwards
et al. 2003). The role of DNA polymerase ε in sister chroma-
tid cohesion establishment merits further exploration. 3 Tof1’s role in topoisomerase I and FACT recruitment In addition to impacting on DNA replication, Tof1 is known
to recruit auxiliary proteins to the replisome. A predicted
unstructured C-terminal Tof1 extension contains the interac-
tion sites for both topoisomerase I and FACT (Shyian et al. 2020; Westhorpe et al. 2020; Safaric et al. 2022) (Fig. 4A). It is conceivable that topological stress at replication forks,
which accumulates in the absence of topoisomerase I,
impairs cohesion establishment. Alternatively, histone clear-
ance and redeposition, facilitated by FACT, might have to be
coordinated with sister chromatid cohesion establishment. DNA unwinding is controlled by the replication check-
point. Rad53 phosphorylates Mrc1 on a number of C-ter-
minal phosphorylation sites to slow down replication fork
progression in response to checkpoint activation (McClure
and Diffley 2021) (Fig. 3A). An MRC1-8D allele in which
8 of these phosphorylation sites were changed to phospho-
rylation-mimicking aspartates imposes constitutive replica-
tion fork slowdown. We therefore analyzed the proficiency
of MRC1-8D cells to establish sister chromatid cohesion. Despite slower DNA unwinding, we did not observe any
cohesion establishment defect in MRC1-8D cells, as 1 3 123 Chromosoma (2023) 132:117–135 35
30
25
0
G1 release into
metaphase arrest (min)
45
60
120
40
1c 2c
1c 2c
1c 2c
1c 2c
WT
MRC1 8D
-FLAG
MRC1
-FLAG
mrc1
WT
mrc1
MRC1-FLAG
MRC1 8D-FLAG
0
10
20
30
40
URA3 -GFP separation
(% of cells)
A
B
ac-Smc3
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
Tubulin
Flag
G1 → M
(min)
WT
MRC1 8D
-FLAG
MRC1
-FLAG
mrc1
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0.0
0.5
1.0
1.5
Time after G1 release into metaphase arrest (min)
ac-Smc3/Tubulin
C
N
C
1
1096
MRC1 8D
* *
* *
* *
* Rad53 phosphosites
**
CMG
helicase speed
MRC1 8D
slow
Fig. 3 Helicase speed and cohesion establishment. A Schematic
depicting the MRC1 8D allele and its effect on the speed of DNA
unwinding. FACS analysis of DNA content as cells of the indicated
genotypes were released from G1 block for synchronous progression
through S phase into nocodazole-imposed mitotic arrest. The wild-
type profile is overlaid onto the others as a dark grey outline. Tof1’s role in topoisomerase I and FACT recruitment B Sister
chromatid cohesion in the same experiment was assessed by the GFP
dot assay at the 120-min time point. Means and individual values of
three independent repeat experiments are shown Unpaired t tests
confirmed a significant cohesion defect in mrc1Δ cells compare
wild type (p = 0.0012), but no significant difference between MR
FLAG and MRC1 8D-FLAG cells. C Smc3 acetylation during
same experiment was analyzed by immunoblotting. Tubulin ser
as a loading control. The ac-Smc3/tubulin ratios were normalize
the highest ratio observed in wild-type cells. Means and individ
values from three independent repeat experiments are shown. T
way ANOVA tests showed that acetylation differences remai
insignificant at p < 0 05 35
30
25
0
G1 release into
metaphase arrest (min)
45
60
120
40
1c 2c
1c 2c
1c 2c
1c 2c
WT
MRC1 8D
-FLAG
MRC1
-FLAG
mrc1
A
N
C
1
1096
MRC1 8D
* *
* *
* *
* Rad53 phosphosites
**
CMG
helicase speed
MRC1 8D
slow 35
30
25
0
G1 release into
metaphase arrest (min)
45
60
120
40
1c 2c
1c 2c
1c 2c
1c 2c
WT
MRC1 8D
-FLAG
MRC1
-FLAG
mrc1 A A
N
C
1
1096
MRC1 8D
* *
* *
* *
* Rad53 phosphosites
**
CMG G1 release into ac-Smc3
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
Tubulin
Flag
G1 → M
(min)
WT
MRC1 8D
-FLAG
MRC1
-FLAG
mrc1
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0.0
0.5
1.0
1.5
Time after G1 release into metaphase arrest (min)
ac-Smc3/Tubulin
C WT
mrc1
MRC1-FLAG
MRC1 8D-FLAG
0
10
20
30
40
URA3 -GFP separation
(% of cells)
B C B Time after G1 release into metaphase arrest (min) Fig. 3 Helicase speed and cohesion establishment. A Schematic
depicting the MRC1 8D allele and its effect on the speed of DNA
unwinding. FACS analysis of DNA content as cells of the indicated
genotypes were released from G1 block for synchronous progression
through S phase into nocodazole-imposed mitotic arrest. The wild-
type profile is overlaid onto the others as a dark grey outline. B Sister
chromatid cohesion in the same experiment was assessed by the GFP
dot assay at the 120-min time point. Tof1’s role in topoisomerase I and FACT recruitment Two-way ANOVA tests confirmed a
significant Smc3 acetylation defect in tof1Δ cells compared to wild
type (p = 0.0001), but no significant difference between TOF1-HA
and tof1-ΔC-HA cells. Acetylation was significantly increased in
top1Δ cells (p = 0.0021) N-terminus at the front of the replisome (Eickhoff et al. 2019; Baretić et al. 2020), a prime location where cohesion
establishment factors would physically encounter cohesin as
the replication fork approaches. We therefore investigated
whether Tof1-Csm3 and Mrc1 engage in protein interactions
with cohesin. fundamental role in relieving topological stress. We were
therefore able to use a strain devoid of topoisomerase I
(top1Δ) to independently address whether this enzyme
contributes to cohesion establishment. We found that topoi-
somerase I is dispensable for cohesion establishment, as seen
in the GFP dot assay (Fig. 4A). We noticed somewhat ele-
vated Smc3 acetylation levels in top1Δ cells, when compared
to wild-type cells (Fig. 4B). The reason for this increase,
like that previously seen in HU-treated cells, remains to be
elucidated. In summary, we conclude that successful cohe-
sion establishment is possible without interactions that the
Tof1 C-terminus provides with auxiliary replication factors,
including topoisomerase I. N-terminus at the front of the replisome (Eickhoff et al. 2019; Baretić et al. 2020), a prime location where cohesion
establishment factors would physically encounter cohesin as
the replication fork approaches. We therefore investigated
whether Tof1-Csm3 and Mrc1 engage in protein interactions
with cohesin. Previous studies using nematode and human cell extracts
have reported an interaction between their respective Tof1
orthologs, TIM-1 and TIMELESS, and cohesin (Chan et al. 2003; Leman et al. 2010). However, a mass spectrometry-
based interaction screen using human cell extracts failed
to confirm a TIMELESS-cohesin interaction (Ivanov et al. 2018). To investigate the possibility of direct Tof1-Csm3 or
Mrc1 interactions with cohesin, we biochemically purified
budding yeast Tof1-Csm3, Mrc1, and cohesin (Yeeles et al. 2017; Minamino et al. 2018). We also included the cohesion
establishment factor Chl1, which was reported to interact
with cohesin in human and yeast cell extracts (Parish et al. 2006; Samora et al. 2016). Tof1’s role in topoisomerase I and FACT recruitment Means and individual values of
three independent repeat experiments are shown. Unpaired t tests confirmed a significant cohesion defect in mrc1Δ cells compared to
wild type (p = 0.0012), but no significant difference between MRC1-
FLAG and MRC1 8D-FLAG cells. C Smc3 acetylation during the
same experiment was analyzed by immunoblotting. Tubulin served
as a loading control. The ac-Smc3/tubulin ratios were normalized to
the highest ratio observed in wild-type cells. Means and individual
values from three independent repeat experiments are shown. Two-
way ANOVA tests showed that acetylation differences remained
insignificant at p < 0.05 To determine whether topoisomerase I and/or FACT
recruitment to the replisome play a role in cohesion estab-
lishment, we made use of a C-terminal Tof1 truncation
mutant (tof1-ΔC, lacking residues 982–1238) that no longer
recruits topoisomerase I and FACT (Westhorpe et al. 2020;
Safaric et al. 2022). We then assessed sister chromatid
cohesion and Smc3 acetylation. The frequency of GFP
dot separation, as well as Smc3 acetylation levels, was indistinguishable between wild-type and tof1-ΔC cells, while
cells lacking Tof1 altogether (tof1Δ) displayed the expected
cohesion defect signature (Fig. 4A, B). These observations
suggest that topoisomerase I or FACT recruitment to the
replisome is dispensable for successful sister chromatid
cohesion establishment. Topoisomerase I is a non-essential protein in bud-
ding yeast, as topoisomerase II can compensate for its 1 3 1 Chromosoma (2023) 132:117–135 124 B
A
N
C
tof1- C
1
1238
981
FACT - interacting region
Top1 interacting region
WT
tof1
tof1- C-HA
top1
0
10
20
30
40
URA3 -GFP separation
(% of cells)
TOF1-HA
ac-Smc3
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
TOF1-HA
t
A
WT
tof1
of1- C-H
top1
HA
Tubulin
G1 → M
(min)
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0.0
0.5
1.0
1.5
Time after G1 release into metaphase arrest (min)
ac-Smc3/Tubulin
Fig. 4 Tof1 and auxiliary replisome components. A Schematic
depicting the Tof1 topoisomerase I and FACT interaction motifs,
as well as the tof1-ΔC truncation. Sister chromatid cohesion was
assessed in cells of the indicated genotypes 120 min after release
from G1 block into nocodazole-imposed mitotic arrest. Means and
individual values of three independent repeat experiments are shown. Tof1’s role in topoisomerase I and FACT recruitment Unpaired t tests confirmed a significant cohesion defect in tof1Δ cells
compared to wild type (p = 0.0001), but no significant difference
between TOF1-HA and tof1-ΔC-HA cells, or between top1Δ and
the wild-type control. B Immunoblotting of samples from the same
experiment at the indicated times to analyze Smc3 acetylation and
Tof1. Tubulin served as a loading control. The ac-Smc3/tubulin
ratios were normalized to the highest ratio observed in wild-type
cells. Means and individual values from three independent repeat
experiments are shown. Two-way ANOVA tests confirmed a
significant Smc3 acetylation defect in tof1Δ cells compared to wild
type (p = 0.0001), but no significant difference between TOF1-HA
and tof1-ΔC-HA cells. Acetylation was significantly increased in
top1Δ cells (p = 0.0021) A
N
C
tof1- C
1
1238
981
FACT - interacting region
Top1 interacting region
WT
tof1
tof1- C-HA
top1
0
10
20
30
40
URA3 -GFP separation
(% of cells)
TOF1-HA B
ac-Smc3
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
TOF1-HA
t
A
WT
tof1
of1- C-H
top1
HA
Tubulin
G1 → M
(min)
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0
30
60
90
120
0.0
0.5
1.0
1.5
Time after G1 release into metaphase arrest (min)
ac-Smc3/Tubulin B A A Time after G1 release into metaphase arrest (min) Fig. 4 Tof1 and auxiliary replisome components. A Schematic
depicting the Tof1 topoisomerase I and FACT interaction motifs,
as well as the tof1-ΔC truncation. Sister chromatid cohesion was
assessed in cells of the indicated genotypes 120 min after release
from G1 block into nocodazole-imposed mitotic arrest. Means and
individual values of three independent repeat experiments are shown. Unpaired t tests confirmed a significant cohesion defect in tof1Δ cells
compared to wild type (p = 0.0001), but no significant difference
between TOF1-HA and tof1-ΔC-HA cells, or between top1Δ and
the wild-type control. B Immunoblotting of samples from the same experiment at the indicated times to analyze Smc3 acetylation and
Tof1. Tubulin served as a loading control. The ac-Smc3/tubulin
ratios were normalized to the highest ratio observed in wild-type
cells. Means and individual values from three independent repeat
experiments are shown. Direct protein interactions between Tof1‑Csm3,
Mrc1, and cohesin The known roles of Tof1-Csm3 and Mrc1 at the repli-
some, which we so far studied, appear unrelated to the
establishment of sister chromatid cohesion. We therefore
hypothesized that Tof1-Csm3 and Mrc1 perform a previ-
ously unknown function in cohesion establishment. Recent
structural studies have placed Tof1-Csm3 and the Mrc1 To investigate the possibility of direct protein interac-
tions, we employed an experimental setup in which a puri-
fied target protein is immobilized on beads, before these are
briefly immersed with candidate binding partners (C. Smith 1 3 125 Chromosoma (2023) 132:117–135 and J. Diffley, personal communication). We immobilized
cohesin using an antibody against a Pk epitope tag fused to
its Smc1 subunit. These cohesin-covered beads, or antibody-
only control beads, were incubated with Tof1-Csm3, Mrc1,
or Chl1. Not reported to interact with cohesin, the GINS
complex was used as a control. Beads were then washed,
bound protein eluted, and analyzed by SDS-polyacrylamide
gel electrophoresis followed by Coomassie Blue staining. Tof1-Csm3, Mrc1, as well as Chl1 were all recovered in
the bead-bound fraction in a cohesin-dependent manner
(Fig. 5A), suggesting that these cohesion establishment fac-
tors directly interact with the cohesin complex. The GINS
complex, on the other hand, did not interact with cohesin.i Mrc1 on beads using an antibody against a Flag epitope
tag on Mrc1. Following incubation with cohesin and
washes, the bound protein was eluted by incubation with
Flag peptide. Mrc1 on beads, but not control beads, inter-
acted with cohesin (Fig. 5B). Addition of both Tof1-Csm3
and cohesin to Mrc1 beads led to the retention of both
complexes, consistent with direct interactions among the
three components. As a control, GINS was not retained on
Mrc1 beads (to detect any possible GINS traces, elution in
this case was with SDS). We also immobilized Tof1-Csm3 on beads, which
specifically interacted with cohesin (Fig. S4A). Direct protein interactions between Tof1‑Csm3,
Mrc1, and cohesin This
interaction was unaltered by the inclusion of benzonase
during the incubations, excluding the possibility that the p
To confirm the interaction of Tof1-Csm3 and Mrc1
with cohesin, we took a reverse approach and immobilized during the incubations, excluding the possibility that the
cc3
Pk IP
250
150
100
75
50
37
25
20
kDa
Smc1/Smc3/Scc3
Scc1
IgG/Psf2
Psf1/Psf3
GINS (Input)
-
+
IgG
Sld5
Pk
GINS
Cohesin
Tof1/Csm3
Mrc1
Chl1
250
150
100
75
50
37
25
20
kDa
Mrc1
Tof1/Csm3
IgG/Psf2
Psf1/Psf3
IgG
Sld5
Csm3
GINS
-
+
Input
Flag IP
-
+
-
+
GINS
Flag
staining. B Interaction screen with Mrc1 as the bait. Mrc1-coated or
control beads were first incubated with or without Tof1-Csm3, before
a further incubation with cohesin or the GINS complex and analyzed
as above A
250
150
100
75
50
37
kDa
Input
-
+
-
+
-
+
Pk IP
Mrc1
Smc1/Smc3/Scc3
Tof1
Chl1
Scc1
Csm3/IgG
Pk
Pk IP
250
150
100
75
50
37
25
20
kDa
Smc1/Smc3/Scc3
Scc1
IgG/Psf2
Psf1/Psf3
GINS (Input)
-
+
IgG
Sld5
Pk
GINS
B
Cohesin
Tof1/Csm3
Mrc1
Chl1
250
150
100
75
50
37
25
20
kDa
Mrc1
Tof1/Csm3
IgG/Psf2
Psf1/Psf3
IgG
Sld5
Csm3
GINS
-
+
Input
Flag IP
-
+
-
+
GINS
Flag
Mrc1
Smc1/Smc3/Scc3
Tof1
Scc1
Csm3
Input
-
+
-
+
-
+
Flag IP
Flag
250
150
100
75
50
37
kDa
g. 5 Protein interactions between cohesion establishment factors
nd cohesin. A Interaction screen with cohesin as the bait. Cohesin-
oated or control beads were incubated with the indicated replisome
omponents. Ten percent of input proteins are loaded next to the
ead-bound fractions. Proteins were visualized by Coomassie Blue
staining. B Interaction screen with Mrc1 as the bait. Direct protein interactions between Tof1‑Csm3,
Mrc1, and cohesin Mrc1-coated
control beads were first incubated with or without Tof1-Csm3, befo
a further incubation with cohesin or the GINS complex and analyz
as above Pk IP
250
150
100
75
50
37
25
20
kDa
Smc1/Smc3/Scc3
Scc1
IgG/Psf2
Psf1/Psf3
GINS (Input)
-
+
IgG
Sld5
Pk
GINS
Cohesin
Tof1/Csm3
Mrc1
Chl1 A
250
150
100
75
50
37
kDa
Input
-
+
-
+
-
+
Pk IP
Mrc1
Smc1/Smc3/Scc
Tof1
Chl1
Scc1
Csm3/IgG
Pk A B
Mrc1
Smc1/Smc3/Scc3
Tof1
Scc1
Csm3
Input
-
+
-
+
-
+
Flag IP
Flag
250
150
100
75
50
37
kDa 250
150
100
75
50
37
25
20
kDa
Mrc1
Tof1/Csm3
IgG/Psf2
Psf1/Psf3
IgG
Sld5
Csm3
GINS
-
+
Input
Flag IP
-
+
-
+
GINS
Flag B staining. B Interaction screen with Mrc1 as the bait. Mrc1-coated or
control beads were first incubated with or without Tof1-Csm3, before
a further incubation with cohesin or the GINS complex and analyzed
as above Fig. 5 Protein interactions between cohesion establishment factors
and cohesin. A Interaction screen with cohesin as the bait. Cohesin-
coated or control beads were incubated with the indicated replisome
components. Ten percent of input proteins are loaded next to the
bead-bound fractions. Proteins were visualized by Coomassie Blue 1 3 126 Chromosoma (2023) 132:117–135 interaction was mediated through DNA that might have
contaminated our purified proteins.f analysis. We immobilized Tof1-Csm3 or Mrc1 on beads that
we then incubated with either purified cohesin tetramer com-
plexes (Smc1-Smc3-Scc1-Scc3), cohesin trimers (Smc1-
Smc3-Scc1), or SMC dimers (Smc1-Smc3). The SMC
dimer interacted with both Tof1-Csm3 and Mrc1 equally, if
not more efficiently, when compared to the cohesin trimer
or tetramer complexes (Fig. 6C). This finding is consistent
with the notion that Tof1-Csm3 and Mrc1 make prominent
contacts with the Smc1-Smc3 dimer. i
So far, we performed our interaction incubations in buffer
containing 100 mM potassium glutamate, conditions close
to a budding yeast physiological environment. To test how
stably Tof1-Csm3 interacts with cohesin, we added increas-
ing NaCl concentrations during the binding incubation. The
interaction was gradually lost in the presence of more than
250 mM NaCl (Fig. S4B), suggesting that polar or charge
interactions contribute. We also performed benzonase treat-
ment and salt titration for the Mrc1-cohesin interaction with
similar outcomes (Fig. S4C,D). Direct protein interactions between Tof1‑Csm3,
Mrc1, and cohesin On the cohesion establishment factor side, Tof1 yielded
the greatest number of cohesin crosslinks, which we mapped
onto its available cryo-EM structure (Fig. 7A) (Baretić et al. 2020). Five of the eight identified crosslinks emanated from
unresolved regions that we filled using AlphaFold (Jumper
et al. 2021) predictions, suggesting a role for these probably
more flexible Tof1 regions in providing cohesin contacts. Though we note that flexible protein regions might also show
a greater propensity for spurious CLMS contacts. To investi-
gate the importance of these interaction sites, we performed
a Tof1 truncation analysis. We used previously characterized
Tof1 C-terminal truncations (Westhorpe et al. 2020) and
measured sister chromatid cohesion in these mutants. We
had earlier tested a C-terminal deletion ending at amino acid
981, which is defective in topoisomerase I recruitment but
was proficient in supporting cohesion establishment. When
Tof1 was further truncated, we started to observe cohesion
defects (Fig. 7B, C). A central Tof1 domain delineated by
this analysis (covering amino acids 628–831) is a focal point
of four of the identified CLMS cohesin contact sites, consist-
ent with the idea that Tof1 contacts cohesin during cohesion
establishment. Additionally, the central Tof1 domain might
be required for Tof1-replisome interactions. Finally, we tested whether complexes formed between
Tof1-Csm3, Mrc1 and cohesin are stable enough to be
characterized by size exclusion chromatography. Following
incubation, individual or combined protein samples were
separated on a gel filtration column. Immunoblot analysis
showed that cohesin and Tof1-Csm3 (visualized by a CBP
tag on Csm3) individually eluted at volumes expected of
their respective sizes (Fig. S5). After co-incubation, a faint
Csm3 band became detectable in earlier fractions where
cohesin elutes, while the bulk of the Csm3 profile remained
unchanged, suggesting that the Tof1-Csm3 interaction with
cohesin is weak. In a similar experiment, Mrc1 mostly
co-eluted with cohesin, suggesting that Mrc1 and cohesin
engage in a more durable interaction. Tof1‑Csm3 and Mrc1 deploy multipronged cohesin
interactions To explore the cohesion establishment factor interactions
with cohesin, we performed protein crosslinking mass
spectrometry (CLMS) to identify interaction sites. In two
repeats of the experiment, we included cohesin, Tof1-Csm3
and Chl1, either with or without Mrc1. Cα atom distances
spanned by crosslinks inside known structured regions of
individual proteins fell within the expected range of the
sulfo-SDA crosslinker (Fig. S6). Furthermore, we identi-
fied numerous crosslinks between subunits of the cohesin
complex, as well as between Tof1 and Csm3, that were con-
sistent with prior structural knowledge (Fig. 6A) (Baretić
et al. 2020; Higashi et al. 2020), thereby overall validating
the CLMS experiment. Inspecting the cohesin crosslinks with Mrc1 revealed an
Smc3 interaction close to where Mrc1 interacts with Tof1
(Fig. 7D) (Baretić et al. 2020). The Mrc1 interactions on
Tof1 in turn lie close to Tof1-Smc3 linkages (Fig. 7B), thus
pinpointing a candidate Smc3-Tof1-Mrc1 interaction hub. Two other cohesin linkages fall into an Mrc1 region between
its reported MCM and Cdc45 contact points. Mrc1 likely
comprises of long unstructured extensions between its repli-
some contacts. Interactions with one of these long loops
may provide a flexible cohesin tether that supports cohesion
establishment. It will be of interest to define the interaction
sites between Tof1-Csm3, Mrc1, and cohesin in more detail
in a future study, which will allow interrogating the contri-
butions of these interactions to sister chromatid cohesion
establishment using site-specific mutations. Additionally, we detected crosslinks between the cohe-
sion establishment factors and cohesin (Fig. 6A). Most prev-
alent were interactions of Tof1 and Mrc1 with Smc1 and
Smc3, with an additional link each between Mrc1 and Scc3,
as well as Chl1 and Scc1. Figure 6B shows these interac-
tions mapped onto a Smc1-Smc3 structural model, revealing
interaction clusters along both SMC coiled coils. To experi-
mentally validate the conclusion that Tof1-Csm3 and Mrc1
preferentially interact with the Smc1-Smc3 dimer within the
cohesin complex, we extended our biochemical interaction 3 A protein interaction between Tof1 and Chl1 C Interaction screen with Tof1-Csm3 (left) or
Mrc1 (right) on beads, comparing cohesin tetramer, cohesin trimer,
and Smc1-Smc3 dimer as binding partners. Ten percent of the input
and the bead bound fractions were analyzed by SDS-polyacrylamide
gel electrophoresis followed by Coomassie Blue staining 250
150
100
75
50
kDa
Smc1/
Smc3/
Scc3
Tof1
Scc1
Csm3
Tof1/Csm3
Input
Csm3 IP
-
+
-
+
-
+
Tof1/Csm3
C
Smc1/Smc3
Smc1/Smc3/Sc
1 C Fig. 6 Multipronged cohesion establishment factor interactions with
the cohesin complex. A Collated circos plot of interprotein crosslinks
detected by CLMS in the two samples. Grey lines represent intersub-
unit crosslinks within the cohesin or Tof1-Csm3 complexes. Linkages
between cohesin and Tof1 (green), Mrc1 (red), and Chl1 (purple) are
highlighted. B Atomic model of the budding yeast Smc1-Smc3 dimer
and Scc1 N-terminus, built using Phyre2 (Kelley et al. 2015) based on a fission yeast cohesin model (Higashi et al. 2020). CLMS linkage
sites are highlighted. C Interaction screen with Tof1-Csm3 (left) or
Mrc1 (right) on beads, comparing cohesin tetramer, cohesin trimer,
and Smc1-Smc3 dimer as binding partners. Ten percent of the input
and the bead bound fractions were analyzed by SDS-polyacrylamide
gel electrophoresis followed by Coomassie Blue staining A protein interaction between Tof1 and Chl1 Cohesion establishment factors at the replication fork
engage in physical contacts with each other. Tof1-Csm3
and Mrc1 form a complex, while Ctf4 interacts with 3 3 127 Chromosoma (2023) 132:117–135 127
Chromosoma (2023) 132:117–135
250
150
100
75
50
kDa
Smc1/
Smc3/
Scc3
Tof1
Scc1
Csm3
Tof1/Csm3
Input
Csm3 IP
-
+
-
+
-
+
Tof1/Csm3
C
250
150
100
75
50
kDa
Mrc1
Smc1/
Smc3/
Scc3
Scc1
Mrc1
Input
Mrc1 IP
-
+
-
+
-
+
Mrc1
Smc1/Smc3
Smc1/Smc3/Scc1
Smc1/Smc3/Scc1/Scc3
A
head
1
1096
1
566
1
1225
1
1150
1
1230
1
317
1
1238
1
861
TOF1
CSM3
SMC1
SMC3
SCC1
SCC3
CHL1
MRC1
B
head
915Smc1 494Mrc1
115Tof1 622Smc3
651Tof1 927Smc3
1212Smc1
872Smc1 595Tof1
860Tof1 387Smc3
1092Tof1
903Smc3
899Smc3
736Tof11003Smc3
390Tof1 96Scc1
291Mrc1 208Smc3
318Tof1
hinge
SMC3
SMC1
SCC1
Fig. 6 Multipronged cohesion establishment factor interactions with
the cohesin complex. A Collated circos plot of interprotein crosslinks
detected by CLMS in the two samples. Grey lines represent intersub-
unit crosslinks within the cohesin or Tof1-Csm3 complexes. Linkages
between cohesin and Tof1 (green), Mrc1 (red), and Chl1 (purple) are
highlighted. B Atomic model of the budding yeast Smc1-Smc3 dimer
and Scc1 N-terminus, built using Phyre2 (Kelley et al. 2015) based
on a fission yeast cohesin model (Higashi et al. 2020). CLMS linkage
sites are highlighted. C Interaction screen with Tof1-Csm3 (left) or
Mrc1 (right) on beads, comparing cohesin tetramer, cohesin trimer,
and Smc1-Smc3 dimer as binding partners. Ten percent of the input
and the bead bound fractions were analyzed by SDS-polyacrylamide
gel electrophoresis followed by Coomassie Blue staining A
1
1096
1
566
1
1225
1
1150
1
1230
1
317
1
1238
1
861
TOF1
CSM3
SMC1
SMC3
SCC1
SCC3
CHL1
MRC1
B
6
86
1092 head
B
head
915Smc1 494Mrc1
115Tof1 622Smc3
651Tof1 927Smc3
1212Smc1
872Smc1 595Tof1
860Tof1 387Smc3
92Tof1
903Smc3
899Smc3
736Tof11003Smc3
390Tof1 96Scc1
291Mrc1 208Smc3
318Tof1
hinge
SMC3
SMC1
SCC1 B A 250
150
100
75
50
kDa
Mrc1
Smc1/
Smc3/
Scc3
Scc1
Mrc1
Input
Mrc1 IP
-
+
-
+
-
+
Mrc1
c1
Smc1/Smc3/Scc1/Scc3
head
head
915Smc1 494Mrc1
115Tof1 622Smc3
51Tof1 927Smc3
1212Smc1
872Smc1 595Tof1
0Tof1 387Smc3
Tof1
903Smc3
899Smc3
736Tof11003Smc3
390Tof1 96Scc1
291Mrc1 208Smc3
318Tof1
hinge
SMC3
SMC1
SCC1
on a fission yeast cohesin model (Higashi et al. 2020). CLMS linkage
sites are highlighted. Discussion Chl1 (Bando et al. 2009; Samora et al. 2016). The human
Tof1 and Chl1 orthologs, TIMELESS and DDX11, have
also been reported to interact (Cortone et al. 2018). To
address whether budding yeast Chl1 and Tof1 interact,
we immobilized Chl1 on beads that we then incubated
with Tof1-Csm3. Tof1-Csm3 was efficiently retained on
Chl1-containing beads, but not on control beads (Fig. S7). Therefore, an interaction between Chl1 and Tof1-Csm3 is
a feature conserved from yeast to human. The roles of Tof1-Csm3 and Mrc1 in sister chromatid
cohesion establishment have been known for almost two
decades, but the basis for their contribution has remained
unknown. In our study, we have systematically ruled out
many of the multiple roles by which Tof1-Csm3 and Mrc1
participate in DNA replication. Instead, our findings open 1 3 128 Chromosoma (2023) 132:117–135 (
)
A
ed
h). nd
y. -
g
-
-
ual
f
ks,
m-
A
90°
115Tof1 622Smc3
860Tof1 387Smc3
651Tof1 927Smc3
1092Tof1 899Smc3
1092Tof1 903Smc3
390Tof1 96Scc1
318Tof1 1212Smc1
736Tof1 1003Smc3
595Tof1 872Smc1
Tof1
(Alphafold)
Tof1-Csm3
PDB 6SKL
MCMs
B
WT
tof1
TOF1-HA
-HA
-HA
tof1( 628-1238)
tof1( 763-1238)
tof1( 831-1238)-HA
ac-Smc3
Tubulin
HA
150 kDa
75 kDa
WT
tof1
)-HA
)-HA
tof1( 628 -1238
tof1( 763 -1238
tof1( 831-1238)-HA
0.0
0.5
1.0
1.5
ac-Smc3/Tubulin
TOF1-HA
C
Smc1
Smc3
Scc1
N
C
TOF1
1
1238
( 628 -1238)
( 763 -1238)
( 831 -1238)
WT
tof1
tof1( 628-1238)-HA
tof1( 763-1238)-HA
tof1( 831-1238)-HA
0
10
20
30
40
URA3 -GFP separation
(% of cells)
TOF1-HA
C
N
MRC1
1
1096
Tof1 Mcm6 Mcm2 Cdc45 Ctf4
Smc3 Smc1 Scc3
D 90°
115Tof1 622Smc3
860Tof1 387Smc3
651Tof1 927Smc3
1092Tof1 899Smc3
1092Tof1 903Smc3
390Tof1 96Scc1
318Tof1 1212Smc1
736Tof1 1003Smc3
595Tof1 872Smc1
WT
tof1
TOF1-HA
-HA
-HA
tof1( 628-1238)
tof1( 763-1238)
tof1( 831-1238)-HA
ac-Smc3
Tubulin
HA
150 kDa
75 kDa
WT
tof1
)-HA
)-HA
tof1( 628 -1238
tof1( 763 -1238
tof1( 831-1238)-HA
0.0
0.5
1.0
1.5
ac-Smc3/Tubulin
TOF1-HA
C A
90°
115Tof1 622Smc3
860Tof1 387Smc3
651Tof1 927Smc3
1092Tof1 899Smc3
1092Tof1 903Smc3
390Tof1 96Scc1
318Tof1 1212Smc1
736Tof1 1003Smc3
595Tof1 872Smc1
Tof1
(Alphafold)
Tof1-Csm3
PDB 6SKL
MCMs Fig. 7 Tof1 regions important
for sister chromatid cohesion. A
CLMS contact sites, highlighted
on the Tof1-Csm3 structure
(green) (Baretić et al. 2020),
with unresolved Tof1 regions
(amino acids 107–115, 305–
328, 605–659, and 782–1000)
filled with their AlphaFold
predictions (cyan, manually
adjusted to avoid a DNA clash). Discussion Mrc1 in turn makes a parallel contri-
bution to cohesion establishment. This contribution could
lie in securing additional cohesin contacts as the replisome
progresses, resulting in a choreographed series of cohesin
interactions that lead to cohesion establishment. the possibility that a series of direct physical interactions
between these replication fork components and the cohesin
complex facilitate successful sister chromatid cohesion
establishment during DNA replication. Discussion Linkages with Smc1, Smc3, and
Scc1 are highlighted in grey,
yellow, and black, respectively. DNA and the MCM hexamer
are shown for context. B Sche-
matic representation of Tof1
and its cohesin crosslinks, as
well as of three Tof1 trunca-
tion mutants. Sister chromatid
cohesion in strains of the
indicated genotypes, including
the three truncation mutants,
was assessed by the GFP dot
assay. Means and individual
values from two independent
repeat experiments are shown. C Smc3 acetylation and Tof1
were analyzed by immunoblot-
ting in the same experiment. Tubulin served as a loading
control. The ac-Smc3/tubulin
ratios were normalized to the
highest ratio observed in wild-
type cells. Means and individual
values from two independent
repeat experiments are shown. D Schematic representation of
Mrc1 and its cohesin crosslinks,
as well as previously reported
crosslinks with replisome com-
ponents (Baretić et al. 2020) A B
Smc1
Smc3
Scc1
N
C
TOF1
1
1238
( 628 -1238)
( 763 -1238)
( 831 -1238)
WT
tof1
tof1( 628-1238)-HA
tof1( 763-1238)-HA
tof1( 831-1238)-HA
0
10
20
30
40
URA3 -GFP separation
(% of cells)
TOF1-HA B B C WT
tof1
TOF1-HA
-HA
-HA
tof1( 628-1238)
tof1( 763-1238)
tof1( 831-1238)-HA
ac-Smc3
Tubulin
HA
150 kDa
75 kDa
WT
tof1
)-HA
)-HA
tof1( 628 -1238
tof1( 763 -1238
tof1( 831-1238)-HA
0.0
0.5
1.0
1.5
ac-Smc3/Tubulin
TOF1-HA
C C
N
MRC1
1
1096
Tof1 Mcm6 Mcm2 Cdc45 Ctf4
Smc3 Smc1 Scc3
D D Ctf4 to a position that likely also points towards the front of
the replisome (Samora et al. 2016). Thus, it could be that the
cohesion establishment factors Tof1-Csm3 and Chl1, which
form one epistasis group for their shared contributions to
sister chromatid cohesion establishment (Xu et al. 2007), are
jointly the first ones to engage with cohesin as the replica-
tion fork approaches. Mrc1 in turn makes a parallel contri-
bution to cohesion establishment. This contribution could
lie in securing additional cohesin contacts as the replisome
progresses, resulting in a choreographed series of cohesin
interactions that lead to cohesion establishment. Ctf4 to a position that likely also points towards the front of
the replisome (Samora et al. 2016). Thus, it could be that the
cohesion establishment factors Tof1-Csm3 and Chl1, which
form one epistasis group for their shared contributions to
sister chromatid cohesion establishment (Xu et al. 2007), are
jointly the first ones to engage with cohesin as the replica-
tion fork approaches. Replisome interactions with the cohesin complex Recent structural insight shows how Tof1 and Csm3 form
a complex at the head of the replisome, preceding the point
where the helicase begins unwinding the DNA double helix
(Eickhoff et al. 2019; Baretić et al. 2020). This places Tof1-
Csm3 at a vantage point to engage with DNA binding pro-
teins as the replication fork approaches. Chl1 is recruited by Might replisome components other than those discussed
above engage with cohesin during sister chromatid cohesion 1 3 129 Chromosoma (2023) 132:117–135 establishment? The presently known “cohesion establishment
factors” are typically non-essential proteins, as systematic
genetic screens for chromosome stability mutants, in which
many cohesion establishment factors were discovered, were
restricted to non-essential genes (Mayer et al. 2004; Warren
et al. 2004). Other screens uncovered the essential compo-
nents of the cohesion machinery (Guacci et al. 1997; Michae-
lis et al. 1997; Tóth et al. 1999). However, the latter screens
would not have uncovered essential replication fork compo-
nents, as no cohesion defects can be measured if there is no
DNA replication. It would be of interest to expand our cohesin
interaction screen to include all of the replisome, consisting of
well over 30 proteins and protein complexes. One candidate
interactor is DNA polymerase ε, which has been implicated
in sister chromatid cohesion (Edwards et al. 2003). here the possible implications of these contacts in the con-
text of current models for cohesion establishment. Struc-
tural and microscopic studies show cohesin often adopting
a folded conformation where the Smc1 and Smc3 coiled
coils bend at their elbow (Sakai et al. 2003; Higashi et al. 2020). We do not currently know what structure cohesin
adopts when bound to chromosomes. If cohesin retains a
folded conformation, transitioning to an open ring-like con-
formation would be necessary if the replisome were to pass
through cohesin rings (Fig. 8A and Fig. S8). In this scenario,
Tof1 and Mrc1 might facilitate the unraveling of the coiled
coils by contacting Smc1 and Smc3. Alternatively, if cohesin is unloaded as the fork
approaches and then reloaded behind the replisome, our
cohesion establishment factors might be assisting in these
unloading and/or reloading reactions. Interestingly, three
Tof1 and Mrc1 contact points lie near the cohesin N-gate,
through which DNA might pass (Fig. S8, cluster i). Replisome interactions with the cohesin complex Even
without active involvement in unloading or reloading, cohe-
sion establishment factors may transiently tether cohesin
while it is being transferred from the front to the back of the
replisome (Fig. 8B). Analogous to the related case of histone Further roles of Tof1‑Csm3 and Mrc1 Tof1-Csm3 and Mrc1 fulfil additional roles at the replica-
tion fork that we have not considered in our current study. A recently described function lies in Mcm7 ubiquitination,
which promotes replisome disassembly during replication
termination. This role diverged among eukaryotes. Tof1
and Csm3 orthologs in C. elegans, TIM-1 and TIPIN-1, are
necessary for Mcm7 ubiquitination, while Mrc1 and Ctf4
orthologs, CLSP-1 and CTF-4, are not (Xia et al. 2021). Conversely, budding yeast Mrc1 and Ctf4 are required for
Mcm7 ubiquitination, while Tof1 and Csm3 are not (Deegan
et al. 2020). As all these factors participate in cohesion
establishment, but only subsets in Mcm7 ubiquitination,
replisome disassembly may not be their main function dur-
ing sister chromatid cohesion establishment. Yeast strains All budding yeast strains used in this study were of W303
background. Gene deletions and epitope tagging were per-
formed by gene targeting using polymerase chain reaction
(PCR) products. For strains with C-terminal truncations of
Tof1 and Mrc1, one-step truncation and epitope tagging at
the C-terminus were combined through gene targeting using
PCR products. To create polε-Δcat (pol2 Δ1-1262) strains
(Kesti et al. 1999; Devbhandari and Remus 2020), In-Fusion
cloning (TaKaRa Bio) was used to add a 371 bp POL2 pro-
moter region in front of a POL2 fragment encompassing its
C-terminal portion (amino acids 1263–1797) in the yeast
shuttle vector YIplac204, followed by a further 226 bp of
POL2 upstream region. The plasmid was linearized with
ApaI, between the end of POL2 and the upstream region for
gene replacement at the POL2 locus. The rad53-11 allele
was introduced into a strain harboring a GFP-marked URA3
locus by crossing strain PP37 (Forey et al. 2020) with our
strain Y194, followed by tetrad dissection. The mrc1AQ-
myc9 allele was constructed based on plasmid pD2043
(McClure and Diffley 2021) adding a myc9 epitope at the
MRC1 gene C-terminus. The resulting plasmid (p1942) was
linearized with BsrGI to integrate at the LEU2 locus in an
mrc1Δ background strain. The MRC1 8D-Flag strain was
constructed by linearizing pD3093 (McClure and Diffley
2021) with XbaI to target the MRC1 locus for gene replace-
ment. Colonies were genotyped using PCR for gene dele-
tions. Epitope tagging was confirmed using immunoblotting
against the respective epitope tags. In case of gene altera-
tions, the genotypes of the final strains were confirmed by
PCR followed by Sanger sequencing. All strains used in this
study and their genotypes are listed in Table S1. A list of
plasmids used in this study and their descriptions is con-
tained in Table S2. Smc3 acetylation during sister chromatid cohesion
establishment Smc3 acetylation by the Eco1 acetyltransferase has been
tied to DNA replication (Moldovan et al. 2006; Minamino
et al. 2023), but how sister chromatid co-entrapment and
Smc3 acetylation are coordinated remains unknown. We
encountered examples where sister chromatid cohesion was
defective, despite efficient Smc3 acetylation. This was the
case in tof1Δ, csm3Δ, and mrc1Δ cells passing through S
phase in the presence of HU. It appears that acetylation in
this case targeted a cohesin pool that does not hold sister
chromatids together. In another example, Smc3 acetylation
levels in eco1-1 mutant cells at permissive temperature are
lower than levels observed in ctf4Δ and chl1Δ cells, despite
a similar resulting strength of sister chromatid cohesion
(Borges et al. 2013). Not all acetylated cohesin in ctf4Δ and
chl1Δ cells is therefore engaged in sister chromatid cohesion. We imagine that challenged replication forks recruit and
acetylate cohesin in support of fork protection (Delamarre
et al. 2020), independently of sister chromatid cohesion. We
also note that Smc3 acetylation defects in mrc1Δ cells often
seemed less pronounced than the accompanying cohesion
defects, maybe for this reason. Our observations emphasize Replisome‑cohesin contacts during cohesion
establishment Our results suggest that Tof1 and Mrc1 make cohesin con-
tacts along the Smc1 and Smc3 coiled coils. How might such
contacts contribute to cohesion establishment? We consider 1 3
Cohesin
Tof1/Csm3
Chl1
Mrc1
A
B
C A B C 3 3 130 Chromosoma (2023) 132:117–135 inheritance during DNA replication (Willhoft and Costa
2021), cohesion establishment factors might act as a con-
veyor belt that transfers cohesin along a moving replisome. the importance of further investigations into Smc3 acetyla-
tion and how cohesion establishment factors contribute to
this reaction. the importance of further investigations into Smc3 acetyla-
tion and how cohesion establishment factors contribute to
this reaction. In a third model of cohesion establishment, cohesin is de
novo recruited to the replisome during S phase and sequen-
tially loaded onto leading and lagging strands. Interactions
with replisome components may in this case attract cohesin
to the replication fork (Fig. 8C). The three models are not
mutually exclusive in explaining sister chromatid cohesion
establishment. Immunoblotting y
p
g
To purify Chl1, an ectopic copy of codon-optimized
Chl1 was overexpressed under control of the GAL1 pro-
moter in budding yeast. Strain Y5562 was grown in YP
+ 2% raffinose at 30 °C to an OD600 = 1.0 in a 100 L fer-
menter. Two percent galactose was added to induce protein
expression for 2 h before harvesting. Cells were washed
with deionized water and resuspended in lysis buffer (50
mM Tris-HCl pH 7.0, 500 mM NaCl, 10% glycerol, 2 mM
MgCl2, 0.1% Triton X-100, 0.5 mM TCEP, 0.5 mM Pefa-
block + cOmplete™ protease inhibitors (Roche)). The sus-
pension was frozen dropwise in liquid nitrogen and then
pulverized in a freezer mill. Cell powder was thawed in
lysis buffer on ice. Cell debris was removed by centrifuga-
tion in a Ti45 rotor at 40,000 rpm for 1 h. The supernatant
was supplemented with benzonase and RNaseA and trans-
ferred onto IgG agarose beads for incubation on a rotating
wheel at 4 °C for 2 h. The beads were then washed and
incubated overnight with 3C protease in the same buffer. The eluate was diluted to 160 mM NaCl and loaded onto a
heparin column equilibrated with 50 mM Tris-HCl pH 7.0,
160 mM NaCl, 10% glycerol, 2 mM MgCl2, and 0.5 mM
TCEP. Protein was eluted with a salt gradient from 160
mM to 1 M NaCl. Chl1-containing fractions were pooled,
concentrated using a 30-kDa cutoff Vivaspin concentrator,
and further purified using a Superdex 200 Increase column
equilibrated in 20 mM Tris pH 7.5, 150 mM NaCl, 10%
glycerol, and 0.5 mM TCEP. The final Chl1 preparation
was again concentrated by ultrafiltration. Aliquots of the culture were harvested at the indicated time-
points during synchronized cell cycle progression and fixed
using cold 20% TCA for at least 1 h. Cells were resuspended
in sample buffer containing 100 mM Tris-HCl pH 6.8, 4%
SDS, 20% glycerol, and 4% β-mercaptoethanol. Bead beat-
ing was used to break cells, before cell debris removal by
centrifugation. Protein concentrations in the extract were
determined using the Bradford assay. Fifteen microgram
protein equivalent of each sample was separated by SDS-
polyacrylamide gel electrophoresis, and gels were trans-
ferred to nitrocellulose membranes. Details of the antibodies
used for immunoblotting can be found in Table S3. Immunoblotting ECL or
ECL Prime reagents (Cytiva) were used to visualize signals
using an Amersham Imager 600 (Cytiva) or ChemiDoc MP
Imaging System (Bio-Rad). Ac-Smc3 signals were quanti-
fied using FIJI software and normalized against tubulin that
served as the loading control. Protein purification The budding yeast cohesin tetramers Tof1-Csm3 and Mrc1
were purified as previously described (Yeeles et al. 2017;
Minamino et al. 2018). Cohesin trimer and the Smc1-
Smc3 dimer were purified following the same protocol as
cohesin tetramers. While a Protein A tag is fused to Scc1
for the purification of the cohesin trimer and tetramers,
a corresponding Protein A tag is contained at the Smc1
C-terminus for purification of the Smc1-Smc3 dimer. In
each case, after adsorption to IgG agarose beads during
the first step of purification, the Protein A tag is removed
by 3C protease cleavage. Yeast culture All cultures were grown in liquid YPD medium at 25 °C in
shaking incubators at 180 rpm unless otherwise stated. Cells
of a mating type were grown overnight in YPD medium and
back-diluted in the morning. Four microgram per milliliter
α-factor was added at OD600 = 0.2 and subsequently twice
more every 55 min to arrest cells in G1. After 2 h 45 min,
cells were released from G1 arrest by filtration and washing 1 3 3 131 Chromosoma (2023) 132:117–135 with 10× culture volume of YP. Cells were then resuspended
in YPD + 6 μg/mL nocodazole to allow them to progress
through S phase followed by arrest in metaphase. Samples
for FACS analysis, for immunoblotting, and for the GFP dot
assay were taken at the indicated timepoints after release
from G1 arrest. Alternatively, cells were released from G1
arrest into YPD + 6 μg/mL nocodazole + hydroxyurea at the
indicated concentrations. were prepared on glass slides. Cells were sonicated and
resuspended in phosphate-buffered saline, and a drop was
applied to the agarose patches. z-stacks with 35 images
at 0.2-μm intervals were acquired using a DeltaVision
Olympus IX70 inverted microscope with a 100× (NA =
1.40) PlanApo objective, deconvolved, and merged using
maximum intensity projection. The number of GFP dots
in each cell were then manually counted. One hundred to
200 cells were scored for each sample in each experiment. Flow cytometry analysis (FACS) One-milliliter culture (OD600 0.2–0.7) was harvested by cen-
trifugation and resuspended and fixed overnight in cold 70%
ethanol. Then, RNA was digested with 0.1 mg/mL RNaseA
for 4 h (or overnight) at 37 °C in 50 mM Tris-HCl pH 7.5
buffer. To stain the DNA, the cells were resuspended in 200
mM Tris-HCl pH 7.5, 210 mM NaCl, 78 mM MgCl2 buffer
containing 0.025 mg/mL propidium iodide. Cells were soni-
cated and diluted in 50 mM Tris-HCl pH 7.5 before analysis
using a FACSCalibur cell analyzer (BD Biosciences). For
each sample, 10,000 cells were counted. Flow cytometry
data was analyzed using FlowJo (v10) software. Protein interaction analyses Cohesin as bait. For each interaction test, 5 μL Protein A
dynabeads were equilibrated with interaction buffer (25 mM
HEPES-KOH pH 7.6, 100 mM potassium glutamate, 10 mM
magnesium acetate, 0.12% NP-40). α-Pk antibody was added
in interaction buffer on a wheel at 4 °C for at least 2 h. The beads were washed; then, 200–600 nM purified cohesin
tetramer was added and adsorbed for 10 min on a thermo-
mixer at 30 °C and 1000 rpm. The beads were washed again
before 200 nM prey proteins were added in interaction buffer
at 30 °C for 5 min. The beads were washed again, and bead-
bound proteins eluted with sample buffer (100 mM Tris-HCl
pH 6.8, 4% SDS, 20% glycerol, and 4% β-mercaptoethanol)
at 95 °C for 5 min, before analysis by SDS polyacrylamide
gel electrophoresis, alongside 10% of input proteins. Input
and recovered proteins were visualized by Coomassie Bril-
liant Blue staining. Precipitated protein samples were resolubilized in diges-
tion buffer (8M urea in 100 mM ammonium bicarbonate) to
an estimated protein concentration of 1 mg/mL. Dissolved
protein sample was reduced by addition of 1 M dithiothrei-
tol (DTT) to a final concentration of 5 mM. The reaction
was incubated at room temperature for 30 min. The free
sulfhydryl groups in the sample were then alkylated by add-
ing 500 mM iodoacetamide (final concentration of 15 mM)
and incubation at room temperature for 20 min in the dark. After alkylation, additional DTT was added to a total con-
centration of 10 mM to quench excess of iodoacetamide. Next, protein samples were digested with LysC (at a 50:1
(m/m) protein to protease ratio) at room temperature for 4
h. The sample was then diluted with 100 mM ammonium
bicarbonate to reach a urea concentration of 1.5 M. Trypsin
was added at a 50:1 (m/m) protein to protease ratio to fur-
ther digest proteins overnight (~ 15 h) at room tempera-
ture. Resulting peptides were desalted using C18 StageTips
(Rappsilber et al. 2007). Tof1-Csm3 as bait. The interaction analysis was carried
out as above, but 3 μL of Calmodulin dynabeads were used,
to which 200–600 nM Tof1/Csm3 were adsorbed. Mrc1 or Chl1 as bait. Four microliter α-Flag M2 mag-
netic beads were used, to which 200–600 nM Flag-tagged
Mrc1 or Chl1 was adsorbed. GFP dot assay Strains harboring tetO repeats at the URA3 locus (at
35-kb distance from the centromere of chromosome V)
and expressing the tetR-GFP fusion protein were harvested
in metaphase, 2 h after G1 release into nocodazole-con-
taining medium. Two-milliliter culture was harvested by
centrifugation and resuspended and fixed in 70% cold
ethanol overnight. For imaging, thin 2% agarose patches 1 3 132 Chromosoma (2023) 132:117–135 A total of 100 μg cohesin, Tof1-Csm3, and Chl1 with or
without Mrc1 at 300–350 nM concentration was incubated
at 30 °C for 5 min with shaking. The crosslinker Sulfo-SDA
was added at a protein to crosslinker weight ratio of 1:0.75,
and the mixtures were left in the dark at 4 °C for 2 h. The
diazirine group in SDA was then photoactivated by UV irra-
diation at 365 nm from an CL-1000 Ultraviolet Crosslinker
(Spectrum). Samples were spread as thin droplets on opened
lids of Eppendorf tubes and placed on ice at a distance of
5 cm from the UV-A lamp and irradiated for 20 min. Unre-
acted NHS ester was then quenched with 20 mM ammonium
bicarbonate at room temperature for 20 min. The proteins
were precipitated with 4 volumes of acetone overnight at
− 20 °C and protein pellets processed for mass spectrometry
analysis. Protein interaction analyses When M2 beads with Mrc1 as
bait were first incubated with Tof1-Csm3 (prey 1), the beads
were washed before incubation with cohesin or GINS (prey
2). As alternative to sample buffer, bead-bound protein was
eluted by competition using 0.5 mg/mL 3× Flag peptide in
interaction buffer. As indicated, benzonase, or increasing
concentrations of NaCl, was included in the binding incuba-
tion with prey. Analytical size exclusion chromatography A Superose 6 Increase 3.2/300 column was equilibrated with
buffer (25 mM HEPES-KOH pH 7.6, 100 mM sodium ace-
tate, 0.5 mM TCEP, 2 mM EDTA). Ten micrograms of pro-
teins at the same molarity (> 1 mM) was incubated together
or separately in the above buffer at 30 °C for 5 min with
shaking, before loading. Thirty microliter fractions were col-
lected and analyzed by polyacrylamide gel electrophoresis
and immunoblotting. For each sample, resulting peptides were fraction-
ated using size exclusion chromatography to enrich for
crosslinked peptides (Leitner et al. 2013). Peptides were
separated using a Superdex™ 30 Increase 3.2/300 column
(Cytiva) at a flow rate of 10 mL/min. The mobile phase con-
sisted of 30% (v/v) acetonitrile and 0.1% trifluoroacetic acid. The earliest six peptide-containing fractions (50 μL each)
were collected. Solvent was removed using a vacuum con-
centrator. The fractions were then analyzed by LC-MS/MS. Protein crosslinking mass spectrometry LC-MS/MS analysis was performed using an Orbitrap
Fusion Lumos Tribrid mass spectrometer (Thermo Fisher
Scientific), connected to an Ultimate 3000 RSLCnano sys-
tem (Dionex, Thermo Fisher Scientific). Each size exclusion
chromatography (SEC) fraction was resuspended in 1.6%
v/v acetonitrile 0.1% v/v formic acid, and each SEC frac-
tion was analyzed with duplicated LC-MS/MS acquisitions. Peptides were injected onto a 50-cm EASY-Spray C18 LC
column (Thermo Scientific) that is operated at 50 °C column
temperature. Mobile phase A consists of water and 0.1% v/v Cohesin and Tof1-Csm3 were purified as above, but the
buffer used during the final size exclusion chromatography
step was 25 mM HEPES-KOH pH 7.6, 150 mM sodium
acetate, 0.5 mM TCEP, and 2 mM EDTA. The final buffer
for Chl1 was 25 mM HEPES-KOH pH 7.6, 100 mM NaCl,
0.5 mM TCEP, and 2 mM EDTA. To prepare Mrc1, 25 mM
HEPES-KOH pH 7.6, 150 mM NaCl, 0.5 mM TCEP, and
2 mM EDTA were used during the final glycerol dialysis. 3 133 Chromosoma (2023) 132:117–135 formic acid, and mobile phase B consists of 80% v/v ace-
tonitrile and 0.1% v/v formic acid. Peptides were loaded and
separated at a flowrate of 0.3 μL/min. Peptides were sepa-
rated by applying a gradient ranging from 2 to 45% B over
90 min. The gradient was optimized for each fraction. Fol-
lowing the separating gradient, the content of B was ramped
to 55% and 95% within 2.5 min each. Eluted peptides were
ionized by an EASY-Spray source (Thermo Scientific) and
introduced directly into the mass spectrometer. Mrc1 protein, and Christopher Smith for his interaction screen proto-
col. We would like to thank Phillippe Pasero for the rad53-11 strain,
Gavin Kelly from the Crick Bioinformatics & Biostatistics Science
Technology Platform and the Crick Fermentation Science Technology
Platform, as well as Alessandro Costa, John Diffley, Hasan Yardimci,
and our past and present laboratory members for discussions and criti-
cal reading of the manuscript. Author contribution Sudikchya Shrestha and Frank Uhlmann con-
ceived the study, Sudikchya Shrestha performed all experiments,
Masashi Minamino contributed the Chl1 expression system and first
observed a direct Chl1-cohesin interaction, Céline Bouchoux provided
the cohesin trimer and Smc1-Smc3 dimer complexes, Zhuo A. Chen
and Juri Rappsilber performed the CLMS analysis, and Sudikchya
Shrestha and Frank Uhlmann wrote the manuscript with input from
all coauthors. MS data was acquired in the data-dependent mode with
the top-speed option. Protein crosslinking mass spectrometry For each 3-s acquisition cycle, the full
scan mass spectrum was recorded in the Orbitrap with a
resolution of 120,000. The ions with a charge state from
3+ to 7+ were isolated and fragmented using higher-energy
collisional dissociation (HCD). For each isolated precursor,
stepped collision energy (26%, 28%, or 30%) was applied. The fragmentation spectra were then recorded in the Orbit-
rap with a resolution of 50,000. Dynamic exclusion was ena-
bled with single repeat count and 60-s exclusion duration. Funding Open Access funding provided by The Francis Crick Institute
This work was supported by a Wellcome Trust Investigator Award
(220244/Z/20/Z) and The Francis Crick Institute, which receives its
core funding from Cancer Research UK, the UK Medical Research
Council, and the Wellcome Trust (cc2137). Juri Rappsilber received
support from the Deutsche Forschungsgemeinschaft under Germany’s
Excellence Strategy EXC 2008/1-390540038. The Wellcome Centre
for Cell Biology receives its core funding from the Wellcome Trust
(203149). Sudikchya Shrestha was supported by a Boehringer Ingel-
heim Fonds PhD fellowship. The data from the two samples were processed separately. MS2 peak lists were generated from the raw mass spectrom-
etry data files using the MSConvert module in ProteoWiz-
ard (v3.0.11729). Precursor and fragment m/z values were
recalibrated. Identification of crosslinked peptides was car-
ried out using xiSEARCH software (v1.7.6.4) (Mendes et al. 2019). The peak lists were searched against the sequences
and the reversed sequences of the cohesin subunits and cohe-
sion establishment factors present in the sample. The follow-
ing parameters were applied for the search: MS accuracy =
3 ppm; MS2 accuracy = 5 ppm; enzyme = trypsin (with full
tryptic specificity); allowed number of missed cleavages =
3; missing monoisotopic peak = 2; crosslinker = SDA (the
reaction specificity for SDA was assumed to be for lysine,
serine, threonine, tyrosine, and protein N termini on the
NHS ester end, and any amino acids for the diazirine end);
fixed modifications = carbamidomethylation on cysteine;
variable modifications = oxidation on methionine, SDA
loop link, hydrolyzed SDA on the diazirine end, acetyla-
tion on lysine, deamidation on asparagine, phosphorylation
on serine, acetone modification on lysine and histidine; and
maximum variable modification per peptide = 2. Data availability The protein crosslink mass spectrometry data reported
in this study has been deposited with the Japan Proteome Standard
Repository jPOST (project ID JPST002006, PRIDE ID: PXD039609). Protein crosslinking mass spectrometry Any unique reagents generated in this study are available from the
authors upon reasonable request. Consent to participate Not applicable. Consent for publication All authors have given consent for publication. Conflict of interest The authors declare no competing interests. Open Access This article is licensed under a Creative Commons Attri-
bution 4.0 International License, which permits use, sharing, adapta-
tion, distribution and reproduction in any medium or format, as long
as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article's Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article's Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. i
Identified crosslinked peptide candidates that had at
least three matched fragment ions (at least two containing
crosslinked residue) in each crosslinked peptide were filtered
using xiFDR (Fischer and Rappsilber 2018). A false discov-
ery rate of 1% on residue-pair level was applied with “boost
between” option selected. Finally, identified crosslinks from
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Can Government Be Self-Organized? A Mathematical Model of the Collective Social Organization of Ancient Teotihuacan, Central Mexico
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Introduction Oztoyahualco 15B apartment compound revealed that it con-
tained three nuclear households [4], but we do not know how
many members each of them had, perhaps five to ten. More
generally, Cowgill [5] estimates that the mean number of domestic
units per compound was between three and five, and that on
average there were between five and twelve persons in a unit. Teotihuacan was a metropolis in the Valley of Mexico. Starting
around 100 AD, it rapidly grew into the largest city of
Mesoamerica of the first millennium. High-resolution chronology
of the city’s center places its collapse around 550 AD [1]. At its
peak Teotihuacan was one of the largest population centers of the
world. Population estimates vary greatly, but a conservative
estimate is that by 150 AD the city’s population had reached a
plateau of between 80,000 and 100,000 inhabitants [2]. It covered
an area of about 20 km2 and was divided into quadrants by its
north-south (‘‘Street of the Dead’’) and east-west axes (‘‘East
Avenue’’ and ‘‘West Avenue’’) [3]. In the beginning construction
focused on the monumental architecture along the Street of the
Dead, in particular on the ‘‘Pyramid of the Sun’’, the ‘‘Pyramid of
the Moon’’, and the ‘‘Feathered Serpent Pyramid’’ (Figure 1). Later, beginning around 200 AD, more than 2000 large-scale
residential
units
were
built
to
accommodate
most
of
the
population, including all socioeconomic statuses [4]. These were
solid, roofed structures with open patios and drainage. Each of the
so-called ‘‘apartment compounds’’ was shared by a number of
households. It is difficult to assess demography in archaeology, and
Teotihuacan is no exception. For example, excavation of the No previous or contemporary population center in Mesoamer-
ica was as urbanized as Teotihuacan [6]. Given that it was so
unlike the centers that came before, it is an interesting question
how we should account for this qualitative transition in social
organization. Parsons has formulated the key problem of the city’s
origins as follows: ‘‘we have to deal with the organizational
mechanisms whereby Early Classic (and even latest Formative)
Teotihuacan pulled in and (or) attracted large masses of people
from considerable distances and coordinated them, and probably
much of the whole of central Mexico, into an effective urban
system which was adaptive for over 500 years’’ ([7]: 876). Abstract Competing Interests: The authors have declared that no competing interests exist. * Email: t.froese@gmail.com Tom Froese1,2*, Carlos Gershenson1,2, Linda R. Manzanilla3 1 Instituto de Investigaciones en Matema´ticas Aplicadas y en Sistemas, Universidad Nacional Auto´noma de Me´xico, Ciudad Universitaria, Mexico City, Distrito Federal,
Mexico, 2 Centro de Ciencias de la Complejidad, Universidad Nacional Auto´noma de Me´xico, Ciudad Universitaria, Mexico City, Distrito Federal, Mexico, 3 Instituto de
Investigaciones Antropolo´gicas, Universidad Nacional Auto´noma de Me´xico, Ciudad Universitaria, Mexico City, Distrito Federal, Mexico Abstract Teotihuacan was the first urban civilization of Mesoamerica and one of the largest of the ancient world. Following a
tradition in archaeology to equate social complexity with centralized hierarchy, it is widely believed that the city’s origin and
growth was controlled by a lineage of powerful individuals. However, much data is indicative of a government of co-rulers,
and artistic traditions expressed an egalitarian ideology. Yet this alternative keeps being marginalized because the problems
of collective action make it difficult to conceive how such a coalition could have functioned in principle. We therefore
devised a mathematical model of the city’s hypothetical network of representatives as a formal proof of concept that
widespread cooperation was realizable in a fully distributed manner. In the model, decisions become self-organized into
globally optimal configurations even though local representatives behave and modify their relations in a rational and selfish
manner. This self-optimization crucially depends on occasional communal interruptions of normal activity, and it is impeded
when sections of the network are too independent. We relate these insights to theories about community-wide rituals at
Teotihuacan and the city’s eventual disintegration. tation: Froese T, Gershenson C, Manzanilla LR (2014) Can Government Be Self-Organized? A Mathematical Model of the Collective
cient Teotihuacan, Central Mexico. PLoS ONE 9(10): e109966. doi:10.1371/journal.pone.0109966 henson C, Manzanilla LR (2014) Can Government Be Self-Organized? A Mathematical Model of the Collective Social Organization o
ntral Mexico. PLoS ONE 9(10): e109966. doi:10.1371/journal.pone.0109966 Editor: Christopher M. Danforth, University of Vermont, United States of America Editor: Christopher M. Danforth, University of Vermont, United States of America Received June 23, 2014; Accepted September 12, 2014; Published October 10, 2014 Copyright: 2014 Froese et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data and information about the
model are within the paper. Funding: TF was partially supported by UNAM PAPIIT grant number IN113013. CG was partially supported by SNI membership 47907 of CONACyT, Mexico. The
work was partially funded by the project ‘‘Guiando Comportamientos para Mejorar la Movilidad Urbana’’ (CONACyT 212802). The funders had no role in study
design, data collection and analysis, decision to publish, or preparation of the manuscript. October 2014 | Volume 9 | Issue 10 | e109966 Introduction We can
divide this problem of origins into two aspects: how to explain the
large-scale relocation of whole communities into the city in a very
short timespan, and how to explain the coordination of all these
diverse people in a way that allowed the city to function in an
adaptive manner. Unfortunately, not much direct information
about Teotihuacan’s social organization is available, especially October 2014 | Volume 9 | Issue 10 | e109966 October 2014 | Volume 9 | Issue 10 | e109966 1 PLOS ONE | www.plosone.org Can Government Be Self-Organized? Figure 1. View of the extensive ceremonial center of Teotihuacan as seen from the top of the Pyramid of the Moon. In front is the
ceremonial plaza of the Pyramid of the Moon with a small ritual platform in the middle. The Street of the Dead can be seen stretching into the
distance, its sides lined with pyramidal platforms that would have originally supported temple structures. Mimicking the mountainous horizon on the
left is the Pyramid of the Sun. (Photo courtesy of Iliana Mendoza). doi:10.1371/journal.pone.0109966.g001 Figure 1. View of the extensive ceremonial center of Teotihuacan as seen from the top of the Pyramid of the Moon. In front is the
ceremonial plaza of the Pyramid of the Moon with a small ritual platform in the middle. The Street of the Dead can be seen stretching into the
distance, its sides lined with pyramidal platforms that would have originally supported temple structures. Mimicking the mountainous horizon on the
left is the Pyramid of the Sun. (Photo courtesy of Iliana Mendoza). doi:10.1371/journal.pone.0109966.g001 about the city’s initial centuries. There is a consensus that there
probably was a collective form of government during the later
periods [8], because during that time ideology deemphasized
individual status and personal achievement in favor of highlighting
universally applicable social and cosmological principles [9]. However, opinions diverge about the city’s social organization
during the earliest periods. Two competing frameworks of
interpretation have been put forward. suggests that these migrations must have been a deliberate
policy that was enforced for the political advantage of
keeping most of the Valley of Mexico’s population under
direct control in the city ([20]: 103), with the rulers using
force where necessary [21,22]. The rapid increase of population in Teotihuacan was
accompanied by the construction of impressive public
structures, including the three main pyramids. Introduction For Millon,
the expenditures of energy manifest in the pyramids and
the Street of the Dead are dramatic demonstrations of the
exercise of power during a time of strong rulers ([21]: 25). Coe and Koontz concur that the pyramids attest the
immense power of the early Teotihuacan hierarchy to call
up corve´e labor ([12]: 109). On the one hand, there are those who argue that Teotihuacan
was centrally controlled by powerful rulers and military elites, who
imposed law and order on the inhabitants of the city and
conquered distant regions to ensure a constant flow of tribute and
sacrificial victims [10–12]. On the other hand, there are those who
argue that the social organization of the city is more accurately
characterized as a decentralized network of diverse semi-autono-
mous communities that were governed in a corporate manner, and
who were more interested in ritual and trade than empire building
[13–15]. We will briefly indicate some of the key arguments of
both approaches. This construction of the city’s extensive ceremonial center,
as well as of all other quarters of Teotihuacan, were
realized in alignment with a specific orientation, namely
around 15u east of true north. This suggests a high degree
of planning, which in turn implies centralized control ([23]:
226). For instance, Cowgill interprets the alignment of the
whole city as another sign of early strength of the central
authority, which suggests the relative weakness of interme-
diate social units, such as large lineages ([8]: 155). In
addition, several authors have argued that a substantial
part of the city was built using a standard unit of
measurement, which has been taken to imply the existence
of a strong overarching central authority that could
override local interests [24]. (iii) (iv) (ii) (iii) Evidence for Collective Government Yet none of the above points is decisive with regard to the
existence of centralized rulership, especially given the prominent
lack of more direct archaeological evidence of the kind that is
sometimes found in the context of Mesoamerican cities, such as
supreme rulers’ tombs, royal portraiture, dynastic stele and
inscriptions. Although differences in social status can be detected
in Teotihuacan, for example in terms of burial practices and access
to resources [31], these differences are neither very remarkable nor
clearly localized. Moreover, the evidence cited above is consistent
with alternative explanations. The wholesale emigration from other contemporary
regional centers and the notably accelerated early
population growth at Teotihuacan coincided with erup-
tions of at least two volcanoes in the southern parts of the
Basin of Mexico. Most importantly, it has been argued that
the cataclysmic eruption of the Popocate´petl volcano,
dated to have occurred during the mid-first century AD,
was responsible for the displacement of some 50,000
people northwards to Teotihuacan [32]. Just around 50
years later, there was another, smaller eruption in the
southern parts, this time from the Chichinautzin volcano,
which further contributed to their depopulation [33]. These migrations could therefore have simply been caused
by geological factors [34]. Later Aztec accounts of the city’s
origins, as recorded by Spanish friars, talk about migrations
but do not mention that they were enforced by
Teotihuacan [35]. Indeed, they explicitly speak of several
old and wise leaders of the new settlers being installed as
rulers, thereby placing Teotihuacan in the classic Mesoa-
merican tradition of co-rulership [22]. If migrations were
enforced by local rulers it is unlikely that migrant rulers
would have been accorded such elevated status. These
Aztec accounts could be rejected as nothing but myth were
it not for the fact that many of Teotihuacan’s temple
complexes were designed according to migrant traditions
(e.g., from Tetimpa, which had been destroyed by the
Popocate´petl eruption [32]). We also note that during later
times the city’s population was highly mobile, ethnically
diverse, and included several foreign enclaves. Many
people were born outside the city and then immigrated
[36]. There is no evidence that these relocations were (i) It seems likely that Teotihuacan’s canonical orientation
was originally a result of the cosmological observations
made with the Pyramid of the Sun, while other principal
constructions followed the Pyramid’s alignments [44]. Can Government Be Self-Organized? Can Government Be Self-Organized? found in the Pyramid of the Sun were of infants and
children [27]. By the time of the Aztecs there was a
tradition of sacrificing children as an offering to the rain
god, but we do not know if this was the case here. There is
evidence that the burials in the Temple of the Feathered
Serpent had militaristic associations [10]. Although their
cause of death is not evident, the fact that their forearms
crossed behind their bodies as if they had been bound at
the wrists suggests that their sacrifice was not voluntary
[28]. More compelling evidence for a practice of fighting
wars to gain captives comes from the Pyramid of the
Moon. Given that most of the people buried there were not
originally from Teotihuacan [29], and some had been
decapitated and deposited casually without regalia or
offerings [30], they are likely to have been sacrificed
captives. These findings support the hypothesis of a
centralized political hierarchy at Teotihuacan because
the origins of human sacrifice have been associated with
the origins of elites in Mesoamerica [17]. involuntary. In addition, the variable acceptance of the
city’s customs and artistic traditions in the foreign enclaves
suggests that enculturation was based on individual agency
rather than centrally enforced (e.g., in Tlailotlacan, a
Zapotec enclave [37]). The more likely scenario is
therefore that the city originated in the coming together
of several disparate and desperate groups, which would
have facilitated the creation of a governing coalition. This
is consistent with the fact that analyses of the density of
potsherds from Teotihuacan’s earliest periods indicate
several regions of dense occupation, rather than a single
center [38]. involuntary. In addition, the variable acceptance of the
city’s customs and artistic traditions in the foreign enclaves
suggests that enculturation was based on individual agency
rather than centrally enforced (e.g., in Tlailotlacan, a
Zapotec enclave [37]). The more likely scenario is
therefore that the city originated in the coming together
of several disparate and desperate groups, which would
have facilitated the creation of a governing coalition. This
is consistent with the fact that analyses of the density of
potsherds from Teotihuacan’s earliest periods indicate
several regions of dense occupation, rather than a single
center [38]. Can Government Be Self-Organized? (ii) Cowgill reviewed the evidence about Teotihuacan’s social
organization and accepted that powerful rulers were
implicated in the construction of the Feathered Serpent
Pyramid, but stops short of generalizing this state of affairs
to earlier periods ([8]: 156). However, the cessation of the
construction of new monumental structures after around
250 AD and the desecration of the Feathered Serpent
Pyramid soon thereafter, which are often interpreted as
due to a political change from a time of powerful rulers to
more collective institutions (e.g., [39]), should also be
reevaluated. It is surely no coincidence that the eruption of
the Xitle volcano in the southwestern part of the Basin of
Mexico, which destroyed the large settlement of Cuicuilco,
has been dated to around 245–315 AD [40]. Additionally,
evidence that some kind of transition took place during
that time is not limited to the monumental core of the city,
but includes diverse termination rituals in neighborhood
centers, such as in Teopancazco [41]. In other words,
although political changes may have been involved in this
transition, we should not ignore the widespread ecological
and religious impact of yet another nearby eruption. Even
the function of the Feathered Serpent Pyramid as a ruler’s
seat of power can be questioned; others view it as a
grandiose ritual stage managed by a group of priests for
events that involved the whole community [42]. And if the
pyramids mainly served a ritual function for the commu-
nity, then they would be better considered as large-scale
public goods on a continuum with the constructions of
large-scale housing for most of the population [43]. It has
been argued that the construction of these apartment
compounds makes this period of Teotihuacan history the
foremost manifestation of collective government in ancient
Mesoamerica ([15]: 9), so it could be expected that there
were some antecedents in the city’s history. (iii) Evidence for Centralized Government Teotihuacan was long viewed as a peaceful theocratic state, but
this view has fallen out of favor in recent decades. In line with a
growing emphasis of the role of war for state formation, including
for the first Mesoamerican states [16,17], instead there is an
emphasis on militarism ([12]: 105–118) and political power
[10,11]. For example, many archaeologists interpret the construc-
tion of the Feathered Serpent Pyramid around 200 AD, and the
large-scale human sacrifices associated with it, in terms of the
material expression of divine authority, be it individual-centered
[10] or of a repressive state [18]. This pyramid was burned down
and its fac¸ade covered with a platform not too long after its
construction. Millon interprets this event as the end of what had
been a continuous reign of powerful rulers ([19]: 112). In addition,
the existence of an early, centralized government seems to be
supported by the following facts: One prominent theme of mural painting during the city’s
later periods is the depiction of processions of richly attired
persons, often armed with shields, arrows and knifes, and
sometimes having bloody hearts impaled on them. This
kind of imagery has been related to coercive violence and
social status, and has been interpreted in terms of powerful
military orders and the large-scale practice of human
sacrifice [25,26]. Hundreds of human burials have been discovered in the
Pyramid of the Sun, the Pyramid of the Moon, and the
Temple of the Feathered Serpent. Many of the burials (i)
During the city’s ascendency there were rapid, large-scale
demographic shifts in the Valley of Mexico, with most of its
population eventually resettling in Teotihuacan [7]. Millon October 2014 | Volume 9 | Issue 10 | e109966 October 2014 | Volume 9 | Issue 10 | e109966 PLOS ONE | www.plosone.org 2 (iii) (ii) Can Government Be Self-Organized? Can Government Be Self-Organized? ceremonial center but included the city as a whole. This is
consistent with the hypothesis that the designs of the city’s
prominent religious structures, namely the Three-Temple
Compound and talud-tablero architecture, first originated
in a domestic context [32], indicating that there was no
clear distinction between sacred and mundane spaces. It
also matches the findings of a space syntax analysis of the
ceremonial core and of several apartment compounds,
which suggest that the community structure of the
apartment compounds and the city’s organization at the
larger civic scale reflect each other [46]. Moreover, it was
found that, contrary to traditional assumptions about the
grid-like regularity of Teotihuacan’s layout, most of its
streets are in reality short and discontinuous rather than
long and straight, and the actual layout therefore does not
fit with expectations of having been designed by one
centralized authority. The situation is no different at the
scale of apartment compounds. During the Teotihuacan
mapping project it was recognized that the internal
arrangement of each apartment compound is unique,
which suggests that these structures were not built by the
state to a uniform plan, ‘‘and there is no special reason to
think that they were owned by the state’’ ([6]: 221). More
detailed investigations of the spatial layout of the
apartment compounds has confirmed this impression of
diversity [47]. Indeed, excavations of a compound has
provided evidence that ‘‘all building activities, including
planning and construction, were initiated by people solely
within the compound and that no help or support in
construction came from outside of the compound’’ ([48]:
102). practice may not have been present in Teotihuacan. For
example, the persons buried in the Temple of the
Feathered Serpent were dressed like people from Teoti-
huacan [10]. An analysis of oxygen-isotope ratios in
skeletal phosphate of 41 of these individuals revealed that
most men had lived in the city for a prolonged period
before their death, some since childhood and others after
having moved from several foreign locations; most women
had also lived in the city all of their lives or had moved
from there to a foreign location as adults [51]. Moreover,
these sacrifices may have been unique events. So far there
is no archaeological evidence for large-scale human
sacrifice outside of the specific context of dedication rituals
during the construction of these three pyramids. Even the
indirect evidence is sparse. Can Government Be Self-Organized? For instance, Teotihuacan did
not have any dedicated I- or T-shaped ball courts, an
otherwise prominent and ubiquitous structure in contem-
porary Mesoamerican cities typically associated with elites
and human sacrifice. Finally, the hypothesis of a coalition government helps to
resolve some of the outstanding puzzles. Specifically, it explains
why direct evidence of individual rulership remains elusive even
after decades of concerted archaeological search. In addition, it
helps to explain the otherwise mysterious fact that the people of
Teotihuacan chose not to make much use of systematic writing on
permanent media, even though they were familiar with the writing
systems of the Zapotecs and Maya [52]. Evidence of notation is so
sparse that it was long thought that writing was absent altogether,
although it now seems that a rudimentary system of signs was
present [53] (e.g., glyphs forming part of mural paintings). What
explains this strange self-limitation? Given that, as Marcus [54]
has suggested, one of the main uses the Maya, Zapotec, and other
cultures made of their writing systems was to exhibit and glorify
the exploits of their royal lineages, e.g. on public stele, the relative
absence of such writing at Teotihuacan is indicative of the relative
absence of such lineage royalty. Similarly, given that the first
instance of Zapotec script we know about was used to record the
name of a captive who was depicted with his heart cut out [17], the
scarcity of writing (and the absence of such graphic depictions in
the city’s arts) indicates that this kind of military subjugation was
assigned little importance. In other words, we can make better
sense of the relative absence of public writing if we hypothesize
that the city’s government consisted of localized forms of social
interaction. We will return to this point in the discussion. (iv) In contrast to the explicit depictions of the gory details of
war, bloody human sacrifice, and the humiliation of
captives by all-powerful rulers, which can be found in the
artistic traditions of other Mesoamerican states, in
Teotihuacan there is not a single piece of art depicting
the subjugation of one person by another person. Instead
there is a striking emphasis of abstractness, multiple
perspectives, impermanence, playfulness, diversity, person-
al anonymity, and collective values [13,49,50]. Indeed, the
most explicit representations of violence are found in a
couple of atypical mural paintings of animals. Can Government Be Self-Organized? In a mural
painting known as ‘‘The Mythological Animals’’ two
feathered serpents seem to be attacked or confronted by
other animals, including felines, canines, and birds. Another mural painting depicts a pair of wolves slaying a
deer and extracting its heart. It has been argued that this
was the elite’s metaphorical justification for the practice of
human heart sacrifice [26], but no archaeological evidence
for this practice has yet been found. It is also worth noting
that some artistic evidence for militarism seems to be more
closely related with religious themes. For example,
although many of the people buried in the Temple of the
Feathered Serpent were decorated with necklaces consist-
ing of human jaws, most of these were actually finely
crafted replicas made from seashell and stucco [28]. Similarly, many of the obsidian arrowheads that were
found scattered around the skeletal remains were unfit for
use in actual battle [10]. These replica and substitutions
suggest a concern for symbolic and ritual practices rather
than an interest in physical power per se. Evidence for Collective Government The
highly prevalent imitation of this orientation could
therefore have resulted from external constraints to ensure
the maximal use of available space in a crowded city and,
most importantly, from the religious significance of
alignment with the city’s principal axis, which probably
represented a vertical axis mundi [45]. In other words, we
need to look no further than the widely accepted idea that
the people of Teotihuacan considered their city to be a
sacred cosmogram and the center of the universe [9]; it is
unlikely that coercion was needed to convince immigrants
to construct their compounds so as to share in this cosmic
power. The fact that even the more distant residential
structures adhere to the canonical orientation implies that
the notion of sacred space was not limited to the October 2014 | Volume 9 | Issue 10 | e109966 October 2014 | Volume 9 | Issue 10 | e109966 PLOS ONE | www.plosone.org 3 3 A Network of Three-Temple Complexes (TTCs) Of course, there are
only problems with this interpretation because Millon assumes that
Teotihuacan was founded by a legendary individual rather than by
the twenty kin groups as a whole, that is, by one coalition. We note
that the latter interpretation of these murals adds independent
support for the model’s assumption of 22 relatively distinct social
groups: even if one does not agree with the hypothesis that the
twenty-something TTCs could have served as neighborhood
centers during the city’s initial stages (e.g., [2]), the coalition
government may have still consisted of roughly this number of
social groups. Nevertheless, although there exist these various strands of
evidence that support the hypothesis of such a highly distributed
collective government, the very possibility of this unusual type of
political organization at the scale of a city often tends to be
rejected on theoretical grounds. This is because the evolution from
relatively egalitarian hunter-gatherer
societies to ‘‘complex’’
societies is traditionally simply defined as the emergence of a
centralized political hierarchy. Accordingly, it is commonly
thought that the management of Teotihuacan necessitated a
powerful centralized state apparatus ([57]: 79). To be fair, this kind
of assumption is valid for many complex societies, and it also has a
compelling theoretical justification: it is closely related to the
problem of collective action, namely that the potential for selfish
behavior would severely limit the possibilities of cooperation
among a group of equals, thus resulting in the tragedy of the
commons [60]. Accordingly, it seems that even if there weren’t any
powerful individual rulers at Teotihuacan, there must have at least
been a strong political class of elite bureaucrats. Blanton and
Fargher, for example, have argued that there is an essential
relationship between the extent of a society’s collectiveness and the
extent of state involvement that is necessary in order to ensure
cooperation ([61]: 29). Figure 2. Architecture of a typical Three-Temple Complex
(TTC). Central part of a mockup of the Street of the Dead (located in
the Museo de Sitio de la Cultura Teotihuacana). The beginning of the
plaza in front of the Pyramid of the Moon can be seen at the very top,
while the Pyramid of the Sun is on the right. A typical TTC can be seen
on the bottom right: three temples face each other around a square
plaza, with the two equally sized temples flanking a larger temple in
their middle. A Network of Three-Temple Complexes (TTCs) p
p
If Teotihuacan was governed by a coalition of representatives,
how many representatives were there? We can derive a rough
estimate by counting the number of administrative architectural
units that were distributed throughout the early city, i.e. the so-
called ‘‘Three-Temple Complex’’ (TTC) [9]. TTCs came in
varying sizes and orientations, while generally conserving the
arrangement of three temples on three sides of a rectangular plaza
(Figure 2). While estimates of their number vary, there were at
least 20 of them [55,56]. It seems reasonable to assume that TTCs
represented an administrative division of the city into neighbor-
hoods during the city’s initial periods, although their exact role
and relative status continue to be debated ([11]: 103–123). It is
possible that the earliest TTCs constructed during the first century
AD were originally elite houses from migrant groups that only
subsequently acquired temple status [32]. Given their uneven
distribution throughout the city, it is likely that they were not the
only form of neighborhood center [11]; during later periods some (v) While it was common practice for other Mesoamerican
civilizations to wage wars with the specific aim of capturing
enemies for use in rituals based on human sacrifice, this October 2014 | Volume 9 | Issue 10 | e109966 October 2014 | Volume 9 | Issue 10 | e109966 PLOS ONE | www.plosone.org 4 Can Government Be Self-Organized? Figure 2. Architecture of a typical Three-Temple Complex
(TTC). Central part of a mockup of the Street of the Dead (located in
the Museo de Sitio de la Cultura Teotihuacana). The beginning of the
plaza in front of the Pyramid of the Moon can be seen at the very top,
while the Pyramid of the Sun is on the right. A typical TTC can be seen
on the bottom right: three temples face each other around a square
plaza, with the two equally sized temples flanking a larger temple in
their middle. Another TTC can be seen in the center of the photo, with
its plaza opening to the Street of the Dead. (Photo courtesy of Iliana
Mendoza). doi:10.1371/journal.pone.0109966.g002 […]. There are problems with this interpretation, however,
because it would not explain why all the figures wear headdresses
symbolizing the Teotihuacan state’’ ([19]: 91). A Network of Three-Temple Complexes (TTCs) Another TTC can be seen in the center of the photo, with
its plaza opening to the Street of the Dead. (Photo courtesy of Iliana
Mendoza). doi:10.1371/journal.pone.0109966.g002 doi:10.1371/journal.pone.0109966.g002 The possibility that a complex social system such as Teotihua-
can could be successfully managed collectively in a self-organized
manner, involving neither powerful leaders nor an extensive
bureaucratic apparatus, therefore goes against ingrained assump-
tions of archaeology. An important part of the problem is the
difficulty of even imagining the alternatives. However, Ostrom
[62] has collected extensive empirical data which demonstrate
that, at least in the case of smaller communities, shared resources
can be effectively managed via self-organized government –
thereby challenging skeptical sociological theories based on
influential models such as the tragedy of the commons, the
prisoner’s dilemma, and the logic of collective action. Thus, if
Teotihuacan consisted of a diverse and polycentric network of
communities, potentially represented by their TTCs, could this
network not also have relied on a form of self-government rather
than on a centralized coercive state? In the following we address
this question by analyzing a minimalist mathematical model that
makes it at least formally conceivable that Teotihuacan’s political
consensus emerged from a distributed coalition that was not
subject to one central institution. of the larger ‘‘apartment’’ compounds may have served a similar
role [14]. Many researchers interpret the TTCs as local manifestations of
the state’s centralized power ([57]: 81–83), for which such
neighborhood organizations would have provided an important
intermediate organizational level ([6]: 225). On the other hand,
Angulo [56] and Pasztory [55] see them as an indication that
Teotihuacan emerged as a voluntary alliance between around
twenty social units, possibly related clans, territorial units, or
ethnic groups. Manzanilla ([58]: 59) agrees that TTCs are perhaps
the earliest manifestation of the different groups that settled in the
Teotihuacan Valley, following the volcanic eruptions of the first
century AD. She argues that the purpose of the TTCs was to serve
as centers of a redistributive circuit that organized economic
surplus, in particular to pay full-time religious specialists and
artisans [59]. This is in agreement with Paulinyi’s [50] extensive
iconographic analysis, which suggests that the dominant layer of
the city was composed of various groups of noble religious
specialists, each based in their own temple, that shared political
power in a distributed manner. Overview of the Model Spatial distribution of 22 Three-Temple Complexes
(TTCs) of Teotihuacan. Only TTCs of an intermediate scale were
selected as neighborhood centers. The Pyramid of the Sun, Pyramid of
the Moon and known examples of smaller TTCs contained within
excavated apartment compounds were excluded because they seem to
belong to different spatial and temporal contexts. The shortest paths
between any two TTCs are approximated as a rectangle because much
of the city is densely occupied and roughly conforms to a grid-like
pattern. Distances between TTCs were measured by hand on Millon’s
[3] map. Minor measurement errors are visible, since the nodes do not
overlap 100%. The effect of these distances on social constraints was
incorporated into a pilot study, but not in the final model (see Methods
section for details). d i 10 1371/j
l
0109966 003 Figure 3. Spatial distribution of 22 Three-Temple Complexes
(TTCs) of Teotihuacan. Only TTCs of an intermediate scale were
selected as neighborhood centers. The Pyramid of the Sun, Pyramid of
the Moon and known examples of smaller TTCs contained within
excavated apartment compounds were excluded because they seem to
belong to different spatial and temporal contexts. The shortest paths
between any two TTCs are approximated as a rectangle because much
of the city is densely occupied and roughly conforms to a grid-like
pattern. Distances between TTCs were measured by hand on Millon’s
[3] map. Minor measurement errors are visible, since the nodes do not
overlap 100%. The effect of these distances on social constraints was
incorporated into a pilot study, but not in the final model (see Methods
section for details). d i 10 1371/j
l
0109966 003 In the Hopfield network model a connection weight vij
represents the relative importance for agent i of satisfying the
constraint that is posed by its interaction with agent j. In other
words, agents will try to choose a behavioral state si that ensures
si*sj*wij.0 for as many connections as possible, but when
satisfying different connections requires contrasting behaviors
those with the stronger weights are given preference. Every weight
vij is a combination of the weight specified by the original network
topology, vO
ij, and the weight changes that accumulate as agents
update the relative strengths of their connections, vL
ij. Agents
adjust their relationships at a slower rate than updating their
behavior; such a separation between slower and faster dynamical
scales is a general condition for self-organization [73,74]. Overview of the Model Possibly the representatives of these different groups were
depicted in the famous Tassel Headdress murals, as described by
Millon: ‘‘The procession of Tassel Headdress figures could
represent the legendary founding of Teotihuacan by someone
bearing the name of the Storm God. This figure might be leading
twenty kin groups […] to the site of the later city. All of the kin
groups could be depicted as participating in the city’s founding There is a growing interest in complementing traditional
archaeological methods with computational tools, including by
modeling social systems. One prominent approach is agent-based
simulation in various grades of realism and predictive power
[63,64], while others prefer to directly model the nonlinear
dynamics of social systems [65]. The lack of realism of the latter
approach is offset by the generality of its explanatory insights. October 2014 | Volume 9 | Issue 10 | e109966 PLOS ONE | www.plosone.org October 2014 | Volume 9 | Issue 10 | e109966 5 Can Government Be Self-Organized? g Figure 3. Spatial distribution of 22 Three-Temple Complexes
(TTCs) of Teotihuacan. Only TTCs of an intermediate scale were
selected as neighborhood centers. The Pyramid of the Sun, Pyramid of
the Moon and known examples of smaller TTCs contained within
excavated apartment compounds were excluded because they seem to
belong to different spatial and temporal contexts. The shortest paths
between any two TTCs are approximated as a rectangle because much
of the city is densely occupied and roughly conforms to a grid-like
pattern. Distances between TTCs were measured by hand on Millon’s
[3] map. Minor measurement errors are visible, since the nodes do not
overlap 100%. The effect of these distances on social constraints was
incorporated into a pilot study, but not in the final model (see Methods
section for details). doi:10.1371/journal.pone.0109966.g003 Another of its advantages is that we can use models that have
already been systematically studied by other disciplines. For
example, insights from the Hopfield neural network [66], which is
isomorphic with the Ising spin model of statistical physics, are
generalizable to distributed social systems [67]. Indeed, the
cyberneticist McCulloch first introduced the concept of ‘‘heter-
archy’’ to describe the organization of the nervous system, and it is
from his work that archaeologists later adopted the concept to
describe complex societies that were not (or at least not exclusively)
hierarchically organized [68]. Overview of the Model y
g
We use a variation of the Hopfield model based on the work by
Watson and colleagues [67,69]. Here we briefly present the basics
of the model; further technical details are provided in the Methods
section at the end of the paper. We assumed that the TTCs
represent the city’s coalition government in its initial form, and
identified 22 of them on Millon’s [3] map of Teotihuacan
(Figure 3). To ensure that the best solutions require cooperation
not only between neighborhood centers but also within neighbor-
hoods, we assumed that there was a representative for each of the
three temples of a TTC (N = 22*3 = 66). Representatives, also
called agents, are modeled as nodes connected into a network. Each agent can be in one of two behavioral states (si = +1/21). Behaviors stand for a choice of social action (do-a/do-b), for
example voting about public good options (vote-a/vote-b). At each
time step a randomly selected agent adopts the behavior that
maximizes its own utility, ui, defined as the weighted sum of its
interactions with all the other members of the network. With respect to the problem of collective action, this means the
model adopts a worst-case scenario: any agent always chooses so as
to satisfy the maximum number of its own social constraints, never
putting the collective good before their personal good. It could be
argued that this scenario is likely to be overly pessimistic, but it has
the advantage that if our model succeeds in self-organizing a
communal consensus under these conditions then it can be
assumed that it will also do so in more pro-social scenarios. Another possible worry is that an agent’s behavior is determined
solely in relation to its current social network, thus neglecting the
role of memory and intrinsic cognitive dynamics. In this respect
the Hopfield network model is no different than most game
theoretic approaches that derive behavior from simple payoff
matrices [70]. Nevertheless, in spite of this simplifying assumption,
they can still provide insights into actual public good dilemmas
[71]. In addition, the inclusion of more complex cognitive
dynamics seems to facilitate the emergence of an even wider
variety of cooperative strategies [72], which leads us to expect that
it might also help to mitigate the worst-case scenario assumed by
our model. This is a prediction that could be investigated by future
work. Figure 3. Can Government Be Self-Organized? Can Government Be Self-Organized? However, this consistency does not mean that a globally optimal
solution is easily found; that depends on the specific way in which
the agents are connected and on their initial distribution of
behavioral states. Weights between two nodes were symmetrical
and no individual node was more powerful than all the rest. We
investigated the impact of modularity (a network of neighbor-
hoods) and nested hierarchies (a network of neighborhoods
embedded within the city’s districts). The number of districts
was equated with quadrants of the city [14], which is a
conservative estimate (cf. [22]). More specifically, we consider
two scenarios, i.e. neighborhoods and districts. In the neighbor-
hoods scenario we assumed it was more crucial for representatives
of each TTC to agree among each other (vij = 1) than to agree
with representatives of other TTCs (vij = 0.01). In the districts
scenario, we assumed the allegiance among a group of TTC’s
representatives was maximal (vij = 1), as well as comparatively
stronger to other representatives of the same district (vij = 0.02)
when compared to other districts (vij = 0.006). For ease of
comparison of utilities, the connection weights were selected such
that the weight sum of the scenarios was equal. Given the original constraints of the neighborhoods scenario,
either preferred outcome is equal to a utility sum of 173.58. However, it is rare for the network of agents to automatically
converge on either of these two solutions – at least without rituals. In a sample of 200 independent random initializations and
convergences, such a global consensus was never found (the
highest utility sum was 166.02 while the average was 146.74 with a
standard deviation of 8.19). This weak form of self-organization is
a clear example of the problems faced by collective action in a
heterarchical system, whereby the competing interests of selfishly
and rationally behaving individuals prevent the emergence of
more preferable solutions dependent on cooperation (Figure 4). However, the results are rather different when the randomiza-
tions and re-convergences are treated as events of the same social
system, namely as a sequence of community rituals. Specifically,
behaviors were occasionally set to arbitrary behavioral choices and
allowed to converge to an equilibrium, as before, but this time
agents’ changes to their connections were allowed to accumulate
over 200 such ritual interruptions. Can Government Be Self-Organized? Under these conditions agents
managed to reach a complete consensus already after the 56th
ritual, and from the 77th ritual onwards this globally optimal
solution to the original problem was consistently reached from
every arbitrary configuration of behaviors (Figure 5). This is a
strong form of self-organization that we refer to as self-
optimization. Archaeologists and art historians are in agreement that
integration of the various groups in Teotihuacan depended on
the public promotion of a collective ideology, and that public ritual
activity was foundational for the establishment of Teotihuacan. Indeed, shared rituals become especially important if we assume a
collective government because it can ensure cooperation: ‘‘ritual
provided the unifying force for peaceful coexistence among the
different ethnic groups during the early years of the protocity’’
[56]. Ritual activity, like symbolic activity more generally, is
primarily based on arbitrary cultural conventions. It is distin-
guished by conventions that do not seem to seem to have any
immediate functional value, for example prohibitions against
eating certain types of food on auspicious days. But the communal
enactment of such non-ordinary behaviors is a transformative
performance that brings the community together. Turner [75]
concluded from his extensive research into tribal rituals that what
is jointly enacted through them is a domain of the ‘‘uncommon
sense’’ in which people are temporarily released from the
pragmatic constraints of daily life. He famously characterized this
liminal phase of the ritual process in terms of ‘‘anti-structure’’ in
order to distinguish it from the structure imposed by mundane
social relations [76]. We compared cooperation in the neighborhoods scenario with
the districts scenario, in which the 22 neighborhoods are
categorized into the four districts of early Teotihuacan. We
measured cooperation as the total number of agreements between
agents’ behavioral states that satisfy the constraints of their
connections; an agreement is counted whenever si*sj*wij.0. The
maximum number of agreements is 4290 ( = 66*66 connections–
66 self-connections). To assess the lasting impact of network self-
optimization we compared the average number of agreements for
both scenarios in 100 separate runs reach. As before, a run
consisted of 200 converges to equilibrium. Thus, we compared a
total of 20,000 independent convergences of the original topology
with 20,000 independent convergences reached after self-optimi-
zation had already been completed (Figure 6). Can Government Be Self-Organized? For purposes of
comparison, the number of agreements reached by the behaviors
of the modified social network is calculated only with respect to the
original weight matrix, vO
ij. j
In the neighborhoods scenario, agents connected in the original
network rarely managed to resolve all of their constraints without
ritual interruption. Out of the 20,000 independent convergences
only 64 reached the 4290 agreements needed for a complete
consensus. Most frequent were medium levels of cooperation, with
an average of 2945 agreements. This problematic state of affairs
was completely transformed by self-optimization, which for each
of the 100 runs consisted of a series of 200 ritual interruptions of
agents’ behaviors while their changes to connections were
preserved. Of the 20,000 independent convergences after self-
optimization only 7 failed to realize the maximum number of
agreements. Remarkably, this outcome of self-optimization does
not depend on the network encountering and remembering
globally optimal configurations. In most cases, the network had
never even visited such an optimum prior to self-optimization,
thereby showing that the process does not depend on a priori
knowledge of the structure of the underlying problem space. We therefore modeled shared rituals as occasional interruptions
to normal behavior, whereby the converged behaviors of all
representatives are simultaneously replaced by arbitrary states
(each behavior is randomly set to either +1 or 21). Each ritualized
interruption is followed by the network’s convergence on another
equilibrium, i.e., a normalization period of sufficient duration such
that constraint-satisfying behaviors have been regained by the
coalition and utilities remain constant once again. This mechanism
of behavior randomization should not be misunderstood as
isolating the individuals from each other in general; rather, it
represents
a
temporary
bracketing
of
their
normal
social
constraints. As we will see below, it is in fact essential that most
of the community takes part in this ritual process at the same time. And, as would be expected, in the long term these interruptions
actually have the spontaneous effect of bringing the individuals
closer together than before, thereby serving as a unifying force. In the districts scenario, agents managed to resolve their
constraints even less often without self-optimization. Out of 20,000
independent convergences only 53 realized a complete consensus. Most frequently there were medium levels of cooperation with an
average of 2563 agreements. Conditions were improved after self- Overview of the Model In
accordance with our assumption of the worst-case scenario, for
each connection agents selfishly assess whether slightly increasing
or decreasing its strength will increase their own utility and change doi:10.1371/journal.pone.0109966.g003 it accordingly (by modifying the learned weight, vL
ij). Changes to
an agent’s state si depend on the original topology and the
modifications that have taken place (i.e., vij = vO
ij+vL
ij), but for
purposes of analysis it is useful to know how well the network’s
current state satisfies the original constraints alone (vO
ij). j
As an illustrative example, we studied the self-organized
coordination of voters into a global political consensus. All weights
were set to positive values, which poses a consistent problem:
configurations exist that allow every agent to satisfy all of their
social constraints (i.e. all agents vote-a or all agents vote-b). October 2014 | Volume 9 | Issue 10 | e109966 PLOS ONE | www.plosone.org 6 Results Formally, there are two globally optimal behavioral configura-
tions depending on the type of complete political consensus; either
all agents vote for option ‘21’ or all agents vote for option ‘1’ October 2014 | Volume 9 | Issue 10 | e109966 October 2014 | Volume 9 | Issue 10 | e109966 7 PLOS ONE | www.plosone.org Can Government Be Self-Organized? Figure 4. Ten examples of self-organized constraint satisfaction before self-optimization. During each behavior update a representative
in the network is randomly selected, and allowed to adjust its behavior (to +1 or 21) if the new choice satisfies more constraints posed by its
connections with all the others. Because a representative’s received utility is calculated as the weighted sum of its satisfied constraints, priority is
given to satisfying more important (more weighty) connections. As representatives repeatedly optimize their choices, the network’s sum of utilities U
increases. However, as shown by these 10 independent trajectories starting from arbitrary initial conditions, this weak form of self-organization
typically becomes trapped in one of several suboptimal behavioral configurations (U = 173.58 for global optima). Only first 400 behavior updates
shown; after that U stays the same until the end of the run in all cases. doi:10.1371/journal.pone.0109966.g004 Figure 4. Ten examples of self-organized constraint satisfaction before self-optimization. During each behavior update a representative
in the network is randomly selected, and allowed to adjust its behavior (to +1 or 21) if the new choice satisfies more constraints posed by its
connections with all the others. Because a representative’s received utility is calculated as the weighted sum of its satisfied constraints, priority is
given to satisfying more important (more weighty) connections. As representatives repeatedly optimize their choices, the network’s sum of utilities U
increases. However, as shown by these 10 independent trajectories starting from arbitrary initial conditions, this weak form of self-organization
typically becomes trapped in one of several suboptimal behavioral configurations (U = 173.58 for global optima). Only first 400 behavior updates
shown; after that U stays the same until the end of the run in all cases. doi:10.1371/journal.pone.0109966.g004 subsections, e.g. whole neighborhoods and/or whole districts,
resisting behavioral alignment with the global consensus. optimization, even if not to the extent of the neighborhoods
scenario: the maximum number of 4290 agreements was reached
7544 times, while the average number of agreements was 3478. Results Self-optimization therefore frequently resulted in network archi-
tectures significantly more capable of cooperation, but the
outcome was more variable. On many occasions the process
converged on suboptimal configurations, even if optimal or at least
better solutions had previously been encountered. Suboptimal
solutions typically consisted in one or more of the network’s Another important issue to consider is the average amount of
time that is typically required to reach a global behavioral
consensus
and to
complete the
process of
structural self-
optimization. The time taken for structural convergence will be
briefly described in the discussion section below. Here we focus on
the time scale of individual behaviors. We calculated the number
of behavior updates taken to reach the final utility value for each of Figure 5. Sum of utilities reached after convergence during a series of 200 ritual interruptions. In contrast to Figure 4, convergences
happen in sequence rather than independently, and only the final sum of utility is plotted (behavioral updates leading to convergence are not
shown). During a ritual interruption all behavioral states are reset to arbitrary states, as before, but agents’ changes to their social connection weights
are retained. To allow comparison with Figure 4, we show the sum of utilities U based on the original network’s constraints only. Self-optimization is
clearly visible: the network is quickly and spontaneously transformed such that updates of behavior, despite being selfish and rational, consistently
converge on a globally optimal configuration (U = 173.58). doi:10.1371/journal.pone.0109966.g005 Figure 5. Sum of utilities reached after convergence during a series of 200 ritual interruptions. In contrast to Figure 4, convergences
happen in sequence rather than independently, and only the final sum of utility is plotted (behavioral updates leading to convergence are not
shown). During a ritual interruption all behavioral states are reset to arbitrary states, as before, but agents’ changes to their social connection weights
are retained. To allow comparison with Figure 4, we show the sum of utilities U based on the original network’s constraints only. Self-optimization is
clearly visible: the network is quickly and spontaneously transformed such that updates of behavior, despite being selfish and rational, consistently
converge on a globally optimal configuration (U = 173.58). doi:10.1371/journal.pone.0109966.g005 Figure 5. Sum of utilities reached after convergence during a series of 200 ritual interruptions. Results In the case of the neighborhoods scenario we found that
before self-optimization there were on average 225 behavior
updates (with Standard Deviation, SD = 82) before reaching
convergence, whereas after self-optimization an average of 264
updates (SD = 82) were needed. In the case of the districts scenario
an average of 225 (SD = 81) and 269 (SD = 86) updates were
required, respectively. We take it for granted that individuals will behave and change
their social ties in a way that primarily benefits themselves
(although in fact this assumption is not necessary for this self-
optimization
[67]). But
the
peculiar
requirement
that
the
behaviors of the social system as a whole are occasionally
interrupted makes a more specific prediction. We can therefore
hypothesize that the ritual calendar of Teotihuacan included
infrequent, large-scale events during which normal social activity
and norms were suspended. Symbolic behavior largely consists of
arbitrary customs, and it therefore makes a suitable candidate for
the process of systemic interruption by means of behavior
randomization in our model. Ritual intoxication, which was a
common practice in ancient Mesoamerica [77], could have further
enhanced the perturbation. It is suggestive that mural paintings in
the apartment compounds of Tepantitla [78] and Atetelco [42]
depict large-scale social events that include intoxication (Figure 7). In both scenarios significantly more updates were taken after
self-optimization, which is to be expected given that the aim of self-
optimization is precisely to avoid premature convergences on only
partially optimal behavioral consensuses. Yet this represents only
around 3 to 4 behavioral updates per individual representative
until system convergence, given that the network consists of 66
representatives. We note that that this is again a worst-case
scenario because it is the time taken to reach a new consensus after
completely scrambling any previously existing behavioral config-
uration. In everyday situations the impact of ritualized challenges
to the social equilibrium is likely to be smaller and recovery of
normal consensus correspondingly faster. However, we emphasize
that it is difficult to give more precise estimates about the time to
convergence in real time, since the model is too abstract to specify
the duration of a unit of behavior. Results In contrast to Figure 4, convergences
happen in sequence rather than independently, and only the final sum of utility is plotted (behavioral updates leading to convergence are not
shown). During a ritual interruption all behavioral states are reset to arbitrary states, as before, but agents’ changes to their social connection weights
are retained. To allow comparison with Figure 4, we show the sum of utilities U based on the original network’s constraints only. Self-optimization is
clearly visible: the network is quickly and spontaneously transformed such that updates of behavior, despite being selfish and rational, consistently
converge on a globally optimal configuration (U = 173.58). doi:10.1371/journal.pone.0109966.g005 October 2014 | Volume 9 | Issue 10 | e109966 PLOS ONE | www.plosone.org 8 Can Government Be Self-Organized? Figure 6. Comparison of the extent of cooperation in four variations of the Teotihuacan network model. We compared cooperation in
two respects: As a function of self-optimization, i.e. whether connection changes were preserved over the preceding 200 ritualized perturbations or
not (red versus blue bars). And as a function of social organization, i.e. whether the network is divided only into 22 neighborhoods or also additionally
into four larger-scale districts (Neighborhoods versus Districts). For each case we assessed the outcomes of 20,000 independent convergences. The
bars show the average number of agreements reached by the social network given the constraints of its original organization (4290 agreements
equal complete consensus). Error bars represent one standard deviation. doi:10.1371/journal.pone.0109966.g006 Figure 6. Comparison of the extent of cooperation in four variations of the Teotihuacan network model. We compared cooperation in
two respects: As a function of self-optimization, i.e. whether connection changes were preserved over the preceding 200 ritualized perturbations or
not (red versus blue bars). And as a function of social organization, i.e. whether the network is divided only into 22 neighborhoods or also additionally
into four larger-scale districts (Neighborhoods versus Districts). For each case we assessed the outcomes of 20,000 independent convergences. The
bars show the average number of agreements reached by the social network given the constraints of its original organization (4290 agreements
equal complete consensus). Error bars represent one standard deviation. doi:10.1371/journal.pone.0109966.g006 has predictive power that can inform archaeological investigation. In the following we explore some of the implications that could be
topics of future research. the 200 behavioral convergences of each of the 100 experimental
runs. Results If we assume that such ritual events took place twice a year, for
example at the start of the rainy and dry seasons, then the
structural self-optimization exhibited by our model would have
always been completed in less than 100 years. This is consistent
with the time it took for Teotihuacan to grow from a small town to
its full extent as a metropolis. For example, the self-optimization of
the coalition network to a globally optimal configuration shown in
Figure 5 would already have been completed after only 38 years,
which presumably is equivalent to one or two generations of
representatives. Small subsequent adjustments to this optimal
configuration could probably happen much faster, thus making the
coalition quite adaptable. Future work could formally investigate
the network’s adaptability to continuously changing optimality
conditions. October 2014 | Volume 9 | Issue 10 | e109966 Discussion To be sure, this assumes an overly
extreme lack of historicity, but it nevertheless suggests that it is
possible for a government to be collective without a powerful
bureaucracy, and by extension, with little need for extensive
record keeping. In addition to the classes of ruler-controlled
centralized states and bureaucracy-controlled collective states
there may also have been a class of ritually self-optimized
collective states whose unique social organization has so far
largely gone unnoticed by archaeology due to a lack of both
individual personality cults and extensive record keeping. Figure 7. Mural painting located in the Tepantitla apartment
compound. This is a representative section of a much more extensive
scene showing a large number of modestly dressed people who are
engaged in a variety of unusual activities. Most of them do not seem to
be engaged in the mundane activities imposed by the needs of daily
life. Some of the people’s interactions with plants and flowers have
been interpreted in terms of the consumption of psychoactive
substances (see, e.g., on the bottom left where a person is eating a
plant while rainbow-like streams are flowing from his head). Scenes
such as these are indicative of large-scale communal rituals or events at
Teotihuacan. (Photo courtesy of Iliana Mendoza). doi:10.1371/journal.pone.0109966.g007 p
y
p g
Finally, we can also draw insights from the model regarding
what may have caused the downfall of Teotihuacan. The districts
scenario showed that ritualized self-optimization struggles to
develop its full potential when sections of the network are too
independent, so the model predicts that parts of the city became
increasingly autonomous before its end. Indeed, this process is
reflected in the archaeological record. Starting in the city’s middle
period, most inhabitants formed small communities that lived in
self-contained apartment compounds. These walled compounds
increased their autonomy towards the city’s end, as indicated by
controlled access points and direct trade routes to foreign partners. In later periods social divisions at the district-level of the city also
became more prominent, which may have further fractured the
city [14]. Given the crucial role played by system-wide ritual
interruptions in this model, we speculate that Teotihuacan’s end
was also precipitated by a decrease in the relative importance of
citywide rituals compared to compound rituals. which suggests that they were elevated platforms for large public
rituals that could be witnessed at ground level [39]. Discussion This is in
contrast to the common Mesoamerican tradition of building small
and enclosed temples on top of the pyramids, whose indoor
activities would have been visible only to a few elites who were
granted access. In particular, the model supports a religious
interpretation of the enormous building complex known as La
Cuidadela, which contains the Feathered Serpent Pyramid and a
huge open plaza. Archaeologists Go´mez-Cha´vez and Gazzola
have argued that, rather than being the political base of a powerful
ruler, La Cuidadela functioned as a grandiose ritual stage that
incorporated the yearly flooding brought on by the rainy season
and in which all of society participated ([42]: 238). Methods In this final section we provide a more detailed description of
the model and the design choices that were made in its creation. The results also speak to another aspect of the problem of
Teotihuacan’s origins. It is difficult to imagine how individual
rulers and/or bureaucrats could have intentionally designed and
managed the rapidly growing city, especially given that there was
no existing tradition of large-scale urbanism to draw on. However,
our model makes no assumptions regarding the explicit knowledge
and managerial capacities of Teotihuacan’s representatives, since
it is a self-managing system whose adaptive order can spontane-
ously emerge from purely localized social interactions given the
right kinds of conditions. Discussion The model is in agreement with the traditional assumption that
collective action is faced by serious problems without centralized
hierarchical control, but it also clearly shows that spontaneous
cooperation is feasible without it. At least in principle, there is no
necessity to assume the existence of a lineage of powerful rulers to
explain the origins of Teotihuacan. A coalition government,
perhaps as expressed by the TTCs, could have been present from
its beginnings. Moreover, given that the model requires certain
conditions in order for the process of self-optimization to be
effective allows us to put forward the hypothesis that similar
conditions were also present at Teotihuacan. The model therefore Fittingly, there are many large open spaces in the city’s
ceremonial core that could have accommodated such ritual events. Excavations at the Pyramid of the Moon found no evidence for
durable structures on top of its successive construction stages, October 2014 | Volume 9 | Issue 10 | e109966 PLOS ONE | www.plosone.org 9 Can Government Be Self-Organized? Figure 7. Mural painting located in the Tepantitla apartment
compound. This is a representative section of a much more extensive
scene showing a large number of modestly dressed people who are
engaged in a variety of unusual activities. Most of them do not seem to
be engaged in the mundane activities imposed by the needs of daily
life. Some of the people’s interactions with plants and flowers have
been interpreted in terms of the consumption of psychoactive
substances (see, e.g., on the bottom left where a person is eating a
plant while rainbow-like streams are flowing from his head). Scenes
such as these are indicative of large-scale communal rituals or events at
Teotihuacan. (Photo courtesy of Iliana Mendoza). doi:10.1371/journal.pone.0109966.g007 without relying on extensive record keeping, especially if it had a
collective government that had to ensure the cooperation of its
members. Such a corporate strategy has been associated with an
increased, rather than reduced, need for bureaucracy [61]. Of
course, it remains possible that extensive record keeping was done
on perishable media that has had little chance of being preserved
over the centuries. Yet our results suggest another possibility,
namely that even record keeping on impermanent media was a
rather minimal practice. This is because in our model the choices
of representatives are guided by immediate concerns only, without
any explicit consideration of past behaviors, future plans, or rules
of reciprocity and punishment. Spatial Considerations We assume that agents are selfish and rational, which means
that for each social connection they systematically assess whether
increasing or decreasing its strength will increase their individual
utility. In other words, the changes implied by both Dvij = +r and
Dvij = –r are considered, and whichever will increase individual
utility the most is accepted. We fixed the learning rate r to be the
same for all of the experiments (r = 0.0015). If neither change
provides an increase the connection remains unchanged. For
convenience a change is only applied once at the end of a
convergence. Similar results would be obtained if a smaller
learning rate were applied continuously as long as the system
spends most of its time in a converged state [69]. Accordingly: We did not modulate the network’s strengths of connections in
relation to the spatial distance of the TTCs from each other (i.e., as
shown in Figure 3), because a pilot study of a 22-node spatially
embedded network, in which strength of connection was inversely
proportional to distance, did not reveal significant effects of space
compared to a non-spatial network. It is possible to attribute this
irrelevance of spatial distance to the spatial distribution of TTCs. When we analyzed the frequency of distances, we found that most
TTCs are very close to each other with only a few long-distance
outliers. In other words, if we take spatial proximity as a marker of
the strength of mutual interconnectivity, then a network of 22
spatially embedded TTCs has more or less the same connectivity
as a fully connected 22-node network. The absence of a scale-free
distribution
of
connectivity
is
interesting
in
itself,
because
archeologists typically view scale-free networks as an indicator of
social hierarchy [81]. u0
iwui[
X
N
j
v0
ijsisjw
X
N
j
vijsisj u0
iwui[
X
N
j
v0
ijsisjw
X
N
j
vijsisj Archaeologically speaking, choosing to investigate a non-spatial
network of neighborhood representatives also makes sense if all
representatives met in a single location to discuss and vote on
policies, which seems to be a reasonable assumption in the case of
a Teotihuacan coalition. Mathematical Details We follow Watson and colleagues [67,69,80] in using the
network architecture first proposed by Hopfield [66]. Each agent
can adopt one of two discrete behavioral states, si = +1/21, which
stands for a choice of action (do-a/do-b). We use an asynchronous
updating rule, which means that for each point in time t an agent
is randomly selected to update its behavior for time step t+1. The
selected agent will adopt the behavior that maximizes its own
utility, ui, which is defined as the weighted sum of its interactions
with other agents, It goes without saying that the possibility of a coalition
government fits with Teotihuacan’s unique artistic tradition and
the notable absence of public dynastic monuments [43]. The
results also suggest an explanation of another one of Teotihuacan’s
unique cultural traits that has remained a mystery so far, namely
the fact that there was not much interest in emulating Maya or
Zapotec literacy. This raises questions about the nature of a society
that largely dispensed with complex writing on a voluntary basis
[79]. To be sure, in the absence of great individual rulers, there
was no need to record dynastic information. But it remains difficult
to imagine how a city of this complexity could have functioned ui~
X
N
i
vijsisj ui~
X
N
i
vijsisj where the connection weight vij represents the importance for
agent i of satisfying the constraint posed by its interaction with
agent j. The more positive vij, the more important it is for agent i
to imitate the behavior of agent j (such that both do-a or both do-
b), while the more negative vij, the more important it is for agent i October 2014 | Volume 9 | Issue 10 | e109966 PLOS ONE | www.plosone.org October 2014 | Volume 9 | Issue 10 | e109966 10 Can Government Be Self-Organized? to complement the behavior of agent j (such that agent i and agent
j either do-a and do-b, or do-b and do-a, respectively). We distinguish two aspects of vij. On the one hand we specify
and retain the original problem space or network topology, vO
ij,
while on the other hand we keep track of the changes that
accumulate as agents selfishly update the weights of their
interactions, vL
ij. These weight changes are equivalent to
simplified Hebbian learning in Hopfield neural networks [80]. Mathematical Details While the original weights vO are static throughout a run, the
learned weights vL depend on the current time step, and both
make up the current weights of the network: vL
ij(tz1)~vL
ij(t)zDvij Following Hopfield’s original proposal [66] we also enforce that
the connection weights are initialized symmetrically, i.e. vij = vji. This ensures that the network will eventually settle into a fixed-
point attractor. Allowing asymmetrical connections (i.e., vij?vji),
for example such that agent i’s next behavioral state is more
dependent on the behavior of agent j than vice versa, does not
alter the general fact that selfish updates of connections resemble
Hebbian learning [69]. However, a network that includes strongly
asymmetrical connections allows for a greater variety of attractor
types; future work should investigate their effects. We also ignore
self-connections, i.e. vii = 0. Their inclusion would simply have the
effect of increasing the system’s total utility U by a constant
amount, since an agent’s behavior will always be identical with its
own behavior. vij(t)~vO
ij zvL
ij(t) While the update of an agent’s state si is determined by how it
evaluates its modified connections, v, it is possible to determine
how this state affects the network’s ability to satisfy the constraints
of the original weight space vO. The sum of utilities given the
original problem space is calculated as follows: Following Watson and colleagues, a new set of random
behavioral states, R = [21|1]N, was assigned every t time steps;
we set t = eN lnN, where N = 66. We set the number of behavior
randomizations to 200, which was sufficient to allow self-
optimization to occur in most cases. October 2014 | Volume 9 | Issue 10 | e109966 The Self-Optimization Process Further work is required to fully understand the mathematical
basis and scope of the self-optimization process [85], but its effects
should be noticeable in any heterarchically organized system that
satisfies the following conditions: (a)
Agents try to optimize their own situation by rapidly
adjusting their behavior in relation to what others are doing, (b)
Agents try to optimize their own situation by slowly adjusting
their social relations in relation to what others are doing, and (c)
Agents, the more the better, occasionally jointly change their
behavior in some arbitrary manner. Accordingly, the model provides a genuinely system-level
explanation of collective government: globally optimal architec-
tures tend to be spontaneously arranged by the agents even though
each agent is behaving selfishly and even though no agent has
knowledge about the structure of the overall problem space. Adaptive management of the social system as a whole emerges out
of the local interactions among members of the social network. It is
noteworthy that this process is based on formal principles that do
not depend on the specifics of the current model. Mathematically speaking, condition (a) causes the social system
to quickly converge on an optimum, although in most cases it will
only be a locally optimal one, (b) causes the system to form an
associative ‘‘memory’’ of the already visited optima, and (c) causes
the system to visit different kinds of optima over longer periods of
time,
thereby
causing
the
associative
memory
to
become
generalized in such a manner that better optima are more easily
encountered [69]. The very possibility that a complex social system as a whole can
self-organize its governmental functions successfully – without
relying on the usual mechanisms of centralized top-down control,
without depending on any explicit representation or knowledge of
the solutions, and more generally while avoiding the extremes of
social stratification – is something to keep in mind when
addressing today’s seemingly insurmountable global crises. It is condition (c) that is of special interest. Selfish updates of
connections after converging on only one attractor would not be
adaptive. Given that in most complex networks the number of
suboptimal attractors by far outnumbers more optimal configura-
tions, it is most likely that the network would become trapped in a
local optimum, and the adjustments of connections would
therefore simply have the undesirable effect of even further
reinforcing the suboptimal attractor. Spatial Considerations For example, a systematic analysis of
surface remains has indicated that during the city’s later years the
interests of the various neighborhoods might have been repre-
sented in apartment compounds that were co-located in a large
forum known as the Great Compound [82], which is situated at
the southern part of the Street of the Dead across from the
Feathered Serpent Pyramid. Another promising location for a
unified seat of the coalition government is a compound known as
Xalla
[83]. This
alternative
hypothesis
is
currently
being
investigated via excavations conducted by Manzanilla as part of
the project ‘‘Teotihuacan: Elite y gobierno’’. It may be questioned whether real agents can behave as
rationally as this, but the assumption of perfect rationality has little
impact on the overall dynamics of the social network. Similar
effects can be obtained by assuming that agents generally behave
in a habitual manner, such that the propensity of agent i to imitate
(or instead to complement) agent j’s behavior will be enhanced if
agent i is currently imitating (or complementing) agent j’s behavior
[67]. Given that social connections cannot be modified infinitely,
we assume that weights are limited to the range [–1, 1] by a linear
threshold function h such that if x.1 then h(x) = 1 or if x,21 then
h(x) = 21. In other words: vij(t)~h vO
ij zvL
ij(t)
h
i Further choices had to be made regarding how to design the
districts scenario. Evidence exists that during later periods of the
city the neighborhood centers were organized into larger districts,
although retaining a strongly heterarchical emphasis [22]: up to
45% of apartment compounds were comparable in type across the
city, overshadowing neighborhood membership, religious affilia- where where PLOS ONE | www.plosone.org October 2014 | Volume 9 | Issue 10 | e109966 11 Can Government Be Self-Organized? tion, and status [84]. Manzanilla [14] has argued that during this
time the city was divided into four separate districts, that is, from
North to South along the Street of the Dead and from West to
East via the Great Compound and the Feathered Serpent
Pyramid. There are indications that during earlier periods the
West-East axis was located further north in the city, across the
Pyramid of the Sun ([58]; R. Cabrera Castro, personal comm.). The Self-Optimization Process However, the outcome is
quite different when we occasionally randomly reset or otherwise
perturb the system, followed by periods of sufficient time to reach
convergence. If the perturbations are sufficiently powerful then the
system will start itinerating over its different attractors, exploring
the attractors that are possible to reach from different initial 5. Cowgill GL (2008) Teotihuacan as an urban place. In: Mastache AG, Cobean
RH, Garcı´a Cook A´ , Hirth K, editors. El Urbanismo en Mesoamerica/
Urbanism in Mesoamerica Volume 2. Mexico City, DF and University Park,
PA: INAH and Pennsylvania State University. 85–112. Spatial Considerations Since we are interested in modeling the initial period of
Teotihuacan, it is this earlier West-East axis that we used in our
model to define the four districts of neighborhoods. We therefore
ended up with the following districts: Northwest (10 TTCs),
Northeast (2 TTCs), Southwest (5 TTCs), and Southeast (5 TTCs). Future modeling work could look at other ways of carving up the
city’s space and at changing the
number of social units
participating in the network. Nevertheless, due to the generality
of the self-optimizing process we expect that at least qualitatively
similar results can be obtained under a variety of conditions. conditions. Watson and colleagues discovered that under these
conditions the system self-optimizes its connections such that
better configurations are more likely to be visited [69]. There are
two aspects to this self-optimization process. Enhanced recall. It has been shown that in this type of
network better (i.e., higher or deeper, depending on the sign used
to define utility) attractors have larger basins of attraction [86]. This entails that the better attractors, although numerically less
common than suboptimal optima, will be visited relatively more
frequently in a long sequence of restarts. The Hebbian-style
learning implemented by the changes to connections will therefore
tend to reinforce the better attractors more strongly, which means
that their likelihood of visitation is further increased until only the
best attractors remain. Generalization. But how do we explain that the network
frequently converges on globally optimal solutions, which it had
never visited before, and much faster than should normally be
expected? This surprising effect can be accounted for by the fact
that the network starts to generalize over all of the attractor
configurations that it has already reinforced. This generalization is
a well-known property of trained Hopfield networks, which exhibit
a type of associative memory [87], except that in this case the set of
training patterns is the set of visited attractors of the network itself. One way to think about this is to consider that when the visited
attractor configurations of the network are decomposable into
shared features, for example due to modularity, the selfish
modifications of connections can lead to the enhancement of
basins of attraction of not-yet visited attractors consisting of better
combinations of those same component features [69]. Author Contributions Conceived and designed the experiments: TF CG LM. Performed the
experiments: TF. Analyzed the data: TF CG. Contributed to the writing of
the manuscript: TF CG LM. 1. Beramendi-Orosco LE, Gonzalez-Hernandez G, Urrutia-Fucugauchi J, Man-
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the Mesoamerican urban center of Teotihuacan derived from Bayesian statistics
of radiocarbon and archaeological data. Quatern Res 71: 99–107. y
y
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zanilla LR, Soler-Arechalde AM, et al. (2009) High-resolution chronology for
the Mesoamerican urban center of Teotihuacan derived from Bayesian statistics
of radiocarbon and archaeological data. Quatern Res 71: 99–107.
2. Cowgill GL (2007) The urban organization of Teotihuacan, Mexico. In: Stone
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Adams. Los Angeles, CA and Chicago, IL: Cotsen Institute of Archaeology and
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Albuquerque, NM: University of New Mexico Press. 205–247.
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demography. Science 162: 872–877.
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Searching for the Bacterial Effector: The Example of the Multi-Skilled Commensal Bacterium Faecalibacterium prausnitzii
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Frontiers in microbiology
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Searching for the Bacterial Effector:
The Example of the Multi-Skilled
Commensal Bacterium
Faecalibacterium prausnitzii
Rebeca Martín, Luis G. Bermúdez-Humarán and Philippe Langella*
National Institute of Agricultural Research, Commensals and Probiotics-Host Interactions Laboratory, Micalis Institute,
AgroParisTech, Paris-Sud University, Jouy-en-Josas, France National Institute of Agricultural Research, Commensals and Probiotics-Host Interactions Laboratory, Micalis Institute,
AgroParisTech, Paris-Sud University, Jouy-en-Josas, France Faecalibacterium prausnitzii represents approximately 5% of the total fecal microbiota
in healthy adults being one of the most abundant bacterium in the human intestinal
microbiota of healthy adults. Furthermore, this bacterium has been proposed to be a
sensor and a major actor of the human intestinal health because of its importance in
the gut ecosystem. In this context, F. prausnitzii population levels have been found
to be reduced in patients suffering from several syndromes and diseases such as
inflammatory bowel diseases. These diseases are characterized by a breakage of the
intestinal homeostasis called dysbiosis and the use of F. prausnitzii as a next generation
probiotic (also called live biotherapeutics) has been proposed as a natural tool to
restore such dysbiosis within the gut. Nevertheless, despite the potential importance
of this bacterium in human health, little is known about its main effectors underlying
its beneficial effects. In this perspective note, we aim to present the actual state in the
research about F. prausnitzii effectors and the future milestones in this field. Edited by:
Vittorio Capozzi,
University of Foggia, Italy p
,
University of Foggia, Italy
Reviewed by:
Francesca Turroni,
Università degli Studi di Parma, Italy
Valerio Iebba,
Sapienza Università di Roma, Italy
*Correspondence:
Philippe Langella
philippe.langella@inra.fr Reviewed by:
Francesca Turroni,
Università degli Studi di Parma, Italy
Valerio Iebba,
Sapienza Università di Roma, Italy Keywords: probiotic, commensal, Faecalibacterium, bacterial effectors, live-biotherapeutics PERSPECTIVE
published: 06 March 2018
doi: 10.3389/fmicb.2018.00346 PERSPECTIVE INTRODUCTION *Correspondence:
Philippe Langella
philippe.langella@inra.fr Nowadays, humans can be considered as “meta-organisms” composed of 10-fold more
microorganisms than human cells (Neish, 2009), which means 150-fold more genes than the
human genome itself (Qin et al., 2010; Bruls and Weissenbach, 2011). These microorganisms,
named microbiota (and by extension all their genes as a whole, named microbiome) are different
depending on the tissue considered. The human gastrointestinal tract (GIT) is one of the most
complex ecosystems. The advances of molecular techniques have shown that the collective adult
human GIT microbiota is composed of up to 1000–1150 bacterial species (Frank et al., 2007;
Qin et al., 2010). The predominant species (46–58%) are those with low GC-content being the
clostridium group the most abundant (Zoetendal et al., 2002; Qin et al., 2010). As a consequence
of the mutualism established between the host and its microbiota, the GIT micro-ecosystem is
key to maintain the homeostasis of a healthy individual (Leser and Molbak, 2009). Indeed, gut
microbiota supplies essential nutrients, metabolize indigestible compounds and protects the host
against colonization by opportunistic pathogens (Leser and Molbak, 2009; Martín et al., 2013). It also contributes to the development of the intestinal architecture as well as several immuno-
modulatory functions (Mazmanian et al., 2005). In some abnormal conditions, an imbalance
in the microbial ecosystem may happen leading to a microbial imbalance known as dysbiosis. This dysbiosis is characterized by the growth of different non-predominant bacteria and/or the Specialty section:
This article was submitted to
Food Microbiology,
a section of the journal
Frontiers in Microbiology
Received: 24 October 2017
Accepted: 13 February 2018
Published: 06 March 2018
Citation:
Martín R, Bermúdez-Humarán LG
and Langella P (2018) Searching
for the Bacterial Effector: The Example Specialty section:
This article was submitted to
Food Microbiology,
a section of the journal
Frontiers in Microbiology Specialty section:
This article was submitted to
Food Microbiology,
a section of the journal
Frontiers in Microbiology
Received: 24 October 2017
Accepted: 13 February 2018
Published: 06 March 2018 Received: 24 October 2017
Accepted: 13 February 2018
Published: 06 March 2018 THE HUMAN GUT MICROBIOTA AS A
SOURCE OF NEXT GENERATION
PROBIOTICS (NGPs) As gut microbiota is now considered as a major actor
underlying health, the idea of using some well-known microbiota
components as next generation probiotics (NGPs) is very
promising. Probiotics are “live microorganisms which when
administered in adequate amounts confer a health benefit on
the host” (FAO/WHO, 2001). Recently, this definition has been
clarified by an expert panel of the International Scientific
Association for Probiotics and Prebiotics (ISAPP) and re-defined
as: “live microorganisms that, when administrated in adequate
amounts, confer a health benefit on the host” (Hill et al., 2014). Most of the traditional probiotics belong to both lactic acid
bacteria (LAB) and Bifidobacteria groups. These novel probiotics
are thus considered as NGPs in contrast to traditional ones, which
were not selected on the basis of human microbiota analysis. To
consider a strain as probiotic, it should be: (i) well-characterized,
(ii) achieve safety requirements, and (iii) confer beneficial effects
on the host. However, a careful selection should be made in each
case as probiotic properties are usually strain specific and cannot
be extrapolated to another strain even belonging to the same
species (Pineiro and Stanton, 2007; Gareau et al., 2010). Similarly, as probiotic therapy is mainly based on restoring
the normal balance of the intestinal ecosystem, we consider that
the use of commensal bacteria as NGPs is a natural way to
restore the dysbiotic situation within the GIT (Miquel et al.,
2015a). Nevertheless, in contrast to most of probiotic lactobacilli
or bifidobacteria strains, these NGPs are not listed neither on
QPS (for Qualified Presumption of Safety), nor on GRAS (for
Generally Recognized As Safe organisms) lists. Furthermore, as
they have not a long record of safe consumption (precisely no
documented safe use in Europe prior to 1997), these NGPs can
only be used either as novel foods or drugs and the requirements
to allow their market in Europe are more severe than for
conventional probiotic strains (Miquel et al., 2015a). Nowadays,
the inclusion of Faecalibacterium on the QPS list might be
difficult due to its lack of a history of safe use. In addition,
full toxicology assays and characterization of the strain are still
needed for regulatory approval (Brodmann et al., 2017). We
have reviewed this problematic in detail elsewhere (Miquel et al.,
2015a). Because
of
its
important
role
in
GIT
homeostasis,
F. prausnitzii is considered today as a potential NGPs. Citation: As a result, this imbalance can also lead to the lack of some
beneficial effects of these commensal bacteria, and thus unchain
pathogenic conditions not only due to pathogen overgrowth
(Martín et al., 2013). THE HUMAN GUT MICROBIOTA AS A
SOURCE OF NEXT GENERATION
PROBIOTICS (NGPs) Its
potential utilization has been already proposed for livestock
animals, for instance the isolation and characterization of
F. prausnitzii strains from stool of calves and piglets have
been already performed (Foditsch et al., 2014). Also a specific
formulation keeping this EOS bacterium alive at ambient air
conditions has been also proposed for patients with intestinal
dysbiosis-associated diseases (Khan et al., 2014). In order
to evaluate its potential beneficial effects as a NGP, we have
successfully used this bacterium in several murine models of
IBD and IBS (Sokol et al., 2008; Martín et al., 2014a; Laval
et al., 2015; Miquel et al., 2016) and other groups have also
clearly demonstrated its beneficial effects in vivo (Carlsson et al.,
2013; Rossi et al., 2015, 2016). Briefly, we have found that mice
treated with either F. prausnitzii A2-165 or its supernatant (SN)
present lower symptoms of inflammation in both acute and
chronic chemically-induced colitis models as well as improved
gut permeability and function in a model of gut impairment
induced by dinitrobenzene sulfonic acid (DNBS) (Sokol et al.,
2008; Martín et al., 2014a, 2015; Laval et al., 2015). We have also
observed that A2-165 strain was able to reduce pain sensibility in Citation: Martín R, Bermúdez-Humarán LG
and Langella P (2018) Searching
for the Bacterial Effector: The Example
of the Multi-Skilled Commensal
Bacterium Faecalibacterium
prausnitzii. Front. Microbiol. 9:346. doi: 10.3389/fmicb.2018.00346 March 2018 | Volume 9 | Article 346 1 Frontiers in Microbiology | www.frontiersin.org Bacterial Effectors of Faecalibacterium prausnitzii Martín et al. most of the data about its physiology are based on metagenomic
studies (Miquel et al., 2013), with some exceptions (Duncan
et al., 2002; Ramirez-Farias et al., 2009; Lopez-Siles et al.,
2012; Foditsch et al., 2014; Martín et al., 2017). F. prausnitzii
is a member of the Clostridium group (phylum Firmicutes,
class Clostridia, family Ruminococcaceae), specifically of the
C. leptum group (Benevides et al., 2017), and represents around
5% of the total fecal microbiota in healthy adults being one
of the most abundant bacterium in the human intestinal
microbiota in healthy conditions (Hold et al., 2003). The first
isolates were classified as Fusobacterium praustnizzi, but latter
on its close relation with members of the C. leptum group
was established thorough analysis of 16S rRNA gene (Miquel
et al., 2014). The establishment of F. prausnitzii along the
GIT may result from a combination of environmental factors
such as other commensal species, redox mediators, oxygen
concentration, mucus layer as well as bile salt concentrations
and pH (Duncan et al., 2009; Lopez-Siles et al., 2012). In early
infancy, F. prausnitzii abundance is very low and increases
after the establishment of primo-colonizing bacteria (Hopkins
et al., 2005). In the last years, this bacterium has been
suggested as a biosensor and a major actor of the human
intestinal health (Miquel et al., 2013). Indeed, F. prausnitzii
levels have been found to be reduced in patients suffering
from several syndromes and diseases such as inflammatory
bowel diseases (IBDs), irritable bowel syndrome (IBS), colorectal
cancer (CRC), obesity, and celiac disease (Balamurugan et al.,
2008; Sokol et al., 2008; Neish, 2009; De Palma et al., 2010;
Furet et al., 2010; Rajilic-Stojanovic et al., 2011) as well as
in frail elderly (van Tongeren et al., 2005). We have more
deeply review F. prausnitzii physiology and beneficial effect
elsewhere (Miquel et al., 2013, 2014). Nevertheless, its EOS
condition, make viable intestinal delivery one of the current
challenges, due to their stringent survival conditions (El Hage
et al., 2017). depletion of commensal ones that can lead to a situation of illness. Butyrate Typically, as F. prausnitzii is one of the most abundant butyrate-
producing species, its beneficial effects have been first attributed
to butyrate. Butyrate is a short chain fatty acid (SCFA) well-
known for its pleiotropic and beneficial effects in the GIT
(Duncan et al., 2009; Macfarlane and Macfarlane, 2011) as well as
its immune-modulatory properties in vitro (Bocker et al., 2003). Furthermore, it is involved in the cross-feeding between butyrate
producer bacteria and Bifidobacterium sp. which favors the co-
existence of bifidobacterial strains with other bifidobacteria and
with butyrate-producing colon bacteria in the human colon
with the consequent benefit in the human health (Riviere et al.,
2016). Microbial butyrate is considered key for colonic health,
as it is an energy source for epithelial cells and is able to
modulate oxidative stress and inflammation (Hamer et al., 2008). However, its role remains controversial as its effects seem to
be dose- and time-dependent (Martín et al., 2013). In this
sense, it also has different effects depending of the cell line
tested (Bocker et al., 2003). For instance, regarding cells from
intestinal origin, butyrate was found to decrease IL-8 secretion in
Caco-2 and human intestinal primary epithelial cells (HIPECs)
and to enhance IL-8 production in both HT-29/p and HT-29
MTX cells (Bocker et al., 2003). Furthermore, not all butyrate
producing-bacteria have the same anti-inflammatory profile
in vitro. For instance, in 6 h TNF-α stimulated HT-29 cells,
the SN of Roseburia intestinalis, a butyrate producer, does not
have anti-inflammatory properties while F. prausnitzii SN does
(Figure 1D). Staring the host pathways involved in the beneficial effects
displayed by F. prausnitzii, in vitro tests have shown that: (i)
although F. prausnitzii itself had no effect on IL-1β-induced
NF-κB activity, its SN abolished it in Caco-2 cells transfected
with a reporter gene for NF-κB activity and (ii) peripheral blood
mononuclear cell (PBMC) stimulation by F. prausnitzii led to
significantly lower IL-12 and IFN-γ production levels and higher
secretion of IL-10 (Sokol et al., 2008). In this sense, human
dendritic cells (DCs) stimulation with A2-165 and HTF-F strains
also induced the production of IL-10 (Rossi et al., 2015). IL-10 has
been established to be an important immune-regulatory cytokine
that successfully suppresses the exacerbated mucosal immune
response associated with colonic inflammation (Schreiber et al.,
2000). This increasing in IL-10 induced by F. praustnizii has also
been observed in vivo (Sokol et al., 2008). Furthermore, Rossi
et al. (Figure 1D). Although the effect of butyrate is clearly present, the anti-
inflammatory capacities of F. prausnitzii do not seem to be
limited to butyrate only. In previous studies, we have shown
that F. prausnitzii-mediated butyrate production is not the only
beneficial bacterial effector linked to this species in colitis models
(Sokol et al., 2008; Martín et al., 2013; Miquel et al., 2015b). For instance, we found that the increase of the presence of 4-
hydroxybutyric acid in the feces, colon and caecum of dixenic
mice colonized with F. prausnitzii A2-165 and a strain of
Escherichia coli compared to monoxenic mice colonized only by
the strain of E. coli was not directly linked to health parameters
in a rat model of acute trinitrobenzenic acid (TNBS)-induced
colitis although the increase of production of butyrate has been
also verified by gas liquid chromatography (Miquel et al., 2015b). These results support previous findings, where we found that
butyrate did not protect mice from TNBS-induced colitis, in
contrast to F. prausnitzii A2-165 SN (Sokol et al., 2008). A FOCUS ON Faecalibacterium
praustnizii As Faecalibacterium prausnitzii is an extremely oxygen sensitive
(EOS) bacterium and difficult to grow (Duncan et al., 2002), March 2018 | Volume 9 | Article 346 Frontiers in Microbiology | www.frontiersin.org 2 Martín et al. Bacterial Effectors of Faecalibacterium prausnitzii Some of the putative bacterial effectors identified until now
are presented in the Figure 2 and are described in the next
paragraphs. partial restraint stress (PRS) and neo-maternal separation (NMS)
murine models (Miquel et al., 2016). Furthermore, Carlsson
et al. (2013) and Rossi et al. (2015, 2016) have found similar
anti-inflammatory results as well as restoration of increased
intestinal permeability in dextran sulfate sodium (DSS) induced
colitis. Faecalibacterium praustnizii
EFFECTORS: WHERE DO WE STAND? Since the first study about F. prausnitzii performed in our
laboratory almost 10 years ago (Sokol et al., 2008), we have
sought to identify the bacterial effectors underlying its beneficial
effects. We have been focused on its SN which showed anti-
inflammatory properties in both in vivo and in vitro experiments
(Sokol et al., 2008; Martín et al., 2013). Our main hypothesis
was that F. prausnitzii can produce an anti-inflammatory soluble
molecule. First, we tried to identify the chemical nature of the
molecule by submitting the SN to several enzymatic and physical
methods and as shown in Figures 1A–C, none of them were
able to suppress the anti-inflammatory “properties” from the SN
pointing out the possible presence of more than one effector
on F. praustnizii SN. Of course, this result can also support
an important role of butyrate in F. prausnitzii effects, although
in vivo and in vitro experiments point out for a more complex
situation (see below). Nevertheless, we need to consider that the
beneficial effects of F. prausnitzii might not be present only in its
SN. For instance, we have found that some beneficial effects, such
as the anti-nociceptive one, are observed only when the animals
where treated with the bacterium but not with its SN (Miquel
et al., 2016). Butyrate (2016) found that the strain of F. prausnitzii A2-165 has
a strong capacity to induce IL-10 in human and murine DCs
and influence the T-cell differentiation in vitro and in vivo. In
this sense, F. prausnitzii is also able to increase lymphocyte T
regulatory (Treg) population in vivo after a colonic chemical
challenge (Martín et al., 2014a) and has been identified as
the major inducer of a specific IL-10 secreting Treg subset
named CD4CD8α lymphocytes present in the human colonic
lamina propria and blood which is deficient in IBD patients
(Sarrabayrouse et al., 2014). Other Active Metabolites The use of a gnotobiotic model including F. prausnitzii A2-165
strain and E. coli allowed us, using a metabolomic approach, to
identify several metabolites that, in contrast to 4-hydroxybutyric
acid, are associated to the beneficial effect of F. prausnitzii
in a TNBS-acute model in rats (Miquel et al., 2015b). These
metabolites were the salicylic acid, shikimic acid and raffinose, March 2018 | Volume 9 | Article 346 Frontiers in Microbiology | www.frontiersin.org 3 Martín et al. Bacterial Effectors of Faecalibacterium prausnitzii FIGURE 1 | In vitro immune-modulatory test on HT-29 cells stimulated with TNF-α. Anti-inflammatory activity of Faecalibacterium prausnitzii A2-165 supernatant
(SN) and culture medium (LYBHI) submitted to different treatments: (A) enzymatic treatments: amylase (red), lipase (green) pepsin (yellow), trypsin (orange) and
pronase (pink), (B) pH modifications: pH 3 (cyan) and pH 8 (brown), and (C) different temperatures: 100◦C (red) and 50◦C (orange). (D) Comparison of Roseburia
intestinalis DSM14610T SN and F. prausnitzii A2-165 SN effect. Bacterial SNs were recovered, filtered and submitted to different treatments (if indicated) before
being tested in co-incubation assays with HT-29 cells challenged with TNF-α. IL-8 concentration was determined by ELISA and used as a read-out of the
inflammatory status of the cells. The results were normalized by the negative control (PBS) (for material and methods details see Supplementary Data 1). ∗p < 0.05
versus LYBHI (bacterial culture medium) (n = 3∗3). FIGURE 1 | In vitro immune-modulatory test on HT-29 cells stimulated with TNF-α. Anti-inflammatory activity of Faecalibacterium prausnitzii A2-165 supernatant
(SN) and culture medium (LYBHI) submitted to different treatments: (A) enzymatic treatments: amylase (red), lipase (green) pepsin (yellow), trypsin (orange) and
pronase (pink), (B) pH modifications: pH 3 (cyan) and pH 8 (brown), and (C) different temperatures: 100◦C (red) and 50◦C (orange). (D) Comparison of Roseburia
intestinalis DSM14610T SN and F. prausnitzii A2-165 SN effect. Bacterial SNs were recovered, filtered and submitted to different treatments (if indicated) before
being tested in co-incubation assays with HT-29 cells challenged with TNF-α. IL-8 concentration was determined by ELISA and used as a read-out of the
inflammatory status of the cells. The results were normalized by the negative control (PBS) (for material and methods details see Supplementary Data 1). ∗p < 0.05
versus LYBHI (bacterial culture medium) (n = 3∗3). among others (Miquel et al., 2015b). Other Active Metabolites Salicylic acid is used
in the pharmaceutical industry to produce the amine derivate
5-aminosalicylic acid (5-ASA or mesalamine) that it is nowadays
used to treat patients suffering from IBD (Messori et al., 1994). Furthermore, shikimic acid is a precursor for the synthesis of several aromatic compounds among which we can also find
the salicylic acid through the achorismate synthase pathway
(Bochkov et al., 2012). These two compounds point out a key role
of F. praustnizii in the biosynthesis of salicylic acid, precursor of
5-ASA, both anti-inflammatory molecules that should be linked March 2018 | Volume 9 | Article 346 Frontiers in Microbiology | www.frontiersin.org 4 Martín et al. Bacterial Effectors of Faecalibacterium prausnitzii FIGURE 2 | Putative effectors of Faecalibacterium prausnitzii and its effects on the host. F. prausnitzii exerts its benefic effects by means of different effectors:
(A) MAM peptides secreted by F. prausnitzii block NF-κB activation induced by a pro-inflammatory stimulus. (B) Butyrate produced by F. prausnitzii inhibits NF-κB
activation and interacts with the intestinal epithelial cells (IEC) driving to the activation of different genes involved on the differentiation, proliferation, and restitution of
enterocytes. It is also involved on the regulation of fatty acid oxidation, electrons transport chain, oxidative stress, and apoptosis. In goblet cells it has been
described to stimulate muc genes allowing a high production of mucus. (C) EPM produced by F. prausnitzii modulates IL-10 cytokine production in antigen
presenting cells. Finally, (D) salicylic and shikimic acids are anti-inflammatory molecules able to block inflammation induced by a pro-inflammatory stimulus while
raffinose is key in maintaining gut permeability. FIGURE 2 | Putative effectors of Faecalibacterium prausnitzii and its effects on the host. F. prausnitzii exerts its benefic effects by means of different effectors:
(A) MAM peptides secreted by F. prausnitzii block NF-κB activation induced by a pro-inflammatory stimulus. (B) Butyrate produced by F. prausnitzii inhibits NF-κB
activation and interacts with the intestinal epithelial cells (IEC) driving to the activation of different genes involved on the differentiation, proliferation, and restitution of
enterocytes. It is also involved on the regulation of fatty acid oxidation, electrons transport chain, oxidative stress, and apoptosis. In goblet cells it has been
described to stimulate muc genes allowing a high production of mucus. (C) EPM produced by F. prausnitzii modulates IL-10 cytokine production in antigen
presenting cells. Other Active Metabolites Finally, (D) salicylic and shikimic acids are anti-inflammatory molecules able to block inflammation induced by a pro-inflammatory stimulus while
raffinose is key in maintaining gut permeability. seven peptides, all derived from the same anti-inflammatory
molecule, a protein of 15 kDa named MAM (ZP05614546.1)
(Quevrain et al., 2016). Due to the difficulties to test directly
the peptides or the protein, mainly due to solubility problems,
indirect strategies were performed to determine their biological
effect. Transfection of MAM cDNA in epithelial cells led to
a significant decrease in the activation of the nuclear factor
(NF)-κB pathway with a dose-dependent effect (Quevrain
et al., 2016). This inactivation of NF-κB pathway was also
observed in vivo using a transgenic model of mice producing
luciferase under the control of NF-κB promoter (Breyner et al.,
2017). Finally, the administration of a food-grade bacterium,
Lactococcus lactis, delivering a plasmid encoding MAM was
able to alleviate DNBS and DSS induced colitis in mice
(Quevrain et al., 2016; Breyner et al., 2017). Although these
promising results point out for the strong role of MAM on
F. prausnitzii SN effect, the persistence of the anti-inflammatory
effect of the SN after proteolytic treatment and the ability of
F. prausnitzii SN to block other inflammatory pathways different
from NF-κB (Martin et al., 2014b) point that MAM is not the to the in vivo anti-inflammatory effect observed in mice treated
with F. praustnizii. In contrast, raffinose is an oligosaccharide
only fermented by the gut microbiota that is not related to anti-
inflammatory effects, but with mucosal permeability, as raffinose
permeation is key in maintaining gut permeability (Dawson et al.,
1988). The raffinose could thus play a role in the improvement
on gut permeability promoted by F. praustnizii (Carlsson et al.,
2013; Laval et al., 2014). In this sense, we have found that
F. praustnizii and its SN are able to counterbalance the increase
in intestinal barrier permeability in a murine model of gut
dysfunction induced by DNBS (Martín et al., 2015). Of note, in
this simplified microbiota, we have identified metabolites that can
be produced either by the host or by the bacteria, and the direct
production of these compounds by F. prausnitzii has not yet been
proved. Microbial Anti-inflammatory
Molecule (MAM) Thanks to a peptidomic analysis using mass spectrometry of
F. prausnitzii A2-165 strain SN, we have successfully identified March 2018 | Volume 9 | Article 346 Frontiers in Microbiology | www.frontiersin.org 5 Bacterial Effectors of Faecalibacterium prausnitzii Martín et al. only bacterial effector mediating F. praustnizii SN beneficial
effects. only bacterial effector mediating F. praustnizii SN beneficial
effects. crosstalk with the host and the possible effectors underlying
these effects. Furthermore, the pangenome analysis have been
performed to get a global view of the genome of this genus. A total of 10,366 core-genome codifying DNA sequences
have been found (3.33-fold the average total number of
genes of the 17 analyzed strains) (Benevides et al., 2017). Nevertheless, up today no extensive genomic description of
this taxon has been finished, being an ongoing task with
a key role in the future perspectives of the analysis of this
genus. CONCLUDING REMARKS In this perspective article, we have tended to highlight
the importance and problematic of asserting the probiotic
characterization of a NGP, a field on the focus of the research
of Frontiers in Microbiology audience. Nowadays, the research
community recognizes the importance of F. prausnitzii in the
human health. A decrease of this bacterium in the GIT has
been linked to several diseases and syndromes but it is still
not clear if this is a cause or a consequence of them. As
mentioned above, this special condition, make this species a
unique bacterial sensor and actor in the human health, mainly
related with intestinal issues, but not only. Taking advantage
of this, its use as NGP is being explored for both human and
animal use. However, more research in its phylogeny, physiology,
safety, and beneficial effects should be performed to fill the lack
that currently exists between the knowledge of the biology of
the bacterium and the medical interest that it produces. As an
example, the analysis of the possible bacterial effectors taking
into account the phylogeny and the biological effects related
should be performed in more detail to find the best probiotic
candidate and refine its use as biomarker in several human
disorders. Extracellular Polymeric Matrix (EPM) Rossi et al. (2015) found that the biofilm-forming strain
F. praustnizii HTF-F was able to attenuate the clinical symptoms
of DSS-induced colitis in a stronger manner than A2-165
strain. Furthermore, the intra-rectal administration of purified
extracellular polymeric matrix (EPM) decrease the disease index
of the mice indicating that it contributes strongly to the protective
effects of HTF-F strain. However, although the authors concluded
that the anti-inflammatory effects of F. prausnitzii HTF-F strain
may be in part be due to the immune-regulating properties
of the EPM, they were not able to rule out other possible
strain differences such as colonization ability, stress resistance
or in vivo fitness, among others. In fact, these parameters
might also contribute to the efficacy of the strain as improved
survival might impact on butyrate or MAM production for
instance. ACKNOWLEDGMENTS RM receives a salary from Danone Nutricia Research in the
framework of a postdoc contract. AUTHOR CONTRIBUTIONS Recently, we have isolated a collection of novel F. prausnitzii
strains form healthy volunteers that we have characterized
(Miquel et al., 2015a) and analyzed for anti-inflammatory
properties with the aim of selecting new NGPs candidates
(Martín et al., 2017). The deeper phylogenic analysis of
the complete genome of this bacterial collection joint to
the genomes already present in the public data bases has
revealed
that
there
are
at
least
three
separate
clusters,
spanning the classical Phylogroups I and II already found
in F. praustnizii (Lopez-Siles et al., 2012, 2016; Martín
et al., 2017) and that some strains appear to represent
a deeper, more divergent branch of the “Faecalibacterium
prausnitzii” taxon (Benevides et al., 2017). This new truth
about F. prausnitzii provides evidence that the phylogeny of
F. praustnizii should be reconsidered. In line with these results,
in the future we should take into account the presence of
at least three different genospecies inside Faecalibacterium
group to better characterize their beneficial effects, their RM,
LB-H,
and
PL
designed
the
perspective
and
the
experiments. RM
wrote
the
manuscript
and
performed
the experiments. LB-H and PL corrected the manuscript. All
the
authors
approved
the
last
version
of
the
manuscript. RM,
LB-H,
and
PL
designed
the
perspective
and
the
experiments. RM
wrote
the
manuscript
and
performed
the experiments. LB-H and PL corrected the manuscript. All
the
authors
approved
the
last
version
of
the
manuscript. FUTURE PERSPECTIVES The lack of clearness about F. prausnitzii effectors is linked to the
lack of knowledge in its biology and phylogeny. Furthermore,
as we mentioned above, probiotic characteristics are strain-
specific, and therefore individual studies should be performed
in order to determine the individual beneficial effects as
well as the individual effectors linked to these effects. If we
compare to a classic probiotic group, lactobacilli, we can find
that although all lactobacilli are able to produce lactic acid
(proved to have beneficial effects in some ecosystems such
as the vaginal), not all of them are equipped with the same
arsenal of bacterial effectors (bacteriocins, biosulfactants, H2O2,
etc. . .) that are strain specific. In the case of Faecalibacterium,
the framework should be similar, as even if all the strains
are
able
to
produce
butyrate,
other
possible
molecules
could be responsible of additional strain-specific beneficial
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online
at:
https://www.frontiersin.org/articles/10.3389/fmicb. 2018.00346/full#supplementary-material March 2018 | Volume 9 | Article 346 Frontiers in Microbiology | www.frontiersin.org 6 Bacterial Effectors of Faecalibacterium prausnitzii Martín et al. REFERENCES doi: 10.1371/journal.pbi Miquel, S., Leclerc, M., Martin, R., Chain, F., Lenoir, M., Raguideau, S., et al. (2015b). Identification of metabolic signatures linked to anti-inflammatory
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commercial or financial relationships that could be construed as a potential conflict
of interest. Rajilic-Stojanovic, M., Biagi, E., Heilig, H. G., Kajander, K., Kekkonen, R. A.,
Tims, S., et al. (2011). Global and deep molecular analysis of microbiota
signatures in fecal samples from patients with irritable bowel syndrome. Gastroenterology 141, 1792–1801. doi: 10.1053/j.gastro.2011.07.043 Copyright © 2018 Martín, Bermúdez-Humarán and Langella. This is an open-access
article distributed under the terms of the Creative Commons Attribution License
(CC BY). The use, distribution or reproduction in other forums is permitted, provided
the original author(s) and the copyright owner are credited and that the original
publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these
terms. Copyright © 2018 Martín, Bermúdez-Humarán and Langella. This is an open-access
article distributed under the terms of the Creative Commons Attribution License
(CC BY). The use, distribution or reproduction in other forums is permitted, provided
the original author(s) and the copyright owner are credited and that the original
publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these
terms. Ramirez-Farias, C., Slezak, K., Fuller, Z., Duncan, A., Holtrop, G., and Louis, P. (2009). Effect of inulin on the human gut microbiota: stimulation of
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541–550. doi: 10.1017/S0007114508019880 March 2018 | Volume 9 | Article 346 Frontiers in Microbiology | www.frontiersin.org 8
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Hacettepe Üniversitesi Edebiyat Fakültesi Dergisi
Hacettepe University Journal of Faculty of Letters
Haziran/June 2022 – 39(1), 227-236
doi:10.32600/huefd.920078
Hakemli Makaleler – Refereed Articles
Geliş Tarihi / Received: 19.04.2021 Kabul Tarihi / Accepted: 20.11.2021
A Heterotopia Divided: Spaces of Labor in Louisa May Alcott’s Little Women
Bölünmüş bir Heterotopya: Louisa May Alcott’un Küçük Kadınlar Romanında Emek
Mekanları Hacettepe Üniversitesi Edebiyat Fakültesi Dergisi
Hacettepe University Journal of Faculty of Letters
Haziran/June 2022 – 39(1), 227-236
doi:10.32600/huefd.920078
Hakemli Makaleler – Refereed Articles
Geliş Tarihi / Received: 19.04.2021 Kabul Tarihi / Accepted: 20.11.2021
A Heterotopia Divided: Spaces of Labor in Louisa May Alcott’s Little Women
Bölünmüş bir Heterotopya: Louisa May Alcott’un Küçük Kadınlar Romanında Emek
Mekanları Hacettepe Üniversitesi Edebiyat Fakültesi Dergisi
Hacettepe University Journal of Faculty of Letters
Haziran/June 2022 – 39(1), 227-236
doi:10.32600/huefd.920078
Hakemli Makaleler – Refereed Articles
Geliş Tarihi / Received: 19.04.2021 Kabul Tarihi / Accepted: 20.11.2021 Hacettepe Üniversitesi Edebiyat Fakültesi Dergisi
Hacettepe University Journal of Faculty of Letters
Haziran/June 2022 – 39(1), 227-236
doi:10.32600/huefd.920078 Hakemli Makaleler – Refereed Articles
Geliş Tarihi / Received: 19.04.2021 Kabul Tarihi / Accepted: 20.11.2021 Hakemli Makaleler – Refereed Articles
Geliş Tarihi / Received: 19.04.2021 Kabul Tarihi / Accepted: 20.11.2021 A Heterotopia Divided: Spaces of Labor in Louisa May Alcott’s Little Women
Bölünmüş bir Heterotopya: Louisa May Alcott’un Küçük Kadınlar Romanında Emek
Mekanları Sinem YAZICIOĞLU* * Asst. Prof. Dr., İstanbul University, Faculty of Letters, Department of Western Languages and Literatures,
Department of American Culture and Literature, E-mail: sinemyaz@istanbul.edu.tr, ORCID: 0000-0003-4092-3152 Abstract In the second half of the nineteenth century, the development of industrialization and the surging waves of immigration
had drastic effects on the condition of the American working class. Coinciding with the Civil War and the following
Reconstruction era, this period saw a stronger and more determined labor movement in organizing trade unions and
resolving several work-related problems. Louisa May Alcott wrote Little Women (1868) in such a social and economic
climate. This essay will explore the novel’s spatial configuration of the existing labor conditions as two different
heterotopias by turning to how Louisa May Alcott organized characters and space in Little Women. Drawing from
Foucault and Harvey’s approaches to space, the essay will argue that the March family home functions as a labor
heterotopia, and Jo March founds a counter-heterotopia against it. In other words, while the March house depicts,
confronts and reverses the conditions of American labor in the Civil War era, Jo March attempts to follow the same
procedure so as to counteract the order in her family home. Jo’s counteraction is determined by her act of writing,
which gives her an individual and independent voice. Yet, more importantly, her authorship that lets her develop her
own working conditions has effects beyond the garret she uses for writing. The purpose of this essay is to re-read Jo
March’s character in terms of her function in both heterotopias, and to show that she constantly negotiates between
these domestic and intellectual labor heterotopias in an attempt to empower her sisters. Keywords: Louisa May Alcott, Little Women, heterotopia, labor, characterization. Öz Introduction In her introduction to the 2001 edition Broadview edition of Little Women, Anne Hiebert Alton
mentions Louisa May Alcott’s style and the significance of the novel, but imediately adds “[a]t the heart of
the story, however, is Jo. [...] Jo delights readers with her boundless energy, her independence, her
stubbornness, and her quirky individuality” (p. 10). Nicole Maruo-Schröder also acknowledges Jo as “the
novel’s most popular character” because Jo’s transformation from a strong-willed and boyish young woman
to the dutiful wife of an aged professor “led to a variety of different, sometimes contradictory readings” (p. 399). Furthermore, Elaine Showalter asserts that “Jo March has become the most influential figure of the
independent and creative American woman” and inspired female authors and intellectuals ever since the
novel was published (p. 42). As an aspiring female author, Jo has also come to voice the struggles of female
authorship. Accordingly, her persistence to write, mature her style and earn a living by publishing stories
have been associated with Alcott’s own experiences. Showalter, for example, writes that the author “takes
Jo through a literary progress that resembles her own pilgrimage” (p. 59); in addition, Christy Rishoi
maintains that the novel “is an idealized version of Louisa May Alcott’s own childhood that vividly
illustrates how girls are socialized to be women, particularly through Alcott’s fictional self, Jo March” (p. 66). The transformation of Jo’s literary style from sensational thrillers in Little Women (1868) to moral
juvenile fiction in Good Wives (1869), therefore, is interpreted by Sandra Gilbert and Susan Gubar as the
author’s self-denial in that “it is hypocritical of her to continue writing such tales herself” (p. 70),
considering that she published such works even after Jo’s aversion from them1. In brief, Alcott’s Little
Women has led feminist literary scholars to concentrate their efforts on studying Jo March as the central
character of the novel. Jo’s seeming centrality is, however, hampered by her liminal position in two workspaces, namely the
domestic space in which she unwillingly continues to do the housework, and her chamber in the garret
which she transforms into an office for writing. In this sense, although she has been considered central, that
does not qualify her as the novel’s hero. It is true that Jo is given a privileged position in terms of character-
space, as her name is mentioned more than any other character in the novel2. 1 The contemporary scholarship on Alcott’s works has led to a revaluation of her publications other than the Little
Women series. It is known that in between the publication of Little Women [1868] and Jo’s Boys [1886], Alcott
continued to publish sensational narratives such as A Modern Mephistopheles [1877] and A Whisper in the Dark [1877].
2 In the first book of Little Women, also known as Meg, Jo, Beth and Amy, Jo’s diminutive and proper names are used
613 times, whereas the same scores for Meg, Beth and Amy are 472, 244 and 229 respectively. Öz On dokuzuncu yüzyılın ikinci yarısında sanayileşmenin gelişimi ve yükselen göç dalgaları Amerikan işçi sınıfının
koşulları üzerinde büyük etkisi olmuştur. Amerikan İç Savaşı ve sonrasındaki yeniden yapılanma süreciyle kesişen bu
dönem, sendikalar örgütlemekte ve çalışma koşullarıyla ilgili birçok sorunu çözmekte daha güçlü ve kararlı bir emek
hareketine tanıklık etmiştir. Louisa May Alcott, Küçük Kadınlar (1868) romanını böyle bir toplumsal ve ekonomik
iklimde yazmıştır. Bu makale, Louisa May Alcott’un Küçük Kadınlar’daki karakterleri ve uzamı nasıl düzenlediğine
bakarak, romanın mevcut emek koşullarının iki farklı heterotopya biçimindeki uzamsal kurgulanışını inceleyecektir. Foucault ve Harvey’nin uzam yaklaşımlarından yararlanan bu makale, March ailesinin evinin bir emek heterotopyası
işlevi gördüğünü, Jo March’ın ise ona karşı kendi karşı-heterotopyasını kurduğunu ileri sürmektedir. Başka bir deyişle,
March ailesinin evi Amerikan İç Savaşı döneminin emek koşullarını ele alır, eleştirir ve tersine çevirirken, Jo March
kendi aile evinin düzenine karşı çıkmak için aynı işlemleri izler. Jo’nun karşı hamlesi, ona özgün ve bağımsız bir ses
kazandıran yazma edimiyle belirlenmiştir. Ancak, daha da önemlisi, kendi çalışma koşullarını geliştirmesini sağlayan
yazarlığının, yazmak için kullandığı tavanarasının ötesinde etkilerinin olmasıdır. Bu makalenin amacı, Jo March’ın 227 Sinem YAZICIOĞLU karakterini her iki heterotopyadaki işlevine göre yeniden okumak ve onun, kız kardeşlerini güçlendirme çabasıyla,
sürekli olarak ev emeği ve düşünsel emek heterotopyaları arasında arabuluculuk yaptığını göstermektir. Anahtar sözcükler: Louisa May Alcott, Küçük Kadınlar, heterotopya, emek, karakter. Introduction Nevertheless, while Jo is
attributed with agency, she is not the subject whose quest for her desire constitutes the plot. Even though
the novel starts with her complaint about the family’s recent economic difficulties arising from the father’s
absence and wartime conditions, she raises the collective voice of the women in the family and emphasizes
the father’s absence as the cause of the family’s collective suffering. At best, in her struggle to develop an
independent voice, she might be the subject of her own subplot never realized and gradually hampered in
the sequels. Conversely, her prominence in the novel comes from her liminality, which gives her a power
emanating from her labor and a negotiating function for easing and regulating the female characters’
survival in what is presented as a decentered family. It is not surprising that Jo’s negotiating function coincides with her father’s absence. His absence
transforms the house into a space in which all social codes are revised. The family’s economic loss created
by this absence is emphasized in Little Women’s first sentence, with Jo’s complaint that “Christmas won’t
be Christmas without any presents” (Alcott, 2008, p. 11), implying the disparity between the family’s
previous and recent economic conditions. Such economic difficulties introduce the concept of work into the
March household, and several labor-related issues of the Civil War era thus enter the novel, albeit in 228 A Heterotopia Divided: Spaces of Labour in Louisa May Alcott’s ‘Little Women’ different forms. In other words, Alcott uses the absence of the father as a juncture for modelling what might
be called a labor heterotopia for middle-class women. In his essay titled “Of Other Spaces”, Michel Foucault
defines the term heterotopia as “counter-sites, a kind of effectively enacted utopia in which the real sites
that can be found within the culture, are simultaneously represented, contested, and inverted” (p. 24). The
March house similarly represents, contests and inverts the labor conditions in the Civil War era through the
novel’s treatment of child labor, apprentice training, closed shops, immigrant laborers (specifically Irish
immigrant workers) and the regulation of work hours. Jo’s negotiating function emerges in the domestic
space as she bargains with the labor heterotopia of the March house to establish and maintain her counter-
heterotopia of intellectual labor. 3 The confusion in the title of Little Women is related to its publication history. Upon the popular success Little Women
received after its publication in the United States, Alcott rapidly completed its sequel as the second volume. When the
novel was published in the United Kingdom in 1880, the two books were printed as a single novel titled Little Women.
In this edition, the novel and its sequel are organized as two parts, titled “Meg, Jo, Beth and Amy” and “Good Wives”
respectively. In this study, I use the title Little Women for the first book in order to follow the original publication of
this volume. Introduction Jo’s authorship requires a space outside the labor heterotopia of the March
house, but simultaneously creates another work environment where she pursues her work in discipline. Jo’s
counter-heterotopia is, therefore, far from offering her absolute liberation from work, since it introduces a
different set of codes and conventions. Her intellectual labor not only gives her empowerment, but also lets
her organize creative practices with her sisters, offers her payment, makes her realize work conditions
outside the March house, and consequently opens the family home to the outside. In this paper, I will examine the heterotopic configurations of labor in the mid-nineteenth-century
United States by turning to how Louisa May Alcott organized characterization and space in Little Women. To this end, I will focus on the first book in Alcott’s series3, since this is the book that initiates and
encapsulates Jo March’s negotiating function. In the following study, I will show that Jo’s characterization
as a negotiator originates in her being a laborer whose ability to work in two sectors gives her a liminal
position. For this, I will explain the labor heterotopia of the March house and Jo’s counter-heterotopia of
intellectual labor. The purpose of this essay is to re-read Jo March’s character in terms of her function in
both heterotopias so as to show that Jo constantly negotiates between domestic and intellectual labor
heterotopias, which results in the potential empowerment of all the young female characters in the novel. The Other Space of Labor The all-female house in Little Women encapsulates the private and domestic space of the Civil War
era, during which the male members of American families were conscripted for military service; yet
simultaneously (and ironically) it is where national, republican and abolitionist ideals are embodied and
realized, despite the fact that the March family home is safe from the war zone and outside the real-political
realm. This convergence of the private and the public is due to the novel’s ironic configuration of the March
house. Far from being a cozy and comfortable nest, Alcott depicts the March house as a workspace, where
all the female members of the family do the housework. Aşkın Çelikkol indicates that the republican ideals
of the American Revolution transformed society not through a novel distribution of wealth and educational
resources, at least initially, but through a revaluation of labor, which aristocracy had long depreciated. Çelikkol writes “[e]ducation, intelligence, decorum and wealth all came to be refigured around this single
principle and benevolent act of laboring. With laboring came equality, and the sovereignty of the public”
(2019, p. 43). The house as a workplace symbolically keeps the house undivided by following Abraham
Lincoln’s metaphor in his famous “A House Divided” speech of 1858 and maintains the one out of many
by gathering the family members for a common cause in accordance with the American motto “E Pluribus
Unum”. Although the March sisters belong to the same family, a binding common cause is deemed
necessary, since the family members are initially presented with a lack of unity in their physical descriptions,
manners and interests. That Meg is “very pretty”, Jo has a “comical nose”, Beth has a “shy manner, a timid
voice” and Amy has “blue eyes and yellow hair” (Alcott, 2008, p. 14) indicate their noticeable differences. 229 Sinem YAZICIOĞLU Furthermore, Meg’s preoccupation with her physical appearance, Jo’s authorship, Beth’s interest in playing
the piano and Amy’s fascination with valuable objects suggest that their interests vary. Housework done
together thus symbolically resolves the problem of potential disintegration in the March family. Furthermore, Meg’s preoccupation with her physical appearance, Jo’s authorship, Beth’s interest in playing
the piano and Amy’s fascination with valuable objects suggest that their interests vary. Housework done
together thus symbolically resolves the problem of potential disintegration in the March family. The Other Space of Labor g
y
y
p
p
g
y
It is not a coincidence that Little Women’s publication corresponds to the rise of industrial labor in
the United States. By the mid-nineteenth century, the United States had already left the Jeffersonian ideal
of agricultural society and begun its transformation towards industrialization. Several technological
innovations had paved the path for the surmounting industrialization that would later define the Gilded Age,
such as Samuel Cunard’s first transatlantic steamship line in 1846 and Samuel Morse’s first electronic
telegraph in 1844. Yet the gradual development of the transcontinental railroad, which took decades to
finalize, marked the potential ramifications of industrialization in the United States. Aside from its
contribution to the Union’s victory in the Civil War by establishing the connection between the Northeast
and the Northwest, the railroad was essential to American industrialization in that it required heavy industry
for the production of railway materials and the manpower for construction. The railroad’s greatest impact
was, however, its transformative power. Hugh Brogan writes that the long period of building the railway
“created and sustained hundreds and thousands of new jobs; new coal and iron mines; new coking plants
[...] new iron and steelworks; new towns, which were also new markets; new skills; and new forms of
financial and industrial organization,” (p. 380) and concludes that it led to the urbanization of the United
States. From this perspective, he considers the railway the American version of the Industrial Revolution. Newly organized agriculture, on the other hand, increased productivity yet significantly dropped the demand
for agricultural work force (Trachtenberg, 2007, p. 53). Alcott’s Little Women was, therefore, composed not
only in the context of the American Civil War, but also in a climate of gradually rising industrialization. The rise of industrialization in the United States led to the revitalization of trade unionism. For
example, Philip Foner documents American laborers’ brewing discontent with low wages, high prices,
dismal working conditions, the introduction of machinery (which narrowed down the number of jobs), and
the increasing number of apprentices as replacement for skilled mechanics (pp. 339-40). Against these
conditions, a gradually increasing number of laborers started to organize in trade unions with the cooperation
of female workers from several trades. The Other Space of Labor The issue is known to 230 A Heterotopia Divided: Spaces of Labour in Louisa May Alcott’s ‘Little Women’ be a severe problem during the rise of industrial labor in Europe and the United States. Friedrich Engels, in
The Condition of the Working Class in England, documents the surging demand for female and child
workers for more intricate jobs in the textile industry, as the muscular power of male workers were
transferred to heavy industries (p. 164-5). Furthermore, describing the condition of the American working
class, Stephan Thernstrom writes “a great wave of working-class children entered the labour market during
the Civil War decade” (p. 108). In line with the reformists of the following decades, known as “child savers”,
the labor heterotopia in Little Women keeps the working children at home under a caretaker’s supervision
and protection. As an alternative model of child labor, this heterotopia makes the March girls engage in
domestic work in limited hours and provides them with enough nourishment. In one instance, the girls set
to work after dinner and from the words of the narrator, “the needles flew as the girls made sheets for Aunt
March. It was uninterested sewing, but to-night no one grumbled” (Alcott, 2008, p. 21). This work stops at
nine o’clock in the evening and the girls start singing by the piano before they go to bed (p. 21). In terms of
child labor, regulated work hours, occasional free time and adequate nourishment are not only improvements
for the mid-nineteenth century, but they also imply a model closer to an apprenticeship program which
prepares the children for their future individual households. p p
Considered an alternative to the limitations of a strictly regulated social order, the term heterotopia
has been positively valued by a number of scholars. For example, in his own formulation of Foucault’s
heterotopia with the term thirdspace, Edward Soja states that “[e]verything comes together in thirdspace:
subjectivity and objectivity, the abstract and the concrete, the real and the imagined, the knowable and the
unimaginable ... the disciplined and the transdisciplinary” (pp. 56-7). Because of its definition of thirdspace
as a potentially creative and imaginative medium, Soja’s conceptual framework attaches positive value to
heterotopia. David Harvey, in particular, criticizes such an approach and turns to Kevin Hetherington, who
defines heterotopia as “spaces of alternate social ordering” (p. The Other Space of Labor viii), and maintains that it regulates a certain
aspect of the social world differently. In Hetherington’s reformulation of heterotopia, Harvey finds a
pertinent remark for order: Heterotopia is not a space of boundless liberation from social order; in contrast,
it is an establishment of an order distinct from the existing one. For this reason, Harvey reminds that
Disneyland and shopping malls are also heterotopias, and maintains that “what at first sight as so open by
virtue of its multiplicity suddenly appears either as banal [...] or as a more sinister fragmentation of spaces
that are closed, exclusionary, and even threatening” (p. 185). Such negative qualities of heterotopia are not
disregarded by Foucault himself. In his essay that treats the concept in full, Foucault lists psychiatric
hospitals and prisons as heterotopias of deviation “in which individuals whose behavior is deviant in relation
to the required mean or norm are placed” (1986, p. 25). Considering that Foucault delivered the speech on
which “Of Other Spaces” is based eight years before the publication of his Discipline and Punish, Harvey
concludes that he abandoned the concept due to its failure to constitute a viable alternative. To be precise,
the closest Foucault gets to heterotopia in his later work is the Mettray penal colony for young offenders. A
concentration of “all the coercive technologies of behavior” (Foucault, 1995, p. 293), this prison model is
shown to embody the function of a family, an army, a workshop, a school and a court. As a heterotopia, the
March house functions similarly: It exerts limits to the girls’ potentials and reforms them as prospective
housewives in a closed workspace of alternative coercion. In the March house, therefore, the sewing and
singing skills are not equally attended. While the girls sew in discipline, they can sing out of tune and
without proper articulation, since only Beth can “get much music out of the old piano” while Amy “chirp[s]
like a cricket” and Jo always comes “out at the wrong place with a crook or a quaver” (Alcott, 2008, p. 21). Deliberately reluctant to invest on the girls’ artistic skills, the March heterotopia is established as an “other
space” to reproduce the labor power of the future housewife. A particular site of struggle for trade unions of the Civil War era was the arrangement of closed shops
where the employer could only agree to recruit union members. The Other Space of Labor With the formation of Worker Women’s Protective Union in 1863,
female workers had the opportunity to learn different skills needed in other trades; they also received further
training for their present skills to increase their wages and helped recruit male and female laborers to several
trade unions (Foner, 1972, pp. 341-2). This combined force started to organize strikes and boycotts, initiated
the Eight Hour Movement to limit work hours, organized the labor press, and sought to establish national
federations for scattered trade unions, although the unionist laborers were demonized on the grounds that
they were immigrants. All these labor-related issues of the era are reverberated in Alcott’s configuration of
the March house. Little Women is not Alcott’s only work that depicts female labor. Before and after Little Women,
Alcott published Hospital Sketches (1863) and Work: A Story of Experience (1875), both of which depict
women who decide to work. The female characters in both novels attach value to labor even at the stage of
taking the decision to work. In the semi-autobiographical Hospital Sketches, for example, Tribulation
Periwinkle’s excitement for a suggestion to “nurse the soldiers” is encapsulated in the exclamation mark as
she says, “I will!” (Alcott, 1993, p. 3). Christie in Work announces her decision to her aunt with great joy
and calls it “a new Declaration of Independence” (Alcott, 1875, p. 1). Yet in these two examples, the female
characters leave their homes for the workplace, and they seek professionalism so that they can be employed
in a regulated work environment where they develop an expertise. While Tribulation does charitable work
and is not paid, she performs the profession of nursing, and Christie enters different work environments
where she is paid for domestic work. In Little Women, however, domestic work is unpaid and calls for
affective labor as well. It is in this very sense that, unlike the settings in Alcott’s other labor-related works,
the March house is a labor heterotopia, because the actual work conditions of the Civil War era (and of the
Reconstruction era in which Alcott published the novel) are inverted and revised in order to form an
alternative space which is not originally a workspace. The March house is obviously all-female, but aside from the mother, the maid and the eldest sister
Meg, it is also all-children. In other words, the house heavily invests on child labor. The Other Space of Labor By symbolically employing its family
members, the March house functions as a closed shop, and protects its members’ rights. In this sense, the
familial model that had been replaced by the workers’ solidarity in the workplace returns to the family
institution in order to reorganize the domestic labor in Little Women. Another important union activity was
striking to improve work conditions. In the chapter titled “Experiments”, the March girls decide to stop
working for a period and spend their entire time with leisure activities. This however proves unfulfilling, 231 Sinem YAZICIOĞLU because the sisters get bored quickly, but what is more, when the mother decides to “finish off the trial in
an appropriate manner” (p. 125), she gives the maid and herself a day off to show them the effects of their
strike on the domestic order. On the last day of the strike, during which they can neither manage without an
employer nor do all the housework, the girls decide to end it. Their determination turns into a discourse of
negotiation, illustrated by Jo’s comment that “it is better to have a few duties, and live a little for others” (p. 131). In this example, Alcott prioritizes the continuity of work, and because of this, considers strike a
destructive and inconsiderate activity which can only be done by young, immature girls. Conversely,
Alcott’s desired labor struggle for girls is negotiation: She lets the girls negotiate with the mother for better
conditions. Depicted as their symbolic employer and introduced as the “most splendid woman in the world”
(p. 17), the mother tells them not to “go to the other extreme, and delve like slaves. Have regular hours for
work and play; make worth of time by employing it well” (p. 133), hence grants them limited rights. The Irish maid employed in the March house responds to the problem of immigrant workers. Describing workers’ conditions in Britain, Engels repeatedly draws attention to Irish immigrants, and
observes that the Irish are the most destitute and dispossessed of all immigrant worker communities, since
they had no specific expertise for better jobs and could only find the poorest means of accommodation in
which they teemed with several other tenants and even their animals (p. 124-5). In the context of the United
States, the black worker in the Northern states “suffered the same fate Irish workers suffered in Britain”
(Çelikkol, 2015, p. The Other Space of Labor 5), and Alcott herself has been criticized for not having even a single black character in
Little Women, which is considered to signify “the white privilege embedded in little womenhood and the
attendant foreclosure of possibilities and fantasies for a heroine who is black” (Alberghene, 1999, p. 354). Nevertheless, reconsidering Little Women’s character distribution from a standpoint that emphasizes the
color line does not necessarily elevate or belittle the condition of the Irish working class of the era. In the
first half of the nineteenth century, the United States witnessed waves of immigrants from Ireland, who
started establishing their Catholic churches and constituted the largest portion of the urban ghetto
(McCaffrey, 1992, p. 164). Female Irish immigrants in particular managed to form solidarity networks by
joining Irish organizations and funding their sisters’ journey to the United States as new immigrants (May,
2011, p. 58). The Irish maid Hannah, in this regard, symbolically resolves the problems of the Irish
immigrant workers in that the March house offers her humane conditions, makes her part of the domestic
space and yet acknowledges her only as an individual, hence severs her social relations with the Irish
immigrant community. Jo’s Counter-heterotopia At her aunt’s, Amy is
made to wash the cups every morning, and polish up the old-fashioned spoons, the fat silver teapot,
and the glasses, till they shone. Then she must dust the room, and what a trying job that was! Not a speck escaped Aunt March’s eye, and all the furniture had claw legs, and much carving,
which was never dusted to suit. Then Polly must be fed, the lap-dog combed, and a dozen trips
upstairs and down, to get things or deliver orders, for the old lady was very lame, and seldom
left her big chair. After these tiresome labors she must do her lessons, which was a daily trial
of every virtue she possessed. (pp. 210-1). Amy’s work follows as she reads aloud to Aunt March and does the sewing and stitching, while the
aunt continues training her during Amy’s free time in the evenings by recounting stories to her from the
past. Amy is not the only employee at Aunt March’s; the French cook Esther is also employed there, and
through her it is revealed that the aunt recruited her on condition that she change her original French name
Estelle if she insists on maintaining her Catholic faith. Although Amy has familial relation to Aunt March,
she and Esther easily connect as two laborers working under the same strict employer. Esther’s
permissiveness lets Amy “roam about the great house, and examine the curious and pretty things stored
away in the big wardrobes and the ancient chests” (p. 212) and observes the extent of Aunt March’s
accumulated wealth. When Amy is attracted to one of the aunt’s old rings, however, she says “I’ll be a lamb,
if I can only have that lovely ring [...] I do like Aunt March, after all” (p. 213), and thus learns to comply
with her aunt in order to inherit part of her fortune. In contrast to Amy’s compliance, Jo’s actions at Aunt March’s have the characteristics of resistance. When she is sent there, she does not work as hard as Amy does, and spares some of her time in the large
library. Although her reading is constantly distracted by the aunt’s calls, her reluctance to be available for
work at all times is also acknowledged by the aunt. In addition, Aunt March does not attempt to change her
looks and behavior. Jo’s Counter-heterotopia Jo destabilizes the labor heterotopia of the March house, because she considers housework worthless. Her complaint about housework is not solely determined by her consequently lessened time for reading and
writing at home. The narrator explains that Mr. March’s loss of property for the sake of supporting a friend’s
finances led the two eldest March sisters to work outside the house. For this reason, Meg starts working for
the rich King family as a governess. However, she does not share Jo’s discomfort with housework, since
her work corresponds with her subsequent marriage plot with Mr. Brooke. Furthermore, her work lets her
have a contact with the social elite; she enjoys the rich family’s luxurious lifestyle (Alcott, 2008, p. 47),
while at times she resents the economic divide between them and her family. The other sister who is sent to
work is Jo, but she is employed by a member of the extended family, namely Aunt March. Considering her
old age and solitary life as well as her younger brother’s relatively poor status, Aunt March initially wants
to adopt Jo. While the family rejects her demand, they eventually have no option but to send Jo to the aunt
for work. Jo’s employment record is thus marked by the threat of losing her family. Aunt March is also
known to have a disagreeable personality. The “occasional tempest” (p. 47) in her house, her insistent
knowingness that demands the March family “take her advice next time” (p. 79) and her interventions in
the family members’ decisions, such as her attempts to change Meg’s mind from marrying Mr. Brooke and
orient her towards making “a rich match” (p. 253) result in her description as “the peppery old lady” (p. 48)
and “cross old soul” (p. 61). When the family decides to send Amy to Aunt March for protecting her from
Beth’s illness, Amy likewise protests by saying “it’s dull at Aunt March’s, and she is so cross” (p. 197). 232 A Heterotopia Divided: Spaces of Labour in Louisa May Alcott’s ‘Little Women’ Amy’s stepping into the aunt’s house reveals Jo’s working conditions and the aunt’s character as an
employer in detail. Since the aunt gathers that Amy is “more docile and amiable than her sister” (p. 210),
she starts training her to reverse the liberties she has enjoyed at the March house. Jo’s Counter-heterotopia Jo’s resistance, therefore, aims to improve the working conditions in the aunt’s house,
and the narrative voice implies that the aunt is harsher to Amy because she does not follow her sister’s steps. Considering that the March girls were able to start a strike, Jo’s actions at Aunt March’s cannot be deemed
rebellious, but she functions as a negotiator in organizing better conditions for the workers the aunt would
recruit in the future. Jo’s negotiating function comes with a repressed discourse that enables her to occupy and resist the
two domestic workplaces at the same time. Such a discourse becomes even more necessary since Jo’s pursuit
of literary writing demands yet another workplace and schedule that contradict domestic labor. In a
particular example, Jo writes a book of fairy tales, but her completed work is burnt up by Amy. Jo feels a
rage against Amy to the point of not saving her when she falls through thin ice while skating on a frozen
pond. Yet Alcott does not let Jo embody class warfare and eventually makes her resent her rage. On anger
in Little Women, Judith Fetterley asserts that “in the world of ‘little women’ female anger is so unacceptable
that there are no degrees to it; all anger leads to ‘murder’” (p. 380). While Fetterley relates Jo’s rage to
female anger, this rage originates not in female sibling rivalry, but in Amy’s depreciation of Jo’s work. To
continue writing and remain part of the March family, Jo not only learns to repress her anger, but also
manages to transform it into an act of imagining beyond the existing heterotopic configurations of labor. Jo’s authorship is one of the key issues feminist literary scholars have focused on. Her passion for
writing has a distinguishing mark on her actions since writing grants her a strong will and an independent
voice. Yet more importantly, her authorship is depicted not simply as a passion but as labor. After Amy
burns up her book it is revealed that Jo’s book was short, but she “had worked over them patiently, putting
her whole heart into her work, hoping to make something good enough to print. She had just copied them
with great care, and had destroyed the old manuscript, so that Amy’s bonfire had consumed the loving work
of several years” (Alcott, 2008, p. 87). Jo’s Counter-heterotopia Jo’s meticulousness, her editing the manuscript and the repeated use 233 Sinem YAZICIOĞLU of the word “work” for her action and its material result show that her writing is characterized as labor. Similarly, for her final preparations before submitting her story to the Spread Eagle journal, she is described
as “very busy up in the garret”, seating herself “on the old sofa writing busily, with her papers spread out
upon a trunk before her” while she is “[q]uite absorbed in her work” and when she finishes writing, she
starts reading “the manuscript carefully through, making dashes here and there, and putting in many
exclamation points” (p. 165). Although the labor heterotopia of the March house forces her to write as free
time activity, Jo rationalizes her practice by occupying a specific space, converting old house furniture to
office furniture, and following a systematic procedure in which she first writes, then organizes her
manuscript and finally edits it. That she writes in the garret also resonates with Gilbert and Gubar’s analysis
of the madwoman in the attic in that Jo’s authorship poses a threat to the established order in the March
house and is considered a deviance from it. As a result, the space she occupies is the inversion of what the
house represents. Furthermore, writing gives her the opportunity for unalienated labor, since the papers, the
work and the desk are all used with personal pronoun. By writing, she owns her labor, unlike the tasks in
Aunt March’s house or in her family home. Besides, in submitting her manuscript to the journal, she means
to sell her labor herself and establish non-familial relations. Jo consequently manages to form a counter-
heterotopia in the garret where she writes. Jo’s counter-heterotopia of labor is equally regulated as the March house, since her professional
attitude gives an order to her social actions as well. Her reading, writing and editing transgress the limits of
her authorship and come to define her character and actions. To illustrate, Jo corrects Amy when the younger
girl mistakes the word “libel for “label” (p. 13) and “vampire” for “samphire” (p. 122). Jo’s alertness for
correct English use might be read through her assuming the father’s role in his absence, hence maintaining
the law of the father. However, with her counter-heterotopia, Jo does not merely attune her language and
discourse to the symbolic order, but also uses language creatively. Jo’s Counter-heterotopia Furthermore, Jo’s interference to her
sister’s use of language extends beyond Amy’s individual errors and transforms the sisters into temporary
literate communities. For example, at the end of the first chapter, the sisters sew a quilt for Aunt March by
taking guidance from Jo, who suggests “dividing the long seams into long parts, and calling the quarters
Europe, Asia, Africa and America” (p. 21) and conversing about the countries in these continents as they
are stitching the parts. Here, Jo’s counter-heterotopia intervenes to the labor heterotopia of the March house. Furthermore, in this example, her creative plan of sewing the quilt includes all the sisters indiscriminately,
assigning each of them a continent in the form of division of labor, so that only their collective action can
complete the work. Yet, even more importantly, by dividing and naming the parts after continents, Jo shows
her sisters a model through which they can create an alternative world themselves. When Jo manages to
publish one of her stories, the sisters similarly form an audience as she reads it aloud. After listening to the
story attentively, Amy criticizes “the artistic parts of the story, and [offers] hints for a sequel”, Beth gets
“excited” and Meg says she liked “the lovering part” (p. 173). The girls are, therefore, tentatively
encouraged to imagine a world beyond the March house. In both cases, it is Jo who introduces alternative
ways of labor to her sisters. In doing so, Jo modifies the inversion of the very heterotopia that keeps the
working children at home, feeds and trains them, but leaves them without proper education that would limit
their socialization to their prospective households. Jo’s linguistic intervention is thus compensatory to the
sisters’ lack of education, and functions for equipping them with the necessary tools to use language properly
without having to comply with the ruling voice. While Jo turns her sisters into an imaginative community within the house, hence inverts the
principles of the labor heterotopia, she also supports them in their socialization outside. Although the sisters
are invited to social gatherings on several occasions, the novel prioritizes the girls’ relation with Laurie, the
grandson of their neighbor Mr. Laurence. The first mention to Mr. Jo’s Counter-heterotopia The egalitarian relation of both sexes thus
illustrates an alternative to the workspace of the March house. Jo’s sexual ambivalence is only a limited illustration of her general attitude: Since Jo is the negotiator,
she is equipped with a bridging function despite her determinate and passionate character. That she is both
inside and outside of the March labor heterotopia and that she establishes her counter-heterotopia in the
garret indicate her liminal position. In the novel, Jo’s passion for inverting the labor heterotopia is spatially
illustrated with her use of the garden as a liminal space. Although the March sisters consider Mr. Laurence’s
house a fantasy space for their individual creative passions, they can visit there only temporarily. However,
the garden that separates (and unites) both houses enables them to work simultaneously as they imagine
their fantasies. When, for example, Laurie spends idle time in his hammock and is curious about what the
girls are doing in the garden, he eventually learns that they are sewing and stitching. In the novel, it is not
clear with whose decision the girls moved to the garden for work. When asked, Beth tells Laurie “we have
tried not to waste our holiday, but each has had a task, and worked at it with a will” (Alcott, 2008, p. 157). She continues, saying “Mother likes to have us out of doors as much as possible; so we bring our work here,
and have nice times [...] we can look far away and see the country where we hope to live some time” (p. 158). Here, Beth explains their working in the garden as their and the mother’s idea at the same time, but
her further description of the country implies that the girls make Jo’s fantasy theirs: “Jo talks about the
country where we hope to live some time; the real country, she means, with pigs and chickens, and
haymaking. It would be nice, but I wish the beautiful country up there was real, and we could ever go to it”
(p. 158). For Jo, the garden is another location for her counter-heterotopia, supporting her imaginative
faculties. Furthermore, she encourages the others to imagine other worlds while they are working, since she
asks, “Wouldn’t it be fun if all the castles in the air which we make could come true, and we could live in
them?” (p. 159). Jo’s Counter-heterotopia Laurence is when he sends the family a
full-fledged supper for Christmas upon hearing that they shared their breakfast with a poor immigrant
family; however, it is Jo who coincidentally meets and befriends Laurie. After their recently established
friendship, the girls’ prejudgment on Mr. Laurence that he does not “like to mix with his neighbors” (p. 32)
and their own lack of socialization with the other sex significantly change. This friendship specifically
enables Jo and Laurie to enter their respective households; in other words, Jo’s intervention folds the outside
into the inside simultaneously as the inside is opened to the outside. This double exchange lets the March 234 A Heterotopia Divided: Spaces of Labour in Louisa May Alcott’s ‘Little Women’ girls into Mr. Laurence’s house, where Meg can visit the conservatory and enjoy the flowers, Jo can borrow
books from the library, and Amy can observe paintings closely. Yet Beth is the sister who most benefits
from her visits, since she can practice and improve her music skills with a properly functioning piano. Mr. Laurence’s house thus physically illustrates the extensions of Jo’s counter-heterotopia. p y
y
p
In Little Women, the communication between Jo and Laurie is made possible by their configuration
as sexually ambivalent characters. Symbolically equalizing both sexes, the characterization of the tomboy
Jo and the effeminate Laurie serves as an opportunity for the sisters’ socialization, since they both include
Laurie to their activities and join him in his activities with his friends. The promise of equality in this relation
resonates with the demands of the women’s labor movement, especially on the subjects of women’s
suffrage, equal pay for equal work and equal opportunities for education. Alcott herself is known for her
advocation of these rights in her letters and newspaper articles. In her letter to Maria S. Porter from 1874,
for example, she sounds as if she is addressing a larger audience, writing, “let us hear no more of ‘women’s
sphere’ either from our wise (?) legislators [...] or from our clergymen [...] Let woman find out her own
limitations [...] Let the professions be open to her” (Porter, 1893, p. 22). In other words, Alcott acknowledges
that the March house fails to resolve the problem of inequality in women’s labor in the nineteenth century
by forming an all-female domestic space of unpaid labor. Jo’s Counter-heterotopia Upon her remark, she forms yet another imaginative community when Laurie responds,
“I’d like to settle in Germany, and have just as much music as I choose”, Meg adds “I should like a lovely
house”, Beth wants to “help take care of the family”, and Amy expresses her wish to “go to Rome, and do
fine pictures, and be the best artist in the whole world” (p. 160). Tentatively moving from the labor
heterotopia and stepping into their own counter-heterotopias, the children make use of the garden’s
liminality for their fantasies. Conclusion In addition,
Meg’s romantic attachment to Mr. Brooke signals their marriage in the second sequel; Amy indeed becomes
a painter, but she leaves for Italy to follow her pursuit, suggesting the impossibility of being an artist in the
March house. To put it in another way, Jo’s counter-heterotopia fails to persist when its rival heterotopia of
the March house also ceases to exist as an inversion of the labor conditions outside and dissolves with the
father’s arrival and the sisters’ departure. More precisely, both heterotopias ultimately disseminate into the
March girls’ future lives outside the house in alternative forms. Through the clash of both heterotopias of
work, Little Women spatially characterizes and symbolically resolves the conditions of American labor in
the second half of the nineteenth century, and critically reconsiders its own resolutions. Conclusion Despite Jo’s efforts to resist the existing labor heterotopia of the March house, her counter-heterotopia
does not survive. Alcott disrupts the prospects of Jo’s counter-heterotopia in the novel’s sequels. For Jo,
this failure amounts to her new disinterest in romance and her new passion for moral tales for children, her
marriage to Professor Bhaer and the subsequent change in her so-called unladylike attitudes. However, there
is an implication in Little Women that Jo will be made to abandon everything that characterizes her, because 235 Sinem YAZICIOĞLU she is left without a strong ally in her counter-heterotopia. In creating a room of her own, Jo does not have
any prior role models, but she attempts to be one for her sisters. However, Beth, who benefits most from
Jo’s interventions and develops a serious interest in piano playing, forgets her passion abruptly; her
imagination suddenly narrows down to the extent of not being able to imagine a life beyond the March
family, and she becomes severely ill. The second novel, Good Wives, depicts her untimely death. In addition,
Meg’s romantic attachment to Mr. Brooke signals their marriage in the second sequel; Amy indeed becomes
a painter, but she leaves for Italy to follow her pursuit, suggesting the impossibility of being an artist in the
March house. To put it in another way, Jo’s counter-heterotopia fails to persist when its rival heterotopia of
the March house also ceases to exist as an inversion of the labor conditions outside and dissolves with the
father’s arrival and the sisters’ departure. More precisely, both heterotopias ultimately disseminate into the
March girls’ future lives outside the house in alternative forms. Through the clash of both heterotopias of
work, Little Women spatially characterizes and symbolically resolves the conditions of American labor in
the second half of the nineteenth century, and critically reconsiders its own resolutions. she is left without a strong ally in her counter-heterotopia. In creating a room of her own, Jo does not have
any prior role models, but she attempts to be one for her sisters. However, Beth, who benefits most from
Jo’s interventions and develops a serious interest in piano playing, forgets her passion abruptly; her
imagination suddenly narrows down to the extent of not being able to imagine a life beyond the March
family, and she becomes severely ill. The second novel, Good Wives, depicts her untimely death. References Alberghene, J. (1999). Autobiography and the boundries of interpretation. In J. Alberghene and B. Lyon Clark (Eds.), Little Women
and feminist imagination: criticism, controversy, personal essays (pp. 347-376). London: Routledge. Alberghene, J. (1999). Autobiography and the boundries of interpretation. In J. Alberghene and B. Lyon Clark (Eds.), Little Women
and feminist imagination: criticism, controversy, personal essays (pp. 347-376). London: Routledge. Alcott, L. M. (1875). Work: a story of experience. Boston: Roberts Brothers. Alcott, L. M. (1875). Work: a story of experience. Boston: Roberts Brothers. Alcott, L. M. (1993). Hospital sketches. Bedford: Applewood. Alcott, L. M. (2008). Little women. London: Vintage. Alcott, L. M. (2008). Little women. London: Vintage. Brogan, H. (2001). The Penguin history of the USA. London: Penguin. Çelikkol, A. (2015). The representation of family in African American literature: A psychoanalytic approach. PhD Dissertation:
Istanbul University. Retrieved from: http://tez.yok.gov.tr/UlusalTezMerkezi (Thesis number: 417827). Çelikkol, A. (2019). Re-publicizing the nation: Slavery and the American Revolution. Litera: Dil, Edebiyat ve Kültür Araştırmaları
Dergisi, 29(1), 41-58. els, F. (1987). The condition of the working class. London: Penguin. Engels, F. (1987). The condition of the working class. London: Penguin. Fetterley, J. (1979). “Little Women”: Alcott’s Civil War. Feminist Studies, 5(2), 369-383. Foner, P. (1972). History of labor movement in the United States. Volume I: From colonial times to the founding of the American
federation of labor. New York: International Publishers. Foucault, M. (1986). Of other spaces. Diacritics, 16 (1), 22-27. Foucault, M. (1986). Of other spaces. Diacritics, 16 (1), 22-27. Foucault, M. (1995). Discipline and punish. New York: Vintage. Foucault, M. (1995). Discipline and punish. New York: Vintage. Gilbert, S. and Gubar, S. (2000). The madwoman in the attic: The Woman writer and the nineteenth-century literary imagination. New Haven and London: Yale Nota Bene. Harvey, D. (2000). Spaces of hope. Edinburgh: Edinburgh University Press. Harvey, D. (2000). Spaces of hope. Edinburgh: Edinburgh University Press. 003). The badlands of modernity: Heterotopia and social ordering. London: Routledge. Hetherington, K. (2003). The badlands of modernity: Heterotopia and social ordering. London: Routledge. Hiebert Alton, A. (2001). Introduction. In A. Hiebert Alton (Ed.), Little Women (pp. 9-27). Peterborough: Broadview Literary Press. Maruo-Schröder, N. (2018). Louisa May Alcott, Little Women (1868). In C. Gerhardt (Ed.), Handbook of American Novel of the
Nineteenth Century (pp. 399-417). Berlin and Boston: De Gruyter. May, V. H. (2011). Unprotected labor: household workers, politics, and middle-class reform in New York, 1870-1940. Alberghene, J. (1999). Autobiography and the boundries of interpretation. In J. Alberghene and B. Lyon Clark (Eds.), Little Women
and feminist imagination: criticism, controversy, personal essays (pp. 347-376). London: Routledge. References Chapell Hill:
University of North Carolina Press. McCaffrey, L. J. (1992). Textures of Irish America. Syracuse: Syracuse University Press. rey, L. J. (1992). Textures of Irish America. Syracuse: Syracuse University Press. Porter, M. S. (1893). Recollections of Louisa May Alcott, John Greenleaf Whittier, and Robert Browning Porter, M. S. (1893). Recollections of Louisa May Alcott, John Greenleaf Whittier, and Robert Browning. Boston: Collins Press. Rishoi, C. (2003). From girl to woman: American women’s coming-of-age narratives. Albany: SUNY Press. Showalter, E. (1991). Sister’s choice: tradition and change in American women’s writing. Oxford: Clarendon P Soja, E. (1996). Thirdspace: Journeys to Los Angeles and other real-and-imagined places. Cambridge, MA: Blackwell. Thernstrom, S. (1994). Poverty and progress. Cambridge, MA and London: Harvard University Press. Trachtenberg, A. (2007). The incorporation of America. New York: Hill and Wang. Trachtenberg, A. (2007). The incorporation of America. New York: Hill and Wang. 236
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Glutamine Metabolism in Both the Oxidative and Reductive Directions Is Triggered in Shrimp Immune Cells (Hemocytes) at the WSSV Genome Replication Stage to Benefit Virus Replication
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Glutamine Metabolism in Both the
Oxidative and Reductive Directions Is
Triggered in Shrimp Immune Cells
(Hemocytes) at the WSSV Genome
Replication Stage to Benefit Virus
Replication 1 Department of Biotechnology and Bioindustry Sciences, College of Biosciences and Biotechnology, National Cheng Kung
University, Tainan, Taiwan, 2 Graduate Institute of Integrated Medicine, China Medical University, Taichung, Taiwan,
3 International Center for the Scientific Development of Shrimp Aquaculture, National Cheng Kung University, Tainan, Taiwan ORIGINAL RESEARCH
published: 04 September 2019
doi: 10.3389/fimmu.2019.02102 White spot syndrome virus (WSSV) is the causative agent of a shrimp disease that
has caused huge global economic losses. Although its pathogenesis remains poorly
understood, it has been reported that in the shrimp immune cells (hemocytes) targeted
by WSSV, the virus triggers both the Warburg effect and glutamine metabolism
at the WSSV genome replication stage (12 h post infection). Glutamine metabolism
follows two pathways: an oxidative pathway mediated by α-KGDH (α-ketoglutarate
dehydrogenase) and an alternative reductive pathway mediated by IDH1 and IDH2
(isocitrate dehydrogenase 1 and 2). Here we used isotopically labeled glutamine
([U-13C]glutamine and [1-13C]glutamine) as metabolic tracers to show that, at the
replication stage, both the oxidative and reductive glutamine metabolic pathways
were activated. We further show that the mRNA expression levels of α-KGDH and
IDH1 were increased in WSSV-infected shrimps and that silencing of α-KGDH,
IDH1, and IDH2 with their respective dsRNAs led to a decrease in WSSV gene
expression and WSSV replication. Taken together, our findings provide new evidence for
WSSV-induced metabolic reprogramming in hemocytes and demonstrate its importance
in virus replication. Edited by:
Shoichiro Kurata,
Tohoku University, Japan
Reviewed by:
Anchalee-Tassanakajon,
Chulalongkorn University, Thailand
Chia-Ying Chu,
National Taiwan University, Taiwan
*Correspondence:
Han-Ching Wang
wanghc@mail.ncku.edu.tw
†These authors have contributed
equally to this work Edited by:
Shoichiro Kurata,
Tohoku University, Japan Reviewed by:
Anchalee-Tassanakajon,
Chulalongkorn University, Thailand
Chia-Ying Chu,
National Taiwan University, Taiwan
*Correspondence:
Han-Ching Wang
wanghc@mail.ncku.edu.tw †These authors have contributed
equally to this work Specialty section:
This article was submitted to
Comparative Immunology,
a section of the journal
Frontiers in Immunology Keywords: white spot syndrome virus, IDHs, oxidative glutaminolysis, reductive carboxylation, hemocytes Received: 25 June 2019
Accepted: 20 August 2019
Published: 04 September 2019 Keywords: white spot syndrome virus, IDHs, oxidative glutaminolysis, reductive carboxylation, hemocytes Experimental Animals and WSSV Inoculum Cloning of Full-Length cDNA of LvGLS1,
LvGLS2, LvIDH1, LvIDH2, and Lvα-KGDH
By using next generation sequencing, an in-house L. vannamei
stomach transcriptomic database was established (data not
shown) and this was used to search for the target genes. Two
contigs,
PVHP259804.2
and
PVHP193998,
were
found to show high homology with Rat GLS (Accession
number:
M65150.1),
and
PVHP193998
also
matched
P. vannamei GLS (Accession number: XP_027218904.1) with
98% identity. These contig sequences were used to design
primer sets to amplify the two GLS isoforms (Table 1)
and these two genes were named LvGLS1(PVHP259804.2)
and
LvGLS2
(PVHP193998). Three
other
contigs,
PVHP107410.1,
PVHP176973.1,
and
PVHP203127.2,
respectively showed high homology with Penaeus vannamei
IDH1
(Accession
number:
XP_027219531.1),
Penaeus
vannamei
IDH2
(Accession
number:
XP_027239404.1),
and
Penaeus
vannamei
α-KGDH
(Accession
number:
XP_027220285.1). These contig sequences were also used
to design primer sets to amplify the corresponding genes
(Table 1). We have recently shown that glutamine anaplerosis also
occurs in WSSV-infected shrimp: we found that WSSV increases
the expression of GDH, an enzyme which converts glutamate
into α-KG (8), and also that direct in vivo replenishment of α-
KG rescued WSSV replication after the down-regulation of GDH
by dsRNA-mediated gene silencing. These results all suggest that,
in conjunction with the WSSV-induced Warburg effect, WSSV-
infected cells may activate glutamine metabolism to fuel the
TCA cycle. However, it was not known whether WSSV triggered
glutamine metabolism in both the oxidative and reductive
directions. In the present study, we therefore look more closely at
the glutamine metabolism induced by WSSV. To do this, we use
LC-ESI-MS and isotopically labeled glutamine (uniformly-13C
[U-13C] glutamine and [1-13C] glutamine) as metabolic tracers. We also provide additional evidence of the importance of the
reductive carboxylation glutaminolysis for virus replication. Experimental Animals and WSSV Inoculum Experimental Animals and WSSV Inoculum
The shrimp (Litopenaeus vannamei) of around 3 g body weight
used in the study were obtained from the International Center
for the Scientific Development of Shrimp Aquaculture, National
Cheng Kung University (NCKU), and the Department of
Aquaculture, National Pingtung University of Science and
Technology (NPUST). Before the experiments, shrimp were
cultured for 1∼3 days in sterilized seawater (30 ppt at
26∼27◦C). The WSSV (Taiwan isolate, GenBank accession no. AF440570) stock (3.3 × 104 WSSV copies/µl) was prepared from
hemolymph of WSSV-infected moribund SPF (specific pathogen
free) shrimp as described previously (6, 9). The viral inoculum
was prepared from the stock for intramuscular injection by
dilution (10−4) with 1x PBS (137 mM NaCl, 2.7 mM KCl, 10 mM
Na2HPO4, 2 mM KH2PO4). The WSSV challenge dosage (100
µl/3 g shrimp) resulted in an ∼50% cumulative mortality at
3 days post WSSV challenge. Shrimp in the control group
were treated with PBS (100 µl/3 g shrimp). At 12 and 24 h
post WSSV challenge, hemocyte samples and stomach tissue
were collected and used for the glutamine-metabolism-related
enzyme activity assays. The hemocyte samples were also used
to measure the expression of host genes and viral genes, for the
stable-isotope metabolic tracing experiments, and to measure the
copy number of the WSSV genomic DNA as described in Su
et al. (9). When aerobic glycolysis is activated, most of the carbon
atoms from the glucose that has been taken up become
diverted into lactate production instead of being routed into
the mitochondrial TCA (tricarboxylic acid) cycle (2, 14). Thus,
in order to allow the TCA cycle to continue to produce
energy and biosynthetic products during aerobic glycolysis,
glutamine, an amino acid that is abundant in the circulation
system, is used as an alternative carbon source in a process
known as glutamine anaplerosis (11, 15, 16). In normal cells,
after glutamine is converted (via glutamate) into α-KG, it
is subsequently metabolized to succinate by α-ketoglutarate
dehydrogenase (α-KGDH) through oxidative glutaminolysis. However, cells undergoing the Warburg effect are also able
to convert α-KG into isocitrate either by cytoplasmic IDH1
or by mitochondrial IDH2 through the reductive glutamine
metabolic pathway (17, 18). Although mammalian studies have
shown that glutamine metabolism can have both immune-related
benefits as well as pathogenesis-related effects (3–5), the pathway
primarily seems to favor virus replication in shrimp infected by
WSSV (8). Citation: He S-T, Lee D-Y, Tung C-Y, Li C-Y and
Wang H-C (2019) Glutamine
Metabolism in Both the Oxidative and
Reductive Directions Is Triggered in
Shrimp Immune Cells (Hemocytes) at
the WSSV Genome Replication Stage
to Benefit Virus Replication. Front. Immunol. 10:2102. doi: 10.3389/fimmu.2019.02102 Although in many kinds of tumors and cancers, metabolic reprogramming has been found to be
crucial for the cell’s aberrant proliferation and potentially unlimited self-renewal (1, 2), immune
cells are also able to control pathogens by triggering particular metabolic pathways to aid immune
responses, such as increasing amino acid catabolism (3). However, there is also increasing evidence
that some vertebrate viruses, especially oncogenic viruses can utilize the reprogramming of host
metabolism to complete their replication cycle (4, 5). Quite recently, the white spot syndrome virus
(WSSV), a large dsDNA virus that is the causative agent of a devastating viral shrimp disease, September 2019 | Volume 10 | Article 2102 Frontiers in Immunology | www.frontiersin.org He et al. WSSV-Induced Glutamine Metabolism MATERIALS AND METHODS became the first invertebrate virus that was shown to induce host
metabolic reprogramming in shrimp immune cells (hemocytes). These changes include the Warburg effect (aerobic glycolysis),
amino acid catabolism (i.e., glutaminolysis [glutamate-driven
anaplerosis]), lipid metabolism, activation of the pentose
phosphate pathway, nucleotide biosynthesis, and amino acid
biosynthesis (6–9). The effects are most noticeable at the genome
replication stage (12 h post infection), and like the metabolic
reprogramming that is seen in cancer cells and cells infected by
some vertebrate viruses (10–13), the WSSV-induced metabolic
changes benefit the virus by meeting the both its energy
requirements and its biosynthetic needs. Frontiers in Immunology | www.frontiersin.org Measurement of Host Genes and the WSSV
Major Structural Gene VP28 by Real-Time
PCR Abbreviations:
WSSV,
white
spot
syndrome
virus;
LC-ESI-MS,
Liquid
chromatography-electrospray ionization mass spectrometry; PBS, phosphate-
buffered saline; GLS, glutaminase; GDH: glutamine dehydrogenase; ASAT,
aspartate
aminotransferase;
IDH,
isocitrate
dehydrogenase;
α-KGDH,
α-ketoglutarate dehydrogenase. Gln, glutamine; Glu, glutamate; α-KG, α-
ketoglutarate; Suc, succinate; Fum, fumarate; Mal, malate; Oac, oxaloacetate; Cit,
citrate; Ict, isocitrate; Lac, lactate; Asp, aspartate. After shrimp tissues were collected at the 12 and 24 hpi time
points, total RNA was extracted and subjected to cDNA synthesis
by using Superscriptase II Reverse Transcriptase (Invitrogen) and
Anchor-dTv primer (Table 1). The cDNA samples were used September 2019 | Volume 10 | Article 2102 Frontiers in Immunology | www.frontiersin.org 2 WSSV-Induced Glutamine Metabolism He et al. He et al. TABLE 1 | Primer sets used in the present paper. Gene
Primer
Primer sequence (5′-3′)a
Usag
EF1α
EF1α-F
5′-ATGGTTGTCAACTTTGCCC-3′
Cloni
EF1α-R
5′-TTGACCTCCTTGATCACACC-3′
Cloni
EF1α-qF
5′-ACGTGTCCGTGAAGGATCTGAA-3′
Real-
EF1α-qR
5′-TCCTTGGCAGGGTCGTTCTT-3′
Real-
LUCIFERASE
Luc-F
5′-CTGAATACAAATCACAGAATC-3′
Cloni
Luc-R
5′-GTAAGACCTTTCGGTACTTCG-3′
Cloni
T7-Luc-F
5′-TAATACGACTCACTATAGGGAGACTGAATACAAATCACAGAATC-3′
dsRN
T7-Luc-R
5′-TAATACGACTCACTATAGGGAGAGTAAGACCTTTCGGTACTTCG-3′
dsRN
GDH
GDH-qF
5′-TGAGGAGAAGCGCAACAAGA-3′
Real-
GDH-qR
5′-TGGCAGGGCTCCATGATC-3′
Real-
GLS1
GLS1-qF
5′-CATTGGCGACACTGACAT-3′
Real-
GLS1-qR
5′-CTGCAGAAGGCCATTGACTA-3′
Real-
GLS2
GLS2-F
5′-GACCGCAAGAACCTCCTCAA-3′
Cloni
GLS2-R
5′-GTGATGACAGAAGCCACGGA-3′
Cloni
T7-GLS2- F
5′-TAATACGACTCACTATAGGGAGAGACCGCAAGAACCTCCTCAA-3′
dsRN
T7-GLS2- R
5′-TAATACGACTCACTATAGGGAGAGTGATGACAGAAGCCACGGA-3′
dsRN
GLS2-qF
5′-AACTACATGGGGATGGAG-3′
Real-
GLS2-qR
5′-GATTGAAATCCAGGCAAAGCTC-3′
Real-
IDH1
IDH1-F
5′-GAGGATTTGCTCATGCTTC-3′
Cloni
IDH1-R
5′-TCAGGCAGTGATCTTCTTCTGC-3′
Cloni
T7-IDH1-F
5′-TAATACGACTCACTATAGGGAGAGAGGATTTGCTCATGCTTC-3′
dsRN
T7-IDH1-R
5′-TAATACGACTCACTATAGGGAGATCAGGCAGTGATCTTCTTCTGC-3′
dsRN
IDH1-qF
5′-GGCATGATGACCTCGGTACTG-3′
Real-
IDH1-qR
5′-GGCAGCCTCAGACTCCAGAGT-3′
Real-
IDH2
IDH2-F
5′-GCAAGAACTACGATGGTG-3′
Cloni
IDH2-R
5′-ATGCAGCCAGCCAGATCCT-3′
Cloni
T7-IDH2-F
5′-TAATACGACTCACTATAGGGAGAGCAAGAACTACGATGGTG-3′
dsRN
T7-IDH2-R
5′-TAATACGACTCACTATAGGGAGAATGCAGCCAGCCAGATCCT-3′
dsRN
IDH2-qF
5′-CCAACCCTGTTGCTTCCATT-3′
Real-
IDH2-qR
5′-AAGCTTGGCACGATGTTCAAG-3′
Real-
α-KGDH
KGDH-F
5′-ATGGGTTTGAGGCATTCTTG-3′
Cloni
KGDH-R
5′-CCTGAGAAAGCAGCATCTCC-3′
Cloni
T7-KGDH-F
5′-TAATACGACTCACTATAGGGAGAATGGGTTTGAGGCATTCTTG-3′
dsRN
T7-KGDH-R
5′-TAATACGACTCACTATAGGGAGACCTGAGAAAGCAGCATCTCC-3′
dsRN
KGDH-qF
5′-TCCAGCCTCGCATTTCCA-3′
Real-
KGDH-qR
5′-GACGGCCAGCATATGAAA-3′
Real-
VP28
vp28-real-F
5′-AGTTGGCACCTTTGTGTGTGGTA-3′
Real-
vp28-real-R
5′-TTTCCACCGGCGGTAGCT-3′
Real-
aThe added T7 promoter sequence is underlined. to quantify the mRNA expression of the target genes by using
l
h
(
)
gene VP28 and EF1α. The specific primer sets for ea
l
d
bl
l
l
d aThe added T7 promoter sequence is underlined. to quantify the mRNA expression of the target genes by using
real-time PCR with KAPA SYBR1 FAST Master Mix (KAPA)
and the Bio-Rad detection system. The gene expression levels
measured in this study were for the two GLS isoforms (GLS1
and GLS2), IDH1, IDH2, α-KGDH, the WSSV major structural to quantify the mRNA expression of the target genes by using
real-time PCR with KAPA SYBR1 FAST Master Mix (KAPA)
and the Bio-Rad detection system. Determination of the Enzyme Activity of
IDH in Shrimp Stomachs and Hemocytes
During WSSV Infection Hemocytes and stomachs from shrimp were collected at 12
and 24 h after WSSV or PBS injection (6 shrimp/pool and
4 pools/group), and the GLS activity was measured with a
commercial Glutaminase Microplate Assay Kit (MyBiosource). The hemocyte and stomach samples were homogenized with 100
and 300 µl ice cold assay buffer, respectively. After centrifugation
at 4◦C at 13,000 g for 10 min, the cell debris was removed and the
protein concentration in the supernatant was determined. The
hemocyte lysates (3∼52 µg) and stomach lysates (6 µg) were
mixed with 200 µl substrate, and the reaction were incubated
at 37◦C for 1 h. The reactions were stopped by adding 300 µl
Stop solution, incubating for 10 min and then centrifuging at
4◦C at 8,000 g for 5 min. One hundred and thirty microliter of
each supernatant was collected and mixed with a 70 µl reaction
mixture containing 50 µl reaction buffer and 20 µl dye reagent. The controls were prepared as per the samples except that
the protein lysates were replaced by distilled water. Standards
and blanks were prepared according to the manufacturer’s
instructions. The absorbance of the final mixtures was measured
at 450 nm. The GLS activity was calculated by the following
equation: GLS activity (U/mg) =
4×T×CStandard× (ODSample−ODControl)
(ODStandard−ODBlank)
CProtein
(note: CStandard: reference standard [1 mg/ ml]; CProtein: protein
concentration; ODblank: buffer only; T: reaction time in hours). Statistical analysis was performed as described above. g
Hemocytes and stomachs from shrimp were collected at 12
and 24 h after WSSV or PBS injection (6 shrimp/pool and 4
pools/group) to measure the activity of IDH. The hemocyte and
stomach samples were homogenized with 100 and 200 µl ice
cold IDH assay buffer, respectively, from a commercial Isocitrate
Dehydrogenase Activity Assay Kit (Sigma). The cell debris was
removed by centrifugation at 4◦C at 13,000 g for 10 min. After
using a Bio-Rad protein assay to measure protein concentrations,
samples with the appropriate amounts of protein (hemocyte
lysate: 15 ∼30 µg; stomach lysate: 10 µg) were collected and
adjusted to a final volume of 50 µl using IDH assay buffer. The
lysates were then mixed with 50 µl reaction mixture containing
38 µl IDH Assay Buffer, 8 µl Developer 2 µl IDH Substrate, and
2 µl NADP+. The final mixtures were incubated at 37◦C and
protected from light. Measurement of the WSSV Genome Copy
Number Using the IQ RealTM WSSV
Quantitative System mixtures were then incubated at 37◦C and protected from light. The NADH standards supplied with the kit were prepared in the
same way as the shrimp tissue samples. α-KGDH activity was
measured at A450 every 3–5 min until the value of the most active
sample was greater than the value for the NADH standard with
the highest concentration (12.5 nmole). The NADH standard
curve at this time point was then used to convert the difference
in absorbance at the initial (Tinitial) and final (Tfinal) time points
to the amount of NADH (B). The activity was calculated by
the following equation: enzyme activity (mU/mg) = B/([Tfinal –
Tinitial] × [total amount of protein in the reaction]). Statistical
analysis was performed as described above. Shrimp hemocytes were collected from each group at the
12 and 24 hpi time points and subjected to genomic DNA
extraction using a DTAB/CTAB DNA extraction kit (GeneReach
Biotechnology Corp.). The viral genome copy numbers were
then quantified by the real-time PCR-based IQ Real
TM WSSV
quantitative system (GeneReach Biotechnology Corp.). Statistical
analysis was performed as described above. Measurement of Host Genes and the WSSV
Major Structural Gene VP28 by Real-Time
PCR The gene expression levels
measured in this study were for the two GLS isoforms (GLS1
and GLS2), IDH1, IDH2, α-KGDH, the WSSV major structural gene VP28 and EF1α. The specific primer sets for each target
gene are listed in Table 1. Data values were normalized to EF1α
cDNA (internal control) and calculated by the 2−1CT method. Statistically significant differences between groups were analyzed
by Student’s t-test as described in Tseng et al. (19). September 2019 | Volume 10 | Article 2102 Frontiers in Immunology | www.frontiersin.org 3 WSSV-Induced Glutamine Metabolism He et al. He et al. Determination of the Enzyme Activity of
IDH in Shrimp Stomachs and Hemocytes
During WSSV Infection The NADH standards supplied with the kit
were prepared in the same way as the tissue samples. IDH activity
was measured at A450 every 3–5 min until the value of the most
active sample was greater than the value of the standard with the
highest concentration (10 nmole). The difference in absorbance
at the initial (Tinitial) and final (Tfinal) time points was converted
to the NADPH amount (B) using the NADH standard curve at
the final time point. The enzyme activity was calculated by the
following equation: IDH activity (mU/mg) = B/([Tfinal – Tinitial]
× [total amount of protein in the reaction]). Statistical analysis
was performed as described above. In vivo Gene Silencing of LvIDH1, LvIDH2,
Lvα-KGDH Mediated by dsRNA
Interference Using Stable Isotope-Labeled Glutamine
Tracer and Liquid Chromatography
Electrospray Ionization Mass Spectrometry
(LC-ESI-MS) to Monitor Metabolites in the
Hemocytes of WSSV-Infected Shrimp
Stable isotope-labeled glutamine can be used together with LC-
ESI-MS to identify and quantify metabolites in the oxidative and
reductive glutamine metabolic pathways that have incorporated
the labeled glutamine carbon atoms. In this study, we used both
[U-13C]glutamine (M5 gln) and [1-13C]glutamine (M1 gln) to
trace the metabolites of interest as shown in Figure 1. As no
shrimp cell line or alternative cell line is presently available, we
established an in vivo, 13C-labeled shrimp hemocyte metabolic
analysis platform ab initio. Based on preliminary tests (data not
shown), our experimental protocol was to treat WSSV-challenged
shrimp with stable isotope-labeled glutamine by haemocoel
injection at 12 and 24 hpi after challenge. At 10 and 30 min
after injection of the M5 or M1 gln, pooled hemocyte samples
(4 pools; 3 shrimp in each pool) were collected and analyzed as
described below. Using Stable Isotope-Labeled Glutamine
Tracer and Liquid Chromatography
Electrospray Ionization Mass Spectrometry
(LC-ESI-MS) to Monitor Metabolites in the
Hemocytes of WSSV-Infected Shrimp y
p
Stable isotope-labeled glutamine can be used together with LC-
ESI-MS to identify and quantify metabolites in the oxidative and
reductive glutamine metabolic pathways that have incorporated
the labeled glutamine carbon atoms. In this study, we used both
[U-13C]glutamine (M5 gln) and [1-13C]glutamine (M1 gln) to
trace the metabolites of interest as shown in Figure 1. As no
shrimp cell line or alternative cell line is presently available, we
established an in vivo, 13C-labeled shrimp hemocyte metabolic
analysis platform ab initio. Based on preliminary tests (data not
shown), our experimental protocol was to treat WSSV-challenged
shrimp with stable isotope-labeled glutamine by haemocoel
injection at 12 and 24 hpi after challenge. At 10 and 30 min
after injection of the M5 or M1 gln, pooled hemocyte samples
(4 pools; 3 shrimp in each pool) were collected and analyzed as
described below. g
Data were acquired by HyStar and micrOTOF control
software (Bruker Daltonics) and processed by DataAnalysis
and TargetAnalysis software (Bruker Daltonics) to generate the
signals corresponding to the integrated areas for each extracted
ion chromatogram. To monitor the change in the quantity of the 13C labeled
metabolites, fold changes in the WSSV group were calculated
relative to the corresponding PBS group (WSSV/PBS group). All the signal counts were normalized by the sample’s weight
(mg). In vivo Gene Silencing of LvIDH1, LvIDH2,
Lvα-KGDH Mediated by dsRNA
Interference Statistically significant differences between WSSV and PBS
groups were analyzed by Student’s t-test as described above. Stable isotope tracer solutions were prepared by dissolving [U-
13C]glutamine (M5 gln; Cambridge Isotope Laboratories, Inc)
or [1-13C]glutamine (M1 gln; Cambridge Isotope Laboratories,
Inc) in PBS. At 12 or 24 h after the experimental shrimp had
been injected with WSSV or PBS, one of the stable isotope
tracer solutions ([U-13C]glutamine: 400 µg /g shrimp; [1-
13C]glutamine: 800 µg /g shrimp) was injected into the shrimp’s
abdominal hemal sinus. At 10 and 30 min after treatment with the
tracer, 4 pooled hemocyte samples (3 shrimp in each pool) were
collected from each group using a cold anticoagulant (1x PBS,
10 mM EDTA, pH8.0). After immediately centrifuging at 1,000x
g for 10 min by using a swinging bucket, the pellet of shrimp
hemocytes from each sample was collected, washed with ice-cold
1x PBS and the hemocytes were lysed with sterilized ddH2O on In vitro Synthesis of LvGLS2, LvIDH1,
LvIDH2, and Lvα-KGDH dsRNAs g
Hemocytes and stomachs from shrimp were collected at 12
and 24 h after WSSV or PBS injection (6 shrimp/pool and 4
pools/group) to measure the activity of α-KGDH. The hemocyte
and stomach samples were homogenized with 100 and 200 µl
ice cold KGDH assay buffer, respectively, from a commercial
α-Ketoglutarate Dehydrogenase Activity Colorimetric Assay Kit
(Sigma). The cell debris was removed by centrifugation at 4◦C
at 13,000 g for 10 min. After using a Bio-Rad protein assay to
measure protein concentrations, samples with the appropriate
amounts of protein (hemocyte lysate: 3.6 ∼50 µg; stomach lysate:
10 ∼20 µg) were collected and adjusted to a final volume of 50 µl
using the KGDH assay buffer. The lysates were then mixed with
50 µl reaction mixture containing 46 µl KGDH Assay Buffer,
2 µl KGDH Developer, and 2 µl KGDH Substrate. The final dsRNAs were prepared as described in our previous study (9,
20). In short, the partial sequences (300∼600 bp) of each host
gene and the non-specific Luciferase control were amplified by
PCR with the following respective primer sets: GLS2-F/GLS2-
R, IDH1-F/IDH1-R, IDH2-F/IDH2-R, α-KGDH-F/α-KGDH-R,
and Luc-F/Luc-R (Table 1). After obtaining the corresponding
PCR amplicons, T7 promoter sequence was added to the 5
′ and
3
′ ends of each amplicon by a second PCR with the primer sets:
T7-GLS2-F/GLS2-R, GLS2-F/ T7-GLS2-R, T7-IDH1-F/IDH1-R,
IDH1-F/T7-IDH1-R, T7-IDH2-F/IDH2-R, IDH2-F/T7-IDH2-
R, T7-α-KGDH-F/α-KGDH-R, α-KGDH-F/T7-α-KGDH-R, T7-
Luc-F/Luc-R, and Luc-F/T7-Luc-R (Table 1). The ssRNAs were
then synthesized by the T7 RiboMAX Express large-scale RNA September 2019 | Volume 10 | Article 2102 Frontiers in Immunology | www.frontiersin.org Frontiers in Immunology | www.frontiersin.org 4 He et al. WSSV-Induced Glutamine Metabolism production system (Promega) and the corresponding ssRNA
pairs were mixed together to form the dsRNAs. After the dsRNAs
were purified by phenol/chloroform extraction, quantified by UV
spectrophotometer and checked by agarose gel electrophoresis,
the final dsRNA products were stored at −80◦C before use. ice. Next, 100% MeOH was added to the cell lysate at a ratio
of 1:3 to quench the metabolic reactions, and the samples were
kept at −80◦C for 10 min. The samples were then centrifuged
at 4◦C at 18,000 g for 10 min, and 75% MeOH was added to
the cell lysate for secondary extraction. The supernatants, which
now contained the metabolites, were then collected, lyophilized,
and stored at −80◦C before being analyzed by LC-ESI-MS as
described previously (9). Frontiers in Immunology | www.frontiersin.org In vivo Gene Silencing of LvIDH1, LvIDH2,
Lvα-KGDH Mediated by dsRNA
Interference p
y
Briefly, for LC-ESI-MS analysis the samples were dissolved
in 35 µl of ddH2O and 5 µl of reaction buffer (0.3 M aniline
[Sigma-Aldrich, USA] in 60 mM HCl), and 5 µl of N-(3-
dimethylaminopropyl)-N’-ethylcarbodiimide
hydrochloride
(EDC; Sigma-Aldrich, USA) was added. After incubating the
mixture at 25◦C for 2 h, 5 µl of 10% ammonium hydroxide was
added to stop the reaction. The derivatives were analyzed on
an LC-ESI-MS system comprising an ultra-performance liquid
chromatography (UPLC) system (Ultimate 3000 RSLC, Dionex)
and a quadrupole time-of-flight (QTOF) mass spectrometer
with an electrospray ionization (ESI) source (maXis HUR-
QToF
system,
Bruker
Daltonics). Reversed-phase
liquid
chromatography (RPLC) on a BEH C18 column (2.1 × 100 mm,
Waters) was used. The elution started from 99% mobile phase
A (0.1% formic acid in ddH2O) and 1% mobile phase B (0.1%
formic acid in ACN), held at 1% B for 0.5 min, raised to 60% B
in 6 min, further raised to 90% B in 0.5 min, held at 90% B for
1.5 min, and then lowered to 1% B in 0.5 min. The column was
then equilibrated by pumping 1% B for 4 min. The flow rate was
set at 0.3 ml/min with an injection volume of 10 µl. LC-ESI-MS
chromatograms were acquired under a capillary voltage of either
4,500 or 3,500 V in negative ion mode, a dry temperature of
190◦C, a dry gas flow maintained at 8 l/min, nebulizer gas at 1.4
bar, and an acquisition range of m/z 100–1,000. Shrimp (∼3 g body weight) were injected with the LvIDH1,
LvIDH2, Lvα-KGDH dsRNAs at a concentration of 1 µg /g
shrimp. Shrimp treated with Luc dsRNA were used as the control. At 72 h (3 days) post the dsRNA injection, shrimp hemocyte
samples were collected from each group and real-time PCR was
used to confirm that the LvIDH1, LvIDH2, and Lvα-KGDH genes
had been specifically silenced by the respective dsRNA. At the
same time, the remaining shrimp in each group were challenged
by injection with WSSV inoculum or PBS. At 24 h post injection,
shrimp hemocyte samples were collected (3 shrimp in each
sample, 4 samples for each group) and used to measure the
expression of host genes, viral genes and the WSSV genome copy
number. Statistical analysis was performed as described above. Effect of the Inhibitors Salirasib (S35),
Torin1, and LY294002 on the mRNA
Expression of Key Host Genes Involved in
Glutamine Metabolism During WSSV
Infection Following the protocol described in previous studies (8, 9,
19), shrimp were intramuscularly injected with 100 µl of the
inhibitors Salirasib, Torin1, and LY294002 or their vehicles 2 h
before being challenged with WSSV. Samples were then collected
at 12 and 24 h post WSSV injection. To evaluate the involvement
of shrimp Ras, shrimp were treated with Salirasib (dissolved in
99% EtOH and diluted with PBS, pH 8.0; 35 µg/g shrimp) or with September 2019 | Volume 10 | Article 2102 5 He et al. WSSV-Induced Glutamine Metabolism He et al. FIGURE 1 | Schematic showing how the metabolites derived from (A) [U-13C]glutamine can be traced through the oxidative and reductive glutamine pathways and
(B) [1-13C]glutamine can be traced through the reductive glutamine pathway. In the Gln-Glu-a-KG pathway, white circles represent 12Carbon, while solid black circles
represent 13Carbon. Blue circles represent 13C in the oxidative glutamine metabolism pathway of the TCA cycle and the citrate shuttle, while red circles represent 13C
in the reductive carboxylation pathways in either mitochondria or cytoplasm. The green arrow indicates the shift toward lipid metabolism. For [U-13C]glutamine in (A),
abbreviations are as follows: for the Gln-Glu-α-KG pathway: Gln, M5 glutamine; Glu, M5 glutamate; α-KG, M5 α-ketoglutarate. For oxidative glutamine metabolism:
Suc, M4 succinate; Fum, M4 fumarate; Mal, M4 malate; Oac, M4 oxaloacetate; Cit, M4 citrate; Lac, M3 lactate. For reductive carboxylation: Ict, M5 isocitrate; Cit, M5
citrate; Oac, M3 oxaloacetate; Asp, M3 aspartate; Fum, M3 fumarate; Mal, M3 malate. Similar abbreviations apply for [1-13C]glutamine (M1 glutamine) in (B). FIGURE 1 | Schematic showing how the metabolites derived from (A) [U-13C]glutamine can be traced through the oxidative and reductive glutamine pathways and
(B) [1-13C]glutamine can be traced through the reductive glutamine pathway. In the Gln-Glu-a-KG pathway, white circles represent 12Carbon, while solid black circles
represent 13Carbon. Blue circles represent 13C in the oxidative glutamine metabolism pathway of the TCA cycle and the citrate shuttle, while red circles represent 13C
in the reductive carboxylation pathways in either mitochondria or cytoplasm. The green arrow indicates the shift toward lipid metabolism. For [U-13C]glutamine in (A),
abbreviations are as follows: for the Gln-Glu-α-KG pathway: Gln, M5 glutamine; Glu, M5 glutamate; α-KG, M5 α-ketoglutarate. For oxidative glutamine metabolism:
Suc, M4 succinate; Fum, M4 fumarate; Mal, M4 malate; Oac, M4 oxaloacetate; Cit, M4 citrate; Lac, M3 lactate. Effect of the Inhibitors Salirasib (S35),
Torin1, and LY294002 on the mRNA
Expression of Key Host Genes Involved in
Glutamine Metabolism During WSSV
Infection For reductive carboxylation: Ict, M5 isocitrate; Cit, M5
citrate; Oac, M3 oxaloacetate; Asp, M3 aspartate; Fum, M3 fumarate; Mal, M3 malate. Similar abbreviations apply for [1-13C]glutamine (M1 glutamine) in (B). target genes, all samples were analyzed by real-time PCR as
described above. vehicle only (0.3% ethanol). Samples from Salirasib-pretreated
shrimp were collected into 4 pooled hemocyte samples, with
each sample being taken from 3 shrimp (19). To investigate
the involvement of the PI3K-mTORC1 pathway, shrimp were
treated with LY294002 (dissolved in 10% DMSO and diluted
with PBS; 0.625 µg/g shrimp) or with vehicle only (0.01%
DMSO). Hemocyte samples from LY294002-pretreated shrimp
were collected individually for each group (8). To observe the
involvement of mTORC1/mTORC2, shrimp were treated with
Torin 1 (dissolved in DMSO and diluted with PEG solvent;
20 µg/g shrimp) or with vehicle only (0.25% PEG, 0.25% Tween
20, and 0.15 M NaCl) five or six pleopod samples were collected
from each group, with each sample being taken from 10
shrimp (9). To determine the mRNA expression levels of the RESULTS In vivo Tracking of [U-13C]
Glutamine-Derived Metabolites Showed
That Both Glutaminolysis and Reductive
Carboxylation Were Activated at the WSSV
Genome Replication Stage (12 hpi)
To investigate the effect of WSSV in WSSV-infected hemocytes,
shrimp were challenged with WSSV, and then injected with [U-
13C]glutamine (M5 gln) to allow tracing of this stable carbon In vivo Tracking of [U-13C]
Glutamine-Derived Metabolites Showed
That Both Glutaminolysis and Reductive
Carboxylation Were Activated at the WSSV
Genome Replication Stage (12 hpi) In vivo Tracking of [U-13C]
Glutamine-Derived Metabolites Showed
That Both Glutaminolysis and Reductive
Carboxylation Were Activated at the WSSV
Genome Replication Stage (12 hpi) p
g
(
p )
To investigate the effect of WSSV in WSSV-infected hemocytes,
shrimp were challenged with WSSV, and then injected with [U-
13C]glutamine (M5 gln) to allow tracing of this stable carbon September 2019 | Volume 10 | Article 2102 Frontiers in Immunology | www.frontiersin.org Frontiers in Immunology | www.frontiersin.org 6 WSSV-Induced Glutamine Metabolism He et al. isotope through the glutamine metabolic pathways as shown
in Figure 1A. reductive carboxylation (Figure 1B). Compared to the PBS-
treated shrimp, there was generally an increase of M1 metabolites
in the glutamine-glutamate-αKG (Gln-Glu-α-KG) pathway in
shrimp hemocytes at the WSSV genome replication stage (12
hpi; Figures 4A,B), and there was also a general increase in
the amounts of the subsequent metabolites in the reductive
carboxylation pathway (Figures 4A,B). As before, these results
suggest that reductive carboxylation is triggered at the WSSV
genome replication stage. However, once again, there was
no clear pattern of increase in these metabolites at the late
stage of WSSV replication (Figures 4C,D). Figures 4E,F provide
graphical summaries of these results. Raw metabolomic data is
given in Table S2. Compared to the PBS treated shrimp, at 12 hpi, there
was an 8-fold increase in the amount of M5 glutamine
in shrimp hemocytes at 10 min after treatment with [U-
13C]glutamine (Figure 2A). At the same point, there was also
a significant increase in the final downstream product of
oxidative glutaminolysis, M3 lactate (Figure 2A). These data
suggest that, at 12 hpi, WSSV infection triggers glutamine
uptake into shrimp hemocytes and that at least some of this
glutamine is converted into lactate via oxidative glutaminolysis. Figure 2A also shows a significant increase in M5 isocitrate
and M5 citrate which suggests that the glutamine-dependent
reductive carboxylation pathway is also active in WSSV-infected
shrimp hemocytes at 12 hpi. RESULTS At 30 min after [U-13C]glutamine
treatment, similarity elevated amounts of the oxidative and
reductive pathway metabolites were still detected (Figure 2B),
but the relatively low levels of glutamine and downstream
products such as M4 citrate and M3 oxaloacetate suggest that
most of the labeled glutamine input might already have flushed
through these pathways. Figure 2C summarizes the changes
in these 13C glutamine metabolites in both the oxidative and
reductive directions at 12 hpi and 10 min after [U-13C]glutamine
treatment. The raw metabolomic data for [U-13C]glutamine and
its metabolites are given in Table S1. In vivo Tracking of [U-13C]
Glutamine-Derived Metabolites Suggests
That Glutaminolysis and Reductive
Carboxylation are No Longer Activated at
the Late Stage of the WSSV Replication
Cycle (24 hpi) In vivo Tracking of [U-13C]
Glutamine-Derived Metabolites Suggests
That Glutaminolysis and Reductive
Carboxylation are No Longer Activated at
the Late Stage of the WSSV Replication
Cycle (24 hpi) For GLS, which functions as the initial enzyme in glutamine
metabolism by converting glutamine to glutamate, we found
that although there was no significant change in the expression
of the two GLS isoforms, GLS1 and GLS2, the enzyme
activity was significantly decreased at both stages of the WSSV
replication cycle (12 and 24 hpi) in both hemocytes and
stomach (Figure 6A). For IDH1 and IDH2, which are the
key enzymes in reductive glutamine metabolism, significant
upregulation of IDH1 mRNA expression was observed at 12
and 24 hpi, while there was no change in IDH2. There was
also a significant increase in the enzyme activity of IDH
in shrimp hemocytes at 12 hpi, and a significant decrease
at 24 hpi; meanwhile in the stomach there was no change
(Figure 6B). For α-KGDH, which is a key enzyme in oxidative
glutamine metabolism, the mRNA levels and enzyme activity
were significantly increased in hemocytes at both 12 and 24
hpi (Figure 6C). α-KGDH enzyme activity was also significantly
increased in shrimp stomach (Figure 6C). Taken together, at
least at 12 hpi, WSSV seems to trigger both IDH1-mediated
reductive glutamine metabolism as well as α-KGDH-mediated
glutamine metabolism. At 24 hpi, although [U-13C]glutamine still seems to be taken up
by the WSSV-infected hemocytes, the elevated accumulation of
M5 α-KG and the reduced levels of the glutamine-derived TCA
intermediates (i.e., the M4 forms of succinate, fumarate, malate,
oxaloacetate, and the M3 forms of lactate) suggest that oxidative
metabolism of [U-13C]glutamine was not triggered at this stage
(Figures 3A,B). Similarly, there was no evidence of reductive
carboxylation, i.e., no obvious increase in the M5 forms of
isocitrate and citrate or the M3 forms of oxaloacetate, aspartate,
fumarate, and malate (Figures 3A,B). Figure 3C provides a
graphical summary of the above results. In vivo Tracking of [1-13C]
Glutamine-Derived Metabolites
Reconfirmed That Reductive Carboxylation
Was Activated at the WSSV Genome
Replication Stage (12 hpi) WSSV Activates Glutamine
Metabolism-Related Genes in L. vannamei
at the Genome Replication Stage Previous reports have shown that at the WSSV genome
replication stage (12 hpi), WSSV activates GDH and ASAT to
induce glutamine metabolism, which in turn fuels the TCA
cycle via α-KG (8). Here, to further investigate the enzymes
involved in WSSV-induced glutamine metabolism, we next
isolated and characterized 5 other important genes involved
in this pathway, LvGLS1, LvGLS2, LvIDH1, LvIDH2, and Lvα-
KGDH (Figure 5). The full-length or partial cDNAs of these 5
genes were identified from our in-house transcriptomic database,
and after amplification by PCR with the corresponding primer set
(Table 1), the amplicons were sequenced and confirmed to match
their respective genes (data not shown). Genes Involved in Oxidative Glutaminolysis
and Reductive Glutamine Metabolism Are
Important for WSSV Replication The above isotope-labeled tracing experiments were repeated
using [1-13C]glutamine instead of [U-13C]glutamine. With [1-
13C]glutamine, the first isotope-labeled carbon is lost during
oxidative conversion from α-KG to succinate, while this
same labeled carbon is kept in the metabolites produced by To further investigate the importance of GLS2, IDH1, IDH2, and
α-KGDH in WSSV replication, we performed dsRNA-mediated September 2019 | Volume 10 | Article 2102 Frontiers in Immunology | www.frontiersin.org 7 He et al. WSSV-Induced Glutamine Metabolism ncreased 13C-labeled metabolites in oxidative glutaminolysis and reductive carboxylation at the WSSV replication stage (12 hpi). At 12 h after
V or PBS, shrimps were injected with [U-13C]glutamine and hemocytes were collected (A) 10 min or (B) 30 min later. Metabolomic analysis of
mples by LC-ESI-Q-TOF-MS was used to calculate the fold change of each 13C metabolite in the WSSV group compared to the corresponding
e PBS group. Each bar represents the mean ± SD. Asterisks indicate statistically significant differences between the WSSV group and the
control group (*p < 0.05, **p < 0.01, ***p < 0.001). (C) Summary of changes in 13C glutamine metabolites in the oxidative and reductive
and 10 min after [U-13C]glutamine treatment. Changes in the WSSV group relative to the corresponding PBS control are color coded as follows:
crease), yellow (no significant difference), red (significant increase), and white (non-detectable). Abbreviations are the same as Figure 1. of these genes with the corresponding dsRNAs. GLS1 dsRNA could not be synthesized because
his gene has not yet been completely determined,
and we failed to find any primer set that could successfully
produce any PCR amplicons.) As Figure 7A shows, at 72 h-
post dsRNA treatment, although the gene expression of GLS2 URE 2 | WSSV increased 13C-labeled metabolites in oxidative glutaminolysis and reductive carboxylation at the WSSV replication stage (12 hpi). At 12 h after
enge with WSSV or PBS, shrimps were injected with [U-13C]glutamine and hemocytes were collected (A) 10 min or (B) 30 min later. Metabolomic analysis of
ed hemocyte samples by LC-ESI-Q-TOF-MS was used to calculate the fold change of each 13C metabolite in the WSSV group compared to the corresponding
metabolite in the PBS group. Each bar represents the mean ± SD. Asterisks indicate statistically significant differences between the WSSV group and the FIGURE 2 | WSSV increased 13C-labeled metabolites in oxidative glutaminolysis and reductive carboxylation at the WSSV replication stage (12 hpi). Genes Involved in Oxidative Glutaminolysis
and Reductive Glutamine Metabolism Are
Important for WSSV Replication At 12 h after
challenge with WSSV or PBS, shrimps were injected with [U-13C]glutamine and hemocytes were collected (A) 10 min or (B) 30 min later. Metabolomic analysis of
pooled hemocyte samples by LC-ESI-Q-TOF-MS was used to calculate the fold change of each 13C metabolite in the WSSV group compared to the corresponding
13C metabolite in the PBS group. Each bar represents the mean ± SD. Asterisks indicate statistically significant differences between the WSSV group and the
corresponding PBS control group (*p < 0.05, **p < 0.01, ***p < 0.001). (C) Summary of changes in 13C glutamine metabolites in the oxidative and reductive
directions at 12 hpi and 10 min after [U-13C]glutamine treatment. Changes in the WSSV group relative to the corresponding PBS control are color coded as follows:
green (significant decrease), yellow (no significant difference), red (significant increase), and white (non-detectable). Abbreviations are the same as Figure 1. FIGURE 2 | WSSV increased 13C-labeled metabolites in oxidative glutaminolysis and reductive carboxylation at the WSSV replication stage (12 hpi). At 12 h after
challenge with WSSV or PBS, shrimps were injected with [U-13C]glutamine and hemocytes were collected (A) 10 min or (B) 30 min later. Metabolomic analysis of
pooled hemocyte samples by LC-ESI-Q-TOF-MS was used to calculate the fold change of each 13C metabolite in the WSSV group compared to the corresponding
13C metabolite in the PBS group. Each bar represents the mean ± SD. Asterisks indicate statistically significant differences between the WSSV group and the
corresponding PBS control group (*p < 0.05, **p < 0.01, ***p < 0.001). (C) Summary of changes in 13C glutamine metabolites in the oxidative and reductive
directions at 12 hpi and 10 min after [U-13C]glutamine treatment. Changes in the WSSV group relative to the corresponding PBS control are color coded as follows:
green (significant decrease), yellow (no significant difference), red (significant increase), and white (non-detectable). Abbreviations are the same as Figure 1. in vivo silencing of these genes with the corresponding dsRNAs. (Unfortunately, GLS1 dsRNA could not be synthesized because
the sequence of this gene has not yet been completely determined, and we failed to find any primer set that could successfully
produce any PCR amplicons.) As Figure 7A shows, at 72 h-
post dsRNA treatment, although the gene expression of GLS2 in vivo silencing of these genes with the corresponding dsRNAs. Genes Involved in Oxidative Glutaminolysis
and Reductive Glutamine Metabolism Are
Important for WSSV Replication (Unfortunately, GLS1 dsRNA could not be synthesized because
the sequence of this gene has not yet been completely determined, September 2019 | Volume 10 | Article 2102 Frontiers in Immunology | www.frontiersin.org 8 He et al. He et al. WSSV-Induced Glutamine Metabolism suppressed oxidative glutaminolysis and reductive carboxylation at the late stage of WSSV replication (24 hpi). At 24 h after challenge with WSSV
ere injected with [U-13C]glutamine and hemocytes were collected (A) 10 min or (B) 30 min later. Metabolomic analysis of pooled hemocyte samples
MS was used to calculate the fold change of each 13C metabolite in the WSSV group compared to the corresponding 13C metabolite in the PBS
presents the mean ± SD. Asterisks indicate statistically significant differences between WSSV group and the corresponding PBS control group (*p
***p < 0.001). (C) Schematic representation of 13C metabolic expression of glutamine metabolism in oxidative and reductive directions at 24 hpi
3C]glutamine treatment. Changes in the WSSV group relative to the corresponding PBS control are color coded as follows: green (significant
o significant difference), red (significant increase), and white (non-detectable). Abbreviations are the same as for Figure 1. relative to PBS control, the gene expressions
and α-KGDH were all specifically decreased. point, the shrimp were then injected with
WSSV and hemocytes were collected 24 h later. We found that
although the mRNA expression of IDH1 and α-KGDH were
still significantly suppressed by the corresponding dsRNAs and FIGURE 3 | WSSV suppressed oxidative glutaminolysis and reductive carboxylation at the late stage of WSSV replication (24 hpi). At 24 h after challenge with WSSV
or PBS, shrimps were injected with [U-13C]glutamine and hemocytes were collected (A) 10 min or (B) 30 min later. Metabolomic analysis of pooled hemocyte samples
by LC-ESI-Q-TOF-MS was used to calculate the fold change of each 13C metabolite in the WSSV group compared to the corresponding 13C metabolite in the PBS
group. Each bar represents the mean ± SD. Asterisks indicate statistically significant differences between WSSV group and the corresponding PBS control group (*p
< 0.05, **p < 0.01, ***p < 0.001). (C) Schematic representation of 13C metabolic expression of glutamine metabolism in oxidative and reductive directions at 24 hpi
at 10 min after [U-13C]glutamine treatment. Changes in the WSSV group relative to the corresponding PBS control are color coded as follows: green (significant
decrease), yellow (no significant difference), red (significant increase), and white (non-detectable). Genes Involved in Oxidative Glutaminolysis
and Reductive Glutamine Metabolism Are
Important for WSSV Replication Abbreviations are the same as for Figure 1. FIGURE 3 | WSSV suppressed oxidative glutaminolysis and reductive carboxylation at the late stage of WSSV replication (24 hpi). At 24 h after challenge with WSSV
or PBS, shrimps were injected with [U-13C]glutamine and hemocytes were collected (A) 10 min or (B) 30 min later. Metabolomic analysis of pooled hemocyte samples
by LC-ESI-Q-TOF-MS was used to calculate the fold change of each 13C metabolite in the WSSV group compared to the corresponding 13C metabolite in the PBS
group. Each bar represents the mean ± SD. Asterisks indicate statistically significant differences between WSSV group and the corresponding PBS control group (*p
< 0.05, **p < 0.01, ***p < 0.001). (C) Schematic representation of 13C metabolic expression of glutamine metabolism in oxidative and reductive directions at 24 hpi
at 10 min after [U-13C]glutamine treatment. Changes in the WSSV group relative to the corresponding PBS control are color coded as follows: green (significant
decrease), yellow (no significant difference), red (significant increase), and white (non-detectable). Abbreviations are the same as for Figure 1. WSSV and hemocytes were collected 24 h later. We found that
although the mRNA expression of IDH1 and α-KGDH were
still significantly suppressed by the corresponding dsRNAs and was unchanged relative to PBS control, the gene expressions
of IDH1, IDH2, and α-KGDH were all specifically decreased. After this time point, the shrimp were then injected with September 2019 | Volume 10 | Article 2102 Frontiers in Immunology | www.frontiersin.org 9 He et al. WSSV-Induced Glutamine Metabolism RE 4 | WSSV activates reductive carboxylation at the WSSV genome replication stage (12 hpi) and suppresses it at the late stage of WSSV replication (24 hpi). h after challenge with WSSV or PBS, shrimps were injected with [1-13C]glutamine and hemocytes were collected (A) 10 min or (B) 30 min later. At 24 h after
enge with WSSV or PBS, shrimps were also injected with [1-13C]glutamine and hemocytes were collected (C) 10 min or (D) 30 min later. Metabolomic analysis of
d hemocyte samples by LC-ESI-Q-TOF-MS was used to calculate the fold change of each 13C metabolite in the WSSV group compared to the corresponding
metabolite in the PBS group. Each bar represents the mean ± SD. Asterisks indicate statistically significant differences between WSSV group and the
sponding PBS control group (*p < 0.05, **p < 0.01, ***p < 0.001). Genes Involved in Oxidative Glutaminolysis
and Reductive Glutamine Metabolism Are
Important for WSSV Replication WSSV-Induced Glutamine Metabolism He et al. FIGURE 5 | Simplified schematic of oxidative and reductive glutamine metabolic pathways. Glutamine metabolism in the oxidative direction (glutaminolysis) is shown
in blue, glutamine metabolism in the reductive direction (reductive carboxylation) is shown in red, and shared metabolic pathways are shown in black. Enzymes: GLS,
glutaminase; GDH, glutamine dehydrogenase; ASAT, aspartate aminotransferase; α-KG, α-ketoglutarate; IDH, isocitrate dehydrogenase; α-KGDH, α-ketoglutarate
dehydrogenase. Metabolites: abbreviations are the same as for Figure 1. FIGURE 5 | Simplified schematic of oxidative and reductive glutamine metabolic pathways. Glutamine metabolism in the oxidative direction (glutaminolysis) is shown
in blue, glutamine metabolism in the reductive direction (reductive carboxylation) is shown in red, and shared metabolic pathways are shown in black. Enzymes: GLS,
glutaminase; GDH, glutamine dehydrogenase; ASAT, aspartate aminotransferase; α-KG, α-ketoglutarate; IDH, isocitrate dehydrogenase; α-KGDH, α-ketoglutarate
dehydrogenase. Metabolites: abbreviations are the same as for Figure 1. the mRNA level of GDH, but also drives an increase in reductive
glutamine metabolism by increasing the mRNA levels of IDH2 at
the WSSV genome replication stage. that GLS2 was also suppressed at this time, the mRNA level of
IDH2 was not significantly different from the PBS or Luciferase
controls (Figure 7B). We further found that there was significant
suppression of WSSV VP28 mRNA expression and WSSV viral
copy numbers in the WSSV-injected groups pretreated with
IDH1, IDH2, and α-KGDH dsRNA, while in the GLS2 group,
VP28 mRNA was significantly suppressed even though the viral
copy number was not (Figures 7C,D). Taken together, these data
suggest that IDH1, IDH2, α-KGDH, and to a lesser extent, GLS
might all be important for WSSV replication. To investigate the role of the PI3K-Akt-mTOR pathway,
shrimps were pretreated with LY294002 to inhibit both PI3K and
mTORC1. In LY294002-treated shrimps, at both 12 and 24 hpi,
there was no significant difference in the mRNA levels of IDH1,
IDH2, or α-KGDH compared to the control group (Figure 8C). In shrimps treated with Torin1, which was used to inhibit both
of the mTOR complexes, there was no impact on any of the
three enzymes at 12 hpi, whereas, at 24 hpi, gene expression
of IDH1 decreased while IDH2 and α-KGDH mRNA increased
(Figure 8D). Taken together, these results suggest that at 12 hpi,
glutamine metabolism is regulated by Ras but not by the PI3K-
Akt-mTOR pathway. Genes Involved in Oxidative Glutaminolysis
and Reductive Glutamine Metabolism Are
Important for WSSV Replication (E,F) Schematic representation of 13C metabolic expression of glutamine metabolism in the
ctive direction at (E) 12 and (F) 24 hpi at 10 min after [1-13C]glutamine treatment. Changes in the WSSV groups relative to the corresponding PBS controls are
coded as follows: green (significant decrease), yellow (no significant difference), red (significant increase), gray (not measured), and white (non-detectable). eviations are the same as for Figure 1. FIGURE 4 | WSSV activates reductive carboxylation at the WSSV genome replication stage (12 hpi) and suppresses it at the late stage of WSSV replication (24 hpi). At 12 h after challenge with WSSV or PBS, shrimps were injected with [1-13C]glutamine and hemocytes were collected (A) 10 min or (B) 30 min later. At 24 h after
challenge with WSSV or PBS, shrimps were also injected with [1-13C]glutamine and hemocytes were collected (C) 10 min or (D) 30 min later. Metabolomic analysis of
pooled hemocyte samples by LC-ESI-Q-TOF-MS was used to calculate the fold change of each 13C metabolite in the WSSV group compared to the corresponding FIGURE 4 | WSSV activates reductive carboxylation at the WSSV genome replication stage (12 hpi) and suppresses it at the late stage of WSSV replication (24 hpi). At 12 h after challenge with WSSV or PBS, shrimps were injected with [1-13C]glutamine and hemocytes were collected (A) 10 min or (B) 30 min later. At 24 h after
challenge with WSSV or PBS, shrimps were also injected with [1-13C]glutamine and hemocytes were collected (C) 10 min or (D) 30 min later. Metabolomic analysis of
pooled hemocyte samples by LC-ESI-Q-TOF-MS was used to calculate the fold change of each 13C metabolite in the WSSV group compared to the corresponding
13C metabolite in the PBS group. Each bar represents the mean ± SD. Asterisks indicate statistically significant differences between WSSV group and the
corresponding PBS control group (*p < 0.05, **p < 0.01, ***p < 0.001). (E,F) Schematic representation of 13C metabolic expression of glutamine metabolism in the
reductive direction at (E) 12 and (F) 24 hpi at 10 min after [1-13C]glutamine treatment. Changes in the WSSV groups relative to the corresponding PBS controls are
color coded as follows: green (significant decrease), yellow (no significant difference), red (significant increase), gray (not measured), and white (non-detectable). Abbreviations are the same as for Figure 1. September 2019 | Volume 10 | Article 2102 10 Frontiers in Immunology | www.frontiersin.org He et al. Genome Replication Stage Previous reports have suggested that the PI3K-Akt-mTOR
pathway is involved in triggering both the WSSV-induced
Warburg effect at 12 hpi (9) and WSSV-induced lipogenesis at 24
hpi (7). Meanwhile, mTORC2 is a key regulator for upregulating
the expression of GDH mRNA independently of PI3K-mTORC1
at 12 hpi (8), while LvRas has been shown to play a positive role
in triggering WSSV-induced PI3K-Akt-mTOR activation (19). To investigate which of these pathways might regulate oxidative
and reductive glutamine metabolism during WSSV infection,
shrimp were treated with three specific inhibitors (Figure 8A)
and the mRNA expression of key genes involved in glutamine
metabolism was monitored. Frontiers in Immunology | www.frontiersin.org Genes Involved in Oxidative Glutaminolysis
and Reductive Glutamine Metabolism Are
Important for WSSV Replication Meanwhile, at 24 hpi, mTORC2 is pre-
dominantly responsible for the down-regulation of glutamine
metabolism, and it acts independently of Ras and the PI3K-
mTORC1 pathway. DISCUSSION We have shown here that, as in other cancer or virally infected
cells that are in a state of aerobic glycolysis (10, 22, 23), both
oxidative glutaminolysis and reductive carboxylation have been
triggered in WSSV-infected shrimp hemocytes (Figures 2–4). The present results are mostly consistent with previous findings
for WSSV-induced glutaminolysis. For example, the significantly
increased levels of M4 citrate in Figure 2A can be accounted for
by the upregulation of citrate synthase at the protein level, as
reported by Su et al. (9). Further, the patterns of changes in the
levels of various metabolites suggest that these pathways might be
more active at the genome replication stage (12 hpi) than in the
late stage (24 hpi). This conclusion is further supported by our In shrimps treated with Salirasib, a Ras inhibitor which
disrupts the spatiotemporal localization of active Ras (21), the
gene expression of GDH and IDH2 was significantly decreased
at 12 hpi (Figure 8B). At the same time, IDH1 mRNA was
significantly increased. At 24 hpi, inhibition of Ras caused no
change in the mRNA levels of these glutamine metabolism-
related genes. These data suggest that Ras not only upregulates September 2019 | Volume 10 | Article 2102 Frontiers in Immunology | www.frontiersin.org 11 WSSV-Induced Glutamine Metabolism He et al. He et al. FIGURE 6 | WSSV induces IDH and α-KGDH expression at the WSSV replication stage (12 hpi) in shrimp hemocytes. (A) The mRNA levels of GLS1 and GLS2 and
the GLS enzyme activity in shrimp hemocytes and stomachs during WSSV infection. (B) The mRNA levels of IDH1 and IDH2 and the IDH enzyme activity in shrimp
hemocytes and stomachs during WSSV infection. (C) The mRNA level and the enzyme activity of α-KGDH in shrimp hemocytes and stomachs during WSSV infection. Each bar represents the mean ± SD. Asterisks indicate statistically significant differences between the WSSV group and the corresponding PBS control group (*p <
0.05, **p < 0.01). FIGURE 6 | WSSV induces IDH and α-KGDH expression at the WSSV replication stage (12 hpi) in shrimp hemocytes. (A) The mRNA levels of GLS1 and GLS2 and
the GLS enzyme activity in shrimp hemocytes and stomachs during WSSV infection. (B) The mRNA levels of IDH1 and IDH2 and the IDH enzyme activity in shrimp
hemocytes and stomachs during WSSV infection. September 2019 | Volume 10 | Article 2102 Frontiers in Immunology | www.frontiersin.org DISCUSSION (C) The mRNA level and the enzyme activity of α-KGDH in shrimp hemocytes and stomachs during WSSV infection. Each bar represents the mean ± SD. Asterisks indicate statistically significant differences between the WSSV group and the corresponding PBS control group (*p <
0.05, **p < 0.01). WSSV replication was shown by the significant reduction in
VP28 mRNA and viral genome copies when α-KGDH expression
was silenced by dsRNA (Figures 7C,D). We note too that since
enzymes such as IDH1 and α-KGDH appear to be necessary for
the virus to replicate, they might also be useful as biomarkers for
developing disease-resistant shrimp. dsRNA silencing results for several key enzymes. In particular,
we found that in L. vannamei hemocytes, IDH activity was
induced at 12 hpi, although only cytoplasmic IDH1 showed an
increase in mRNA expression, while the mitochondrial IDH2
was unchanged (Figure 6). Shrimp IDH1 silencing also led
to stronger suppression of the WSSV genome copy number
(Figure 7). From this we infer that cytoplasmic IDH1 is likely
to play a relatively more important role in triggering WSSV-
induced reductive carboxylation. We also note that IDH1 plays
a key role in NADPH production and acts as a potential regulator
of lipid metabolism in cancer cells and in virus-infected cells
with reductive metabolism (23, 24). At the same time (12 hpi),
there was also an increase in α-KGDH activity (Figure 6B),
suggesting that the TCA cycle was also upregulated in the
oxidative direction. Again, the importance of this enzyme to While oxidative glutamine metabolism provides an alternative
mechanism to produce ATP and, like glycolysis, results
in the accumulation of lactate as an end product, the
reductive carboxylation pathway is important for providing the
macromolecular precursors of lipid synthase (18, 23, 24). In a
previous study, we found that lipogenesis, which is essential for
the creation of the lipid-containing fraction of the WSSV viral
envelope, was induced at 24 hpi (7). Here, however, our results
suggest that reductive carboxylation might be more active at 12 September 2019 | Volume 10 | Article 2102 Frontiers in Immunology | www.frontiersin.org 12 He et al. He et al. WSSV-Induced Glutamine Metabolism FIGURE 7 | IDH1, IDH2, α-KGDH, and to a lesser extent, GLS are involved in WSSV replication. DISCUSSION WSSV-Induced Glutamine Metabolism FIGURE 8 | Ras is a primary regulator of reductive glutamine metabolism at the WSSV genome replication stage. (A) Schematic representation of the Ras and
PI3K-Akt-mTOR pathways and the three inhibitors used in this experiment (black boxes). Shrimp were treated with the various inhibitors or vehicle only 2 h before
WSSV infection. (B) Real-time PCR analysis shows that the RAS inhibitor Salirasib had a significant effect on the expression of GDH, IDH1, and IDH2 in shrimp
hemocytes after WSSV infection. (C) Real-time PCR analysis shows that the PI3K-mTOR inhibitor LY29400 had no significant effect on the expression of IDH1, IDH2,
and α-KGDH in shrimp hemocytes after WSSV infection. (D) Real-time PCR analysis shows that the mTORC1/C2 inhibitor Torin 1 had a significant effect on the
expression of IDH1, IDH2, and α-KGDH in shrimp pleopods after WSSV infection. Bars represent the mean ± SD. Asterisks indicate statistically significant differences
in WSSV-injected shrimp between the inhibitor treated groups and the corresponding vehicle-only control (*p < 0.05, **p < 0.01). synthesis in WSSV-infected cells that are in a state of aerobic
We found in an earlier study that although WSSV stimulates FIGURE 8 | Ras is a primary regulator of reductive glutamine metabolism at the WSSV genome replication stage. (A) Schematic representation of the Ras and
PI3K-Akt-mTOR pathways and the three inhibitors used in this experiment (black boxes). Shrimp were treated with the various inhibitors or vehicle only 2 h before
WSSV infection. (B) Real-time PCR analysis shows that the RAS inhibitor Salirasib had a significant effect on the expression of GDH, IDH1, and IDH2 in shrimp
hemocytes after WSSV infection. (C) Real-time PCR analysis shows that the PI3K-mTOR inhibitor LY29400 had no significant effect on the expression of IDH1, IDH2,
and α-KGDH in shrimp hemocytes after WSSV infection. (D) Real-time PCR analysis shows that the mTORC1/C2 inhibitor Torin 1 had a significant effect on the
expression of IDH1, IDH2, and α-KGDH in shrimp pleopods after WSSV infection. Bars represent the mean ± SD. Asterisks indicate statistically significant differences
in WSSV-injected shrimp between the inhibitor treated groups and the corresponding vehicle-only control (*p < 0.05, **p < 0.01). FIGURE 8 | Ras is a primary regulator of reductive glutamine metabolism at the WSSV genome replication stage. (A) Schematic representation of the Ras and
PI3K-Akt-mTOR pathways and the three inhibitors used in this experiment (black boxes). DISCUSSION (A) Gene expression of GLS2, IDH1, IDH2, and α-KGDH in shrimp
hemocytes was analyzed by real-time PCR at 72 h-post injection of the corresponding dsRNA and before WSSV challenge. (B) Gene expression of the above genes
was measured again in dsRNA-treated shrimp at 24 h post WSSV injection. (C,D) The effect of gene silencing of GLS2, IDH1, IDH2, and α-KGDH on the expression
of the WSSV gene VP28 and WSSV genome copy numbers at 24 h post WSSV injection. Groups treated with PBS only or with non-specific luciferase (Luc) dsRNA
were used as control groups. Each bar represents the mean ± SD. Asterisks indicate statistically significant differences between the indicated groups (*p < 0.05,
**p < 0.01, ***p < 0.001). hpi rather than 24 hpi (Figures 2 4)
hich
o ld impl
that
follo ing M nger et al (25)
ho sho ed that HCMV
as able FIGURE 7 | IDH1, IDH2, α-KGDH, and to a lesser extent, GLS are involved in WSSV replication. (A) Gene expression of GLS2, IDH1, IDH2, and α-KGDH in shrimp
hemocytes was analyzed by real-time PCR at 72 h-post injection of the corresponding dsRNA and before WSSV challenge. (B) Gene expression of the above genes
was measured again in dsRNA-treated shrimp at 24 h post WSSV injection. (C,D) The effect of gene silencing of GLS2, IDH1, IDH2, and α-KGDH on the expression
of the WSSV gene VP28 and WSSV genome copy numbers at 24 h post WSSV injection. Groups treated with PBS only or with non-specific luciferase (Luc) dsRNA
were used as control groups. Each bar represents the mean ± SD. Asterisks indicate statistically significant differences between the indicated groups (*p < 0.05,
**p < 0.01, ***p < 0.001). following Munger et al. (25), who showed that HCMV was able
to divert glucose-derived carbon away from lactate and into
fatty acid synthesis, it is also possible that in the same way,
the glycolysis pathway might act as the carbon source for lipid hpi rather than 24 hpi (Figures 2–4), which would imply that
although lipogenesis might still occur at the late stage, the lipid
macromolecular precursors are in fact being produced at an
earlier time point than was previously proposed. Alternatively, September 2019 | Volume 10 | Article 2102 Frontiers in Immunology | www.frontiersin.org 13 He et al. He et al. DISCUSSION Further, although our experiments here
demonstrate that labeled glutamine can be used as the carbon
source, as noted above, our previous study found that WSSV-
induced glutaminolysis was driven by glutamate rather than
glutamine (8). This is consistent with the observed down-
regulation of GLS activity (Figure 6A), because although this
enzyme would still provide some of the carbon input, it would
no longer be essential for driving this metabolic pathway. Figure 9
provides
a
comprehensive
schematic
that
summarizes the increased mRNA levels of all of the above
enzymes in shrimp hemocytes at 12 hpi when the Warburg
effect is activated. The same figure also shows the larger context,
including the PI3K-Akt-mTOR pathway [by which the Warburg
effect is triggered; (9)] and the means by which WSSV is able to
replenish the TCA cycle by triggering glutamine metabolism, i.e.,
the Gln-Glu-α-KG pathway, oxidative glutamine metabolism
and reductive carboxylation. the WSSV-infected hemocytes. The first step in this metabolic
pathway is the conversion of glutamine to glutamate by GLS,
and it is interesting to note that while WSSV upregulates the
activity of both GDH and ASAT (8), we found here that the
mRNA expression levels of shrimp GLS were unchanged and its
enzyme activity was actually reduced (Figure 6A). This is also in
contrast to other similar instances of metabolic reprogramming
during aerobic glycolysis, such as HCMV for example, where
glutaminolysis is associated with the increased activity of both
GLS and GDH (26). In the case of WSSV, we therefore
hypothesize that the increased uptake of glutamine might instead
be driven by the high demand created by the conversion of
glutamate to α-KG. Further, although our experiments here
demonstrate that labeled glutamine can be used as the carbon
source, as noted above, our previous study found that WSSV-
induced glutaminolysis was driven by glutamate rather than
glutamine (8). This is consistent with the observed down-
regulation of GLS activity (Figure 6A), because although this
enzyme would still provide some of the carbon input, it would
no longer be essential for driving this metabolic pathway. In our previous research, we found that WSSV induces
glutamine metabolism independently of the PI3K-Akt-mTORC1
pathway and instead uses mTORC2 to up-regulate GDH
mRNA (8). DISCUSSION Shrimp were treated with the various inhibitors or vehicle only 2 h before
WSSV infection. (B) Real-time PCR analysis shows that the RAS inhibitor Salirasib had a significant effect on the expression of GDH, IDH1, and IDH2 in shrimp
hemocytes after WSSV infection. (C) Real-time PCR analysis shows that the PI3K-mTOR inhibitor LY29400 had no significant effect on the expression of IDH1, IDH2,
and α-KGDH in shrimp hemocytes after WSSV infection. (D) Real-time PCR analysis shows that the mTORC1/C2 inhibitor Torin 1 had a significant effect on the
expression of IDH1, IDH2, and α-KGDH in shrimp pleopods after WSSV infection. Bars represent the mean ± SD. Asterisks indicate statistically significant differences
in WSSV-injected shrimp between the inhibitor treated groups and the corresponding vehicle-only control (*p < 0.05, **p < 0.01). synthesis in WSSV-infected cells that are in a state of aerobic
glycolysis. Unfortunately, in the present study, we were unable
to resolve this question because the Ac-CoA metabolite could
not be detected, but we are currently working on a new set
of experiments that use 13C-labeled glucose to trace Ac-CoA’s
carbon source. We found in an earlier study that although WSSV stimulates
glutamine metabolism to fuel the TCA cycle via α-KG (8),
the carbon source that was taken up by the WSSV-infected
hemocytes was glutamate rather than glutamine. In the present
study, our stable isotope results (Figures 2–4) clearly show
that glutamine is also being taken up and metabolized by September 2019 | Volume 10 | Article 2102 Frontiers in Immunology | www.frontiersin.org 14 He et al. He et al. WSSV-Induced Glutamine Metabolism FIGURE 9 | Schematic representation of metabolism in WSSV-infected hemocytes showing the aerobic glycolysis, glutamine metabolism and lipogenesis pathways FIGURE 9 | Schematic representation of metabolism in WSSV-infected hemocytes showing the aerobic glycolysis, glutamine metabolism and lipogenesis pathways
at the WSSV genome replication stage (12 hpi). This figure was compiled from data obtained in the present study and previous studies (7–9, 19). Enzymes are shown
in boxes where red indicates an upregulated enzyme and yellow indicates the enzyme was unchanged. Oxidative glutamine metabolism and reductive carboxylation
are indicated by blue and red arrows, respectively. Green arrows represent aerobic glycolysis and the pentose phosphate pathway (PPP). WSSV-triggered lipogenesis,
which is triggered at the late stage, is shown in purple. Round boxes and gray arrows indicate enzyme activity and signaling pathways. Pyr, pyruvate; CS, citrate
synthase; FA, fatty acids. DISCUSSION All other abbreviations are the same as for Figure 1. FIGURE 9 | Schematic representation of metabolism in WSSV-infected hemocytes showing the aerobic glycolysis, glutamine metabolism and lipogenesis pathways
at the WSSV genome replication stage (12 hpi). This figure was compiled from data obtained in the present study and previous studies (7–9, 19). Enzymes are shown
in boxes where red indicates an upregulated enzyme and yellow indicates the enzyme was unchanged. Oxidative glutamine metabolism and reductive carboxylation
are indicated by blue and red arrows, respectively. Green arrows represent aerobic glycolysis and the pentose phosphate pathway (PPP). WSSV-triggered lipogenesis,
which is triggered at the late stage, is shown in purple. Round boxes and gray arrows indicate enzyme activity and signaling pathways. Pyr, pyruvate; CS, citrate
synthase; FA, fatty acids. All other abbreviations are the same as for Figure 1. FIGURE 9 | Schematic representation of metabolism in WSSV-infected hemocytes showing the aerobic glycolysis, glutamine metabolism and lipogenesis pathways
at the WSSV genome replication stage (12 hpi). This figure was compiled from data obtained in the present study and previous studies (7–9, 19). Enzymes are shown
in boxes where red indicates an upregulated enzyme and yellow indicates the enzyme was unchanged. Oxidative glutamine metabolism and reductive carboxylation
are indicated by blue and red arrows, respectively. Green arrows represent aerobic glycolysis and the pentose phosphate pathway (PPP). WSSV-triggered lipogenesis,
which is triggered at the late stage, is shown in purple. Round boxes and gray arrows indicate enzyme activity and signaling pathways. Pyr, pyruvate; CS, citrate
synthase; FA, fatty acids. All other abbreviations are the same as for Figure 1. the WSSV-infected hemocytes. The first step in this metabolic
pathway is the conversion of glutamine to glutamate by GLS,
and it is interesting to note that while WSSV upregulates the
activity of both GDH and ASAT (8), we found here that the
mRNA expression levels of shrimp GLS were unchanged and its
enzyme activity was actually reduced (Figure 6A). This is also in
contrast to other similar instances of metabolic reprogramming
during aerobic glycolysis, such as HCMV for example, where
glutaminolysis is associated with the increased activity of both
GLS and GDH (26). In the case of WSSV, we therefore
hypothesize that the increased uptake of glutamine might instead
be driven by the high demand created by the conversion of
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his helpful criticism of the manuscript. We thank Mr. Paul Barlow, National Cheng Kung University, for
his helpful criticism of the manuscript. FUNDING This study was supported financially by the Ministry of Science
and Technology (MOST 107-2313-B-006-006-MY3, MOST 107-
3017-F-006-001, MOST 108-2314-B-006-096-MY3, MOST 107-
2311-B-039-001, and CMU-107-N-16). DATA AVAILABILITY The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fimmu. 2019.02102/full#supplementary-material All datasets generated for this study are included in the
manuscript/Supplementary Files. DISCUSSION Here we further showed that although Ras can
sometimes act as an upstream regulator of the PI3K-Akt-
mTOR and Raf-MEK-ERK pathways (27, 28), in WSSV-infected
hemocytes, it acts independently to significantly upregulate
the gene expression of GDH (Figure 8B). Taken together,
it thus appears that Ras and mTORC2 may both regulate
the gene expression level of GDH. As shown in Figure 9,
however, their exact relationship remains unclear. Although September 2019 | Volume 10 | Article 2102 Frontiers in Immunology | www.frontiersin.org 15 He et al. WSSV-Induced Glutamine Metabolism AUTHOR CONTRIBUTIONS it is possible that Ras acts directly as a GDH regulator, in
Dictyostelium it promotes cell migration via the activation of
mTORC2 (29). mTORC2 has also been reported to encode a
Ras binding domain that may be correlated to Ras regulation
(30), and in cancer research, oncogenic Ras binds to mTORC2
and stimulates its activation to promote cell proliferation and
tumorigenesis (31). Further research will be needed to establish
whether or not the effect of Ras on GDH is mediated by
mTORC2 in a similar way. Lastly, there remaining the question
of which viral factors might be involved in this metabolic
reprogramming. In the case of dengue virus, which leads to
increased glycolysis in the host, Allonso et al. found that
viral NS1 protein interacts with glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) and upregulates its activity (32). To
address the question of how WSSV might regulate glutamine
metabolism, we are now working with a yeast two-hybridization
platform that has so far been used to select several viral factor
candidates that can interact with enzymes related to this pathway. We hope to further explore these potential mechanisms in a
future study. S-TH, C-YT, and C-YL designed and performed in vivo animal
experiments and analyzed data. D-YL performed LC-ESI-MS-
based isotopic labeled metabolomic analysis. S-TH, D-YL,
and H-CW wrote the manuscript. H-CW conceived the idea,
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conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. y
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essential for human cytomegalovirus infection. J Virol. (2010) 84:1867–73. doi: 10.1128/JVI.02123-09 Copyright © 2019 He, Lee, Tung, Li and Wang. This is an open-access article
distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the
original author(s) and the copyright owner(s) are credited and that the original
publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these
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19:25–31. doi: 10.1016/j.semcancer.2008.11.010 September 2019 | Volume 10 | Article 2102 Frontiers in Immunology | www.frontiersin.org 17
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https://openalex.org/W4377012299
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https://zenodo.org/records/7946614/files/Fazilova%20Nurxon.pdf
|
English
| null |
"LOST IN TRANSLATION: THE INFLUENCE OF CULTURE ON FILM TRANSLATION"
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Zenodo (CERN European Organization for Nuclear Research)
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cc-by
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"LOST IN TRANSLATION: THE INFLUENCE OF CULTURE ON FILM
TRANSLATION" https://doi.org/10.5281/zenodo.7946614 Fazilova Nurxon
Master's degree student in Uzbekistan World Languages University Key words. Humor, satire, idioms, cultural sensitivity and awareness, Cultural Differences in Film Translation. Abstract. Abstract. The film industry is an ever-growing global market, with movies being watched and
appreciated across the world. However, with diverse cultures and languages, translating
films for international audiences can be a challenging task. Culture plays a significant role
in the translation process, and understanding the influence of culture on film translation is
essential. This article aims to explore the impact of culture on film translation and how
cultural considerations affect the final product. Publishing centre of Finland Cultural Differences in Film Translation. Films reflect the values, beliefs, and social norms of the culture in which they
are created. These cultural differences can create challenges when translating films
for international audiences. For instance, humor and satire in films are often
culturally specific and do not always translate well into other languages. Translators must ensure that these elements are preserved and conveyed to the
target audience effectively. Similarly, cultural references, idioms, and expressions used in films can be
challenging to translate. The use of language in films can convey cultural values
and social norms, which can be lost in translation. For example, the film "Pulp
Fiction" uses extensive profanity, which is culturally acceptable in American society
but may not be suitable for all cultures. Translators must consider these cultural
differences and adjust the language to make it more acceptable for the target
audience. The cultural implications of a film's title are significant, as different cultures
have varying perceptions and connotations of certain words and phrases. For
example, the American film "The Shawshank Redemption" was translated into
French as "Les Évadés," which translates to "The Escaped." This title change was
necessary as the French language does not have an equivalent word for 778 Publishing centre of Finland International Journal of Education, Social Science & Humanities. Finland Academic Research Science Publishers
ISSN: 2945-4492 (online) | (SJIF) = 7.502 Impact factor
Volume-11| Issue-5| 2023 Published: |22-05-2023|
"Shawshank," and the concept of redemption is not as prominent in French culture. Thus, the French title better reflects the story's central theme of escape. International Journal of Education, Social Science & Humanities. Finland Academic Research Science Publishers
ISSN: 2945-4492 (online) | (SJIF) = 7.502 Impact factor
Volume-11| Issue-5| 2023 Published: |22-05-2023|
"Shawshank," and the concept of redemption is not as prominent in French culture. Thus, the French title better reflects the story's central theme of escape. "Shawshank," and the concept of redemption is not as prominent in French culture. Thus, the French title better reflects the story's central theme of escape. Another example is the film "The Hangover," which was translated into
Chinese as "Do Not Think About Tomorrow." This title change reflects the Chinese
cultural emphasis on moderation and responsible behavior, as the original title did
not resonate with Chinese audiences who may have viewed excessive drinking as
problematic behavior. Challenges in Film Translation: The translation of films requires not only linguistic expertise but also cultural
understanding. Cultural Differences in Film Translation. Translators must ensure that the translation accurately conveys the
original meaning and stays true to the film's cultural context. The challenge is to
preserve the cultural nuances and maintain the film's artistic integrity while
ensuring that it resonates with the target audience. Additionally, some languages have different sentence structures, word order,
and expressions that can make translations challenging. Translators must be aware
of these differences and ensure that the translation is grammatically correct and
flows naturally. The Importance of Cultural Considerations in Film Translation: Cultural considerations are essential in film translation to ensure that the
film's message and artistic value are conveyed to the target audience accurately. A
poorly translated film can affect the audience's perception of the film and impact its
commercial success. Cultural sensitivity and awareness are crucial for translators to
create a translation that accurately represents the film's cultural context. Conclusion: In conclusion, cultural differences play a significant role in film translation,
and understanding the impact of culture is crucial for successful film translation. Translators must navigate cultural nuances and linguistic differences to create a
translation that resonates with the target audience while preserving the film's
original message and artistic value. By considering cultural factors, translators can
create a high-quality film translation that enhances the film's appeal and
contributes to its success in the global market. Publishing centre of Finland 1.
Komisserov, V, Teaching Translation, Linguistica Pravda, Kiev. 1981
2.
Arnold I.V. The value of a strong position for the interpretation of a
literary text. Ц Foreign languages at school. 1978. Publishing centre of Finland International Journal of Education, Social Science & Humanities.
Finland Academic Research Science Publishers
ISSN: 2945-4492 (online) | (SJIF) = 7.502 Impact factor
Volume-11| Issue-5| 2023 Published: |22-05-2023|
3.
Ivanov P.V. Models of the translation feature films // Uch. Rec.
Leningrad, state, region. Zap them. AS Pushkin. St. Petersburg, 2005.
4.
http://www.llas.ac.uk/re/42
5.
https://www.researchgate.net/publication/313315503_THE_ISSUES_
ON_TRANSLATION_OF_FILM_TITLES_AND_PUBLICISTIC_HEADLINES5 LITERATURE: 1. Komisserov, V, Teaching Translation, Linguistica Pravda, Kiev. 1981
2. Arnold I.V. The value of a strong position for the interpretation of a
literary text. Ц Foreign languages at school. 1978. 779 Publishing centre of Finland International Journal of Education, Social Science & Humanities. Finland Academic Research Science Publishers
ISSN: 2945-4492 (online) | (SJIF) = 7.502 Impact factor
Volume-11| Issue-5| 2023 Published: |22-05-2023|
3. Ivanov P.V. Models of the translation feature films // Uch. Rec. Leningrad, state, region. Zap them. AS Pushkin. St. Petersburg, 2005. 4. http://www.llas.ac.uk/re/42
5. https://www.researchgate.net/publication/313315503_THE_ISSUES_
ON_TRANSLATION_OF_FILM_TITLES_AND_PUBLICISTIC_HEADLINES5 International Journal of Education, Social Science & Humanities. Finland Academic Research Science Publishers
ISSN: 2945-4492 (online) | (SJIF) = 7.502 Impact factor
Volume-11| Issue-5| 2023 Published: |22-05-2023|
3. Ivanov P.V. Models of the translation feature films // Uch. Rec. Leningrad, state, region. Zap them. AS Pushkin. St. Petersburg, 2005. 4. http://www.llas.ac.uk/re/42
5. https://www.researchgate.net/publication/313315503_THE_ISSUES_
ON_TRANSLATION_OF_FILM_TITLES_AND_PUBLICISTIC_HEADLINES5 5. https://www.researchgate.net/publication/313315503_THE_ISSUES_
ON_TRANSLATION_OF_FILM_TITLES_AND_PUBLICISTIC_HEADLINES5 780
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STATISTIK KO'RSATKICHLAR MOHIYATI VA TURLARI
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Baxshillayeva Shaxnoza Said qizi Toshkent davlat iqtisodiyot universiteti 2- kurs statistika ta’lim yo'nalishi talabasi. Annontatsiya:Ushbu maqolada statistik ko’rsatkichlar mohiyati va turlari haqida
fikr yuritilgan. Kalit so’zlar::Statistika,guruhlash,mohiyat,vazifa,tur,ma’lumot,jarayon. STEP KONFERENCE 2022 STEP KONFERENCE 2022 2022 Kalit so’zlar::Statistika,guruhlash,mohiyat,vazifa,tur,ma’lumot,jarayon. Statistika ijtimoiy – iqtisodiy hodisa va jarayonlarni o‘rganadi. Ijtimoiy voqea –
hodisalarga aholining soni, uning xarakati, sog‘ligi, ta’lim olishi, turmush darajasi
bilan bog‘liq jarayonlar kiradi. Iqtisodiy voqea – hodisalarga mahsulot ishlab
chiqarish, xizmat ko‘rsatish (sanoat, qishloq xo‘jaligi, qurilish, transport, savdo,
sog‘liqni saqlash va shu kabi tarmoqlarda sodir bo‘layotgan jarayonlar), tayyor
mahsulotlarni taqsimlash, ayirboshlash va iste’mol qilish bilan bog‘liq jarayonlar
kiradi. Ijtimoiy – iqtisodiy voqea – hodisalar holati va harakati ma’lum
ko‘rsatkichlarda ifodalanadi. Statistik ko‘rsatkichlar ijtimoiy – iqtisodiy voqea – hodisalarning miqdoriy
tomonlarini ularning sifat mohiyati bilan uzviy bog‘liqlikda ifodalaydi. Voqea –
hodisalarning miqdoriy tomoni deganda, ularning soni, hajmi, o‘lchami, qiymati
kabi miqdorlar tushuniladi. Sifat esa voqea – hodisalarning ichki qiyofasi,
mohiyatidan kelib chiqadi. Statistikada ko‘rsatkichlar miqdoriy va sifat tomondan
chambarchas bog‘liqlikda o‘rganiladi. Ijtimoiy – iqtisodiy voqea – hodisalar
ma’lum makon va zamonda sodir bo‘ladi. 1 STEP KONFERENCE 2022 Shuning uchun ham statistik ko‘rsatkichlar ularni makon (korxona, firma,
birlashma, tuman va mamlakat miqyosida) va davrlar (kunlar, oylar, choraklar, yil
va yillar) bo‘yicha o‘rganadi. Demak, statistik ko‘rsatkichlar deb, ommaviy
ijtimoiy – iqtisodiy jarayonlarning ma’lum makon va zamondagi holatini,
xarakatini, rivojlanishini, tarkibiy tuzilishini ifodalovchi me’yorlarga aytiladi. Ijtimoiy – iqtisodiy voqea – hodisalar holati va xarakati, ularni kuzatishdan
(o‘rganishdan) olingan miqdorlar (raqamlar) bilan ifodalanadi. Statistik ko‘rsatkichlarning turlari hodisalar kabi ko‘p bo‘lganligi sababli, ularni
biror belgilari bo‘yicha tasniflash lozim bo‘ladi. Statistik ko‘rsatkichlar voqea –
hodisalar to‘plamini qamrab olishiga qarab, makroiqtisodiy va mikroiqtisodiy
ko‘rsatkichlarga guruhlanadi. Mikroiqtisodiy ko‘rsatkichlar faoliyat yurutuvchi
sub’ektlarda (firma, korxona ularning bo‘limlari, birlashmalar, koorporatsiyalar va
shu
kabilar)
sodir
bo‘layotgan
jarayonlarni
ifodalaydi. Makroiqtisodiy
ko‘rsatkichlar mamlakat yoki uning hududlaridagi ijtimoiy – iqtisodiy jarayonlar
holati va xarakatini ifodalaydi. Angren ko‘mir konida qazilgan ko‘mir, biror
fermer xo‘jaligida terilgan paxta, biror korxonada ishlayotgan xodimlar soni va shu
kabilar mikroiqtisodiy ko‘rsatkichlardir. Mamlakat miqyosida qazib olingan
ko‘mir, viloyat yoki mamlakat miqyosida tayyorlangan paxta, ishlayotgan
xodimlar soni va shu kabilar makroiqtisodiy ko‘rsatkichlardir. Statistik
ko‘rsatkichlar miqdori raqamlar bilan ifodalanadi. Miqdorlar mohiyatiga ko‘ra mutloq, nisbiy va o‘rtacha miqdorlarga bo‘lishi
mumkin. Ularning mohiyati keyingi savol va mavzularda yoritiladi. Statistik
ko‘rsatkichlar
olish
usuliga
qarab birlamchi va
ikkilamchi
(hosilaviy)
ko‘rsatkichlarga guruhlanadi. Birlamchi ko‘rsatkichlar hodisa va jarayonlarni
kuzatishdan olingan, hamda ular asosida jamg‘arilgan ko‘rsatkichlardir. Ikkilamchi
(hosilaviy) ko‘rsatkichlar birlamchi ko‘rsatkichlar asosida matematik amallarni
qo‘llab topilgan, hosil bo‘lgan ko‘rsatkichlardir. Mehnat unumdorligi, hosildorlik, 2 2 2 STEP KONFERENCE 2022 STEP KONFERENCE 2022 korxona
(firma)
rentabelligi
kabi
ko‘rsatkichlar
ikkilamchi
(hosilaviy)
ko‘rsatkichlarga misol bo‘ladi. korxona
(firma)
rentabelligi
kabi
ko‘rsatkichlar
ikkilamchi
(hosilaviy)
ko‘rsatkichlarga misol bo‘ladi. Demak, statistik ko‘rsatkichlarda: ko‘rsatkichlarning nomi (voqea – hodisalar,
ob’yektning nomi), ularning qachon, qayerda bo‘lganligi, o‘lchov birligi va
raqamda ifodalangan voqea – hodisaning miqdori bo‘lar ekan. Iqtisodchi mutaxassislar bu raqamlarni o‘qib, ularni tilga kiritib, mohiyatiga
tushunib, hodisa va jarayonlarning holatiga va davr mobaynida qanday
o‘zgarganligiga baho berar ekan.Ijtimoiy va iqtisodiy hodisalar, jarayonlar tegishli
ko‘rsatkichlarda ifodalanadi. Iqtisodchi mutaxassislar bu raqamlarni o‘qib, ularni tilga kiritib, mohiyatiga
tushunib, hodisa va jarayonlarning holatiga va davr mobaynida qanday
o‘zgarganligiga baho berar ekan.Ijtimoiy va iqtisodiy hodisalar, jarayonlar tegishli
ko‘rsatkichlarda ifodalanadi. Bu ko‘rsatkichlar turli xil miqdorlarda, ma’lumotlarda berilib, hujjatlarda qayd
qilinadi. Statistik kuzatish natijasida miqdorlar yig‘ilib, tegishli ko‘rsatkichlar
bo‘yicha guruhlanib, o‘rganiladi. Miqdorlarda ifodalangan iqtisodiy va ijtimoiy
ko‘rsatkichlarni umumlashtirib , tegishli xulosalar chiqarish statistikaning asosiy
vazifasidir. Xodisa va jarayonlar dastlab mutloq miqdorlarda qayd qilinadi, so‘ngra
esa ular asosida nisbiy va o‘rtacha miqdorlar hisoblab topiladi. Demak mutloq
miqdorlar har qanday statistik ko‘rsatkichlarning dastlabki (boshlang‘ich)
shaklidir. Miqdorlar qo‘shish, ayirish, o‘lchash kabi amallarni bajarish bilan
aniqlanadi. Aholining umumiy soni, shu jumladan erkaklar va ayollar soni,
korxonalarda xodimlar soni, ishlab chiqarilgan stanoklar soni - “bir”, ”ikki”, “uch”
kabi sonlarda qayd qilinadi. Ularning to‘plamlari ming kishi, ming dona kabi son
ko‘rsatkichlarda ifodalanadi. Qurilgan yo‘llar uzunligi, daryolarning uzunligi,
bosib o‘tilgan yo‘l - metr, kilometr kabi uzunlik o‘lchovi birliklari bilan
ifodalanadi. Murakkab xodisalarni ifodalovchi ko‘rsatkichlar kompleks o‘lchov birliklarida
ifodalanadi. Bunday hodisalar ikki va undan ortiq o‘lchov birliklarining o‘zaro
birikmasi bilan tavsiflanadi. Masalan: tashilgan yuk - tonna/ kilometrda, ishlab 3 udayberdiev U.X. Statistika. Ma’ruza matnlari. Samarqand, SamISI, 2011 STEP KONFERENCE 2022 STEP KONFERENCE 2022 chiqarilgan elektr quvvati kilovatt - soatda, xodimlarning ishlagan ish vaqtlari -
kishi / soatda. Lekin natura o‘lchovida xususiyatlari bilan har xil bo‘lgan hodisa va
jarayonlar umumlashtirib ko‘rsatilmaydi. Shuning uchun ham qiymat (pul)
o‘lchovi ishlatiladi. Barcha hodisa va jarayonlarni pul o‘lchovida ya’ni yagona
o‘lchovda umumlashtirib ko‘rsatish mumkin. FOYDALAANILGAN ADABIYOTLAR RO’YXATI: 1.Теория статистики. Учебник. Под ред. Р.А.Шмойловой. – М.: Финансы и
статистика, 2005. 1.Теория статистики. Учебник. Под ред. Р.А.Шмойловой. – М.: Финансы и
статистика, 2005. 2.Tuxliyev N. va boshqalar. O‘zbekiston iqtisodiyoti asoslari. T.: “O‘zbekiston
milliy ensiklopediyasi”, 2006. 2.Tuxliyev N. va boshqalar. O‘zbekiston iqtisodiyoti asoslari. T.: “O‘zbekiston
milliy ensiklopediyasi”, 2006. 3. XudayberdievU.X., Xоliqulоv A.N., TurdibekоvYU.I. Statistika. Oquv
uslubiyqollanma. Samarqand, SamISI, 2012. 4. Xudayberdiev U.X., Xоliqulоv A.N., Bаbаnаzаrоvа S.А. Statistika. Oquv
uslubiyqollanma. (2-qism) Samarqand, SamISI, 2014. 5.Xudayberdiev U.X. Statistika. Ma’ruza matnlari. Samarqand, SamISI, 2011 5.Xudayberdiev U.X. Statistika. Ma’ruza matnlari. Samarqand, SamISI, 2011
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Newman Error Analysis Siswa Madrasah dalam Menyelesaikan Soal Cerita Matematika
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Jurnal cendekia : jurnal pendidikan matematika/Jurnal Cendekia
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E-ISSN : 2579-9258
P-ISSN : 2614-3038 E-ISSN : 2579-9258
P-ISSN : 2614-3038 E-ISSN : 2579-9258
P-ISSN : 2614-3038 Journal Cendekia: Jurnal Pendidikan Matematika
Volume 03, No. 02, Agustus 2019, pp. 379-388 Abstract Students’ mistakes in solving the mathematical word problem is still a problem so it must be identified for each
stage of solving. It is done so that the solutions offered are more effective. One way to identify the stage of
solving of mathematical problem is the Newman Error Analysis stage. So the purpose of this research is to
describe the ability of students of Islamic Junior High School to solve mathematical problems in the form of
word problem and to describe the mistakes of high-ability Islamic High School students in mathematical word
problem based on Newman Error Analysis. The approach used is qualitative descriptive. The research subject is
the high-ability in Islamic Junior High School of Jambi City. Students are asked to work on solving questions in
the form of word problems. Then the student interviewed about the mistakes that were made when solving the
word problems given for each of the stages. The results showed 56% of high-ability students encountered an
error while solving the algebra operation and 44% of high-ability students encountered an error while solving
Pythagoras theorem. As for the errors that occur in high-ability students if in the analysis based on the stage of
Newman Error Analysis occurs at the stage of understanding the problem (comprehension) and the problem
transformation (transformation). Of course, errors in the comprehension and transformation cause errors at a
later stage so that the solution or answer found is worth wrong. g
g
Keywords: Error, Newman Error Analysis, Mathematical Word Problem. NEWMAN ERROR ANALYSIS SISWA MADRASAH DALAM
MENYELESAIKAN SOAL CERITA MATEMATIKA Marni Zulyanty
haha Saifuddin Jambi, Jln. Jambi Ma. Bulian KM 16 Sei. Duren Jambi
marnizulyanty@uinjambi.ac.id Marni Zulyanty
Universitas Islam Negeri Sulthan Thaha Saifuddin Jambi, Jln. Jambi Ma. Bulian KM 16 Sei. Duren Jambi
marnizulyanty@uinjambi.ac.id Abstrak Kesalahan siswa dalam menyelesaikan soal cerita matematika masih menjadi masalah sehingga harus di
identifikasi untuk setiap tahapan penyelesaiannya. Hal ini dilakukan agar solusi yang ditawarkan menjadi lebih
efektif. Salah satu cara mengidentifikasi tahapan penyelesaian soal cerita matematika adalah tahapan Newman
Error Analysis. Sehingga tujuan dari penelitian ini adalah mendeskripsikan kemampuan siswa madrasah dalam
memecahkan masalah matematika dalam bentuk soal cerita dan mendeskripsikan kesalahan siswa madrasah
berkemampuan tinggi dalam menyelesaikan soal cerita matematika berdasarkan Newman Error Analysis. Pendekatan yang digunakan adalah kualitatif deskriptif. Adapun subjek penelitian adalah siswa Madrasah
Tsanawiyah Negeri Kota Jambi yang berkemampuan tinggi. Siswa diminta mengerjakan lembar soal pemecahan
masalah dalam bentuk soal cerita. Kemudian siswa diwawancara seputar kesalahan yang dilakukan saat
menyelesaikan soal cerita yang diberikan untuk setiap tahapannya. Hasil penelitian menunjukkan 56% siswa
kemampuan tinggi mengalami kesalahan saat menyelesaikan soal materi operasi aljabar dan 44% siswa
kemampuan tinggi mengalami kesalahan saat menyelesaikan soal materi teorema Pythagoras. Adapun kesalahan
yang terjadi pada siswa kemampuan tinggi jika di analisis berdasarkan tahapan Newman Error Analysis terjadi
pada tahap memahami masalah (comprehension) dan tahap transformasi masalah (transformation). Tentunya
kesalahan pada tahap comprehension dan transformation ini menyebabkan kesalahan pada tahap selanjutnya
sehingga solusi atau jawaban yang ditemukan bernilai salah. Kata kunci: Kesalahan, Analisis Kesalahan Newman, Soal Cerita Matematika. Matematika sangat erat dengan kehidupan sehari-hari. Bahkan matematika diajarkan kepada manusia
baik secara formal melalui sekolah dan secara tidak formal melalui kehidupan. Di jenjang sekolah,
matematika adalah salah satu mata pelajaran yang dipelajari siswa mulai dari jenjang terendah sampai
tertinggi. Namun faktanya banyak kesulitan yang dialami siswa dalam belajar matematika. Salah satu
aspek kesulitan yang dialami siswa dalam belajar matematika adalah dalam memecahkan masalah
matematika. Faktanya memecahkan masalah adalah tujuan yang paling penting dalam pembelajaran
matematika (B, Sahar & AT, Rohani, 2010), dan untuk meningkatkan kemampuan memecahkan
masalah digunakan masalah matematika (Adebola & Sakiru, 2012). Selain itu, menurut NCTM (2000) terdapat lima standar proses dalam pembelajaran 379 Jurnal Cendekia: Jurnal Pendidikan Matematika, Volume 03, No. 02, Agustus 2019, pp. 379-388 380 matematika, dan salah satunya adalah memecahkan masalah matematika (mathematical problem
solving). Namun faktanya kemampuan siswa Indonesia dalam memecahkan masalah masih rendah jika
dibandingkan dengan negara lain. Oleh karenanya perlu adanya peningkatan kemampuan pemecahan
masalah siswa di Indonesia. Bentuk dari soal pemecahan masalah matematika adalah soal cerita
matematika (D, Setyono & S, Sutarni, 2013). Masalah matematika dapat berupa soal non rutin yaitu
soal cerita yang membutuhkan penyelesaian yang melibatkan tingkat penalaran lebih lanjut. Abstrak Mengetahui letak dan bentuk kesalahan siswa dalam menyelesaikan soal cerita akan dapat
membantu guru dalam meningkatkan kemampuan pemecahan masalah siswa. Untuk mengetahui letak
dan bentuk kesalahan tersebut, dapat dilakukan dengan menganalisis kesalahan siswa dengan
menggunakan Newman Error Analysis. Newman Error Analysis dipilih karena memiliki kredibilitas
yang tinggi (A.L. White, 2005). Menurut A.L. White (2010) terdapat 5 tahap dalam Newman Error
Analysis, yaitu membaca masalah (reading), memahami masalah (comprehension), transformasi
masalah (transformation), keterampilan proses (process skill), dan penulisan jawaban akhir (encoding). Diharapkan dengan menggunakan Newman Error Analysis letak dan bentuk kesalahan siswa dalam
menyelesaikan soal cerita tergambar jelas dan detail. Penelitian sebelumnya yang relevan dilakukan oleh Tello, E.A (2010) menunjukkan bahwa
siswa dapat sukses dalam problem solving dengan membuat keputusan yang berhubungan dengan
informasi dari masalah yang diberikan, memilih pendekatan yang dibutuhkan untuk membantu
memecahkan masalah dan akhirnya menemukan solusi dari masalah. Sehingga proses yang dilakukan
untuk memecahkan masalah tidak hanya membuat model matematika dari masalah yang diberikan
melainkan menemukan solusi dari masalah. Selain itu hasil penelitian Sajadi M, Amiripour P, & Malkhalifeh M.R (2013) menunjukkan
bahwa Problem solving adalah suatu proses yang lebih daripada sekedar membuat model matematika
dari suatu situasi. Situasi yang dimaksud adalah masalah atau persoalan yang penyelesaianya
berhubungan dengan matematika. Dan oleh karenanya dalam pembelajaran matematika Problem
solving ini perlu diajarkan dan dilatih kepada siswa. Berdasarkan uraian di atas, maka tujuan penelitian ini adalah mendeskripsikan kemampuan
siswa madrasah dalam memecahkan masalah matematika dalam bentuk soal cerita dan mendeskripsikan
kesalahan siswa madrasah berkemampuan tinggi dalam menyelesaikan soal cerita matematika
berdasarkan Newman Error Analysis. METODE Penelitian ini menggunakan pendekatan kualitatif dengan jenis penelitian deskriptif. Pendekatan kualitatif ditujukan untuk mengungkapkan suatu masalah dan mengembangkannya secara
detail untuk memahami pusat fenomena dari suatu masalah (Creswell, 2012). Adapun subjek penelitian
adalah siswa Madrasah Tsanawiyah Negeri yang ada di Kota Jambi yang berkemampuan tinggi. Karena
penelitian ini menggunakan pendekatan penelitian kualitatif maka instrumen pengumpulan data terbagi Newman Error Analysis Siswa Madrasah dalam Menyelesaikan Soal Cerita Matematika, Marni Zulyanty 381 dua yaitu instrumen utama yakni peneliti sendiri dan instrumen pendukung yakni lembar soal
pemecahan masalah matematika dan pedoman wawancara. Sementara analisis data dalam penelitian ini
menggunakan analisis data Creswell yang terdiri atas enam tahap yakni (1) menyiapkan dan
mengumpulkan data untuk dianalisis, (2) mengembangkan dan mengkode data, (3) membuat kode
berdasarkan deskripsi-deskripsi, (4) menyajikan dan melaporkan hasil yang ditemukan, (5)
menginterpretasikan hasil yang ditemukan, (6) memvalidasi keakuratan dari hasil yang ditemukan. Hasil Pengerjaan Lembar Soal Pemecahan Masalah Matematika Hasil pengerjaan lembar soal pemecahan masalah ini terdiri dari lima kriteria penilaian, yaitu benar
secara utuh (B), benar tapi tidak menemukan solusi (BTS), salah secara utuh (S), salah langkah
pengerjaan (SL), dan tidak jawab (TJ). Adapun hasil pengerjaan lembar soal pemecahan masalah
matematika siswa seperti pada tabel 1 berikut. HASIL Instumen lembar soal pemecahan masalah matematika digunakan untuk melihat kemampuan
siswa dalam memecahkan masalah matematika. Selain itu lembar soal juga dapat memberikan data
kesalahan yang dilakukan siswa dalam memecahkan masalah matematika. Lembar soal pemecahan
masalah matematika ini disajikan dalam bentuk soal cerita dan disusun pada materi operasi aljabar dan
aplikasi Pythagoras. Masalah operasi aljabar dan masalah aplikasi Pythagoras yang diberikan ini
merupakan soal yang sesuai dengan Kompetensi Inti ranah keterampilan (KI-4) karena sudah
memerlukan strategi dan pola pikir panjang dalam menyelesaikan masalah tersebut. Selanjutnya
instrumen pedoman wawancara berguna untuk membimbing peneliti dalam memastikan terjadinya
tahapan Newman Error Analysis saat siswa memecahkan masalah matematika yang diberikan. Instumen
pedoman wawancara ini disusun berdasarkan tahapan Newman Error Analysis dalam memecahkan
masalah matematika. Penelitian ini dilakukan di Madrasah Tsanawiyah Negeri Kota Jambi. Terdapat 6 sekolah
Madrasah Tsanawiyah Negeri Kota Jambi yaitu MTs Negeri 1 Kota Jambi, MTs Negeri 2 Kota Jambi,
MTs Negeri 3 Kota Jambi, MTs Negeri 4 Kota Jambi, MTs Negeri 5 Kota Jambi, dan MTs Negeri 6
Kota Jambi. Setiap sekolah diambil 3 siswa dengan kriteria siswa berkemampuan tinggi. Jadi total
subjek penelitian 18 siswa berkemampuan tinggi. Tabel 2 Tabel 2
Persentase Kesalahan Subjek
Soal
Frekuensi
%
Operasi Aljabar
10
56
Teorema Pythagoras
8
44 Persentase Kesalahan Subjek
Soal
Frekuensi
%
Operasi Aljabar
10
56
Teorema Pythagoras
8
44 Persentase Kesalahan Subjek Hasil Penelitian Subjek Kemampuan Tinggi Subjek kemampuan tinggi dalam penelitian ini terdiri dari 18 siswa. Dari persentase kesalahan
seperti pada Tabel 2 ada 56% siswa kemampuan tinggi yang mengalami kesalahan saat menyelesaikan
soal materi operasi aljabar. Selain itu ada 44% siswa kemampuan tinggi yang mengalami kesalahan saat
menyelesaikan soal materi teorema Pythagoras. Kesalahan siswa kemampuan tinggi dalam
menyelesaikan soal operasi aljabar dan teorema Pythagoras berdasarkan tahapan Newman Error
Analysis tergambar seperti pada Tabel 3 beikut. Tabel 1 Hasil Pengerjaan Lembar Soal Pemecahan Masalah Matematika Subjek
Soal
B
%
BTS
%
S
%
SL
%
TJ
%
Operasi Aljabar
7
39
0
0
10 56
0
0
1
6
Teorema Pythagoras
7
39
3
17
8
44
0
0
0
0 Hasil Pengerjaan Lembar Soal Pemecahan Masalah Matematika Subjek 382 Jurnal Cendekia: Jurnal Pendidikan Matematika, Volume 03, No. 02, Agustus 2019, pp. 379-388 Dari Tabel 1 terlihat bahwa persentase kesalahan yang dialami siswa saat menyelesaikan lembar
soal matematika masih tinggi. Dalam penelitian ini kategori siswa dikatakan mengalami kesalahan jika
penilaian terhadap lembar jawaban adalah kriteria salah secara utuh (S) dan salah langkah pengerjaan
(SL). Tabel 2 berikut menyajikan persentase kesalahan subjek saat menyelesaikan lembar soal
pemecahan masalah matematika. Tabel 3 Kesalahan Siswa Kemampuan Tinggi
Tahapan
Operasi Aljabar
Teorema Pythagoras
%
Jenis Kesalahan
%
Jenis Kesalahan
Reading
-
-
Comprehension
50
Memaknai
-
Transformation
100
Penjumlahan
100
Rumus L
Bilangan kelipatan 8
Penjumlahan
pengurangan
Pembagian perkalian
Process skill
-
-
Encoding
30
Memaknai
87.5
Memaknai Kesalahan Penyelesaian Soal Materi Operasi Aljabar Terdapat 10 subjek siswa kemampuan tinggi yang mengalami kesalahan saat menyelesaikan soal
operasi aljabar. Selanjutnya kesepuluh subjek ini disebut KTA1, KTA2, …, KTA10. Kesalahan ini
terlihat jelas pada lembar jawaban subjek dan saat wawancara subjek. Secara umum kesalahan ini
dialami oleh siswa kemampuan tinggi di tiga MTs Negeri Kota Jambi dari enam MTs Negeri Kota
Jambi. Berdasarkan lembar jawaban subjek, kesalahan yang dialami ini bersumber pada satu hal yaitu
pemahaman soal dan kegagalan dalam memilih rumus yang tepat untuk menyelesaikan soal. Newman Error Analysis Siswa Madrasah dalam Menyelesaikan Soal Cerita Matematika, Marni Zulyanty Newman Error Analysis Siswa Madrasah dalam Menyelesaikan Soal Cerita Matematika, Marni Zulyanty 383 Terkait tahapan Newman Error Analysis subjek saat menyelesaikan lembar soal pemecahan masalah
matematika dapat dilihat pada lembar jawaban. Indikator pertama tahapan Newman Error Analysis
yaitu membaca soal dengan baik telah dipenuhi dan dilalui oleh semua subjek siswa kemampuan tinggi
(KTA1, KTA2, …, KTA10). Hal ini dapat dibuktikan dengan adanya usaha yang ditulis subjek di
lembar jawaban sesuai dengan semesta soal yang diberikan. Hal ini seperti pada Gambar 1 berikut. Selain itu dalam wawancara subjek juga menjelaskan bahwa tidak mengalami kesulitan saat membaca
soal. Sehingga subjek siswa kemampuan tinggi telah memenuhi indikator membaca soal dengan baik
saat menyelesaikan soal materi operasi aljabar. Gambar 1. Jawaban Subjek KTA1 untuk Soal No. 1 Gambar 1. Jawaban Subjek KTA1 untuk Soal No. 1 Selanjutnya untuk indikator kedua adalah identifikasi ciri masalah juga telah dilalui dan dipenuhi
oleh subjek KTA1, KTA2, …, KTA10 dengan baik. Namun kebenaran dari identifikasi ciri masalah
yang dilalui subjek tidak sepenuhnya benar, ada 50% dari subjek yang mengalami kesalahan melakukan
identifikasi ciri masalah dengan benar yaitu KTA6, KTA7, KTA8, KTA9, dan KTA10, sedangkan 50%
lagi masih mengalami kegagalan dalam mengidentifikasi masalah yaitu KTA1, KTA2, KTA3, KTA4,
dan KTA5. Hal ini terlihat dari lembar jawaban soal dan wawancara. Subjek dalam melakukan
identifikasi ciri masalah dapat menentukan informasi atau yang diketahui pada soal dan apa yang
ditanyakan pada soal. Subjek yang mengalami kegagalan dalam mengidentifikasi masalah ini
disebabkan karena subjek menganggap keterangan pada soal yang mengatakan bahwa “… jumlah dari
masing dua bilangan tersebut secara berturut-turut adalah 28, 36, 44” memberikan informasi bahwa
nilai 28, 36, dan 44 adalah nilai dari masing-masing bilangan yang ditanyakan. Hal ini dapat dilihat
pada Gambar 2.1 di atas. Dengan demikian subjek siswa kemampuan tinggi telah memenuhi indikator
identifikasi ciri masalah dalam menyelesaikan soal materi operasi aljabar. Indikator ketiga tahapan Newman Error Analysis yaitu merencanakan aktivitas pemecahan masalah
telah dilalui dan dipenuhi oleh subjek KTA1, KTA2, …, KTA10 dengan baik. Hal ini dapat dilihat dari
lembar jawaban subjek dan dari wawancara. Subjek dalam merencanakan aktivitas pemecahan masalah
ini telah dapat menemukan model matematika dari masalah operasi aljabar yang diberikan dan
menemukan rumus yang sesuai dengan model matematika yang dibuat. Kesalahan dalam menentukan Jurnal Cendekia: Jurnal Pendidikan Matematika, Volume 03, No. 02, Agustus 2019, pp. Newman Error Analysis Siswa Madrasah dalam Menyelesaikan Soal Cerita Matematika, Marni Zulyanty 379-388 384 model dan rumus untuk menyelesaikan soal ini di alami subjek karena berdasarkan identifikasi ciri
masalah yang telah dilewati sebelumnya memang ada kesalahan sehingga untuk tahap berikutnya juga
mengalami kesalahan. Sehingga subjek siswa kemampuan tinggi telah memenuhi indikator
merencanakan aktivitas pemecahan masalah dalam menyelesaikan soal materi operasi aljabar. Berikutnya indikator memecahkan masalah juga telah dipenuhi dan dilalui subjek KTA1, KTA2,
…, KTA10 dengan baik. Hal ini terlihat pada lembar jawaban dan dalam wawancara. Dari lembar
jawaban terlihat bahwa subjek telah memecahkan masalah operasi aljabar yang diberikan sesuai dengan
model dan rumus yang didapat pada tahap sebelumnya. Saat memecahkan masalah ini subjek tidak
mengalami kesulitan dan langkah pengerjaan yang telah dilakukan juga sudah sesuai dengan model dan
rumus yang ditentukan sebelumnya walaupun rumus dan model yang digunakan bernilai salah. Berdasarkan uraian diatas maka subjek siswa kemampuan tinggi telah memenuhi indikator
memecahkan masalah dalam menyelesaikan soal materi operasi aljabar. Indikator selanjutnya adalah kesimpulan proses pemecahan masalah, pada indikator ini terdapat
30% subjek gagal dalam menuliskan kesimpulan yaitu KTA1, KTA7, dan KTA10. Sedangkan 70%
subjek telah melalui dan memenuhi indikator kesimpulan proses pemecahan masalah ini. Kesimpulan
yang dituliskan oleh 70% subjek ini sesuai dengan semesta masalah soal yang diberikan, walaupun
kesimpulan yang dituliskan bernilai salah. Sementara untuk 30% subjek yang gagal menuliskan
kesimpulan ini mengatakan bahwa subjek lupa untuk menuliskan kesimpulan karena subjek terbiasa
menyelesaikan soal cerita hanya sebatas penemuan nilai jawaban. Hal ini dapat dilihat dari lembar
jawaban subjek dan wawancara. Karena persentase subjek yang memenuhi tahap kesimpulan proses
pemecahan masalah lebih besar daripada yang tidak memenuhi maka subjek siswa kemampuan tinggi
telah memenuhi indikator kesimpulan proses pemecahan masalah dalam menyelesaikan soal materi
operasi aljabar. Kesalahan Penyelesaian Soal Materi Teorema Pythagoras Terdapat 8 subjek siswa kemampuan tinggi yang mengalami kesalahan saat menyelesaikan soal
teorema Pythagoras. Selanjutnya kedelapan subjek ini disebut KTP1, KTP2, …, KTP8. Kesalahan ini
terlihat jelas pada lembar jawaban subjek dan saat wawancara subjek. Secara umum kesalahan ini
dialami oleh siswa kemampuan tinggi di tiga MTs Negeri Kota Jambi dari enam MTs Negeri Kota
Jambi. Berdasarkan lembar jawaban subjek, kesalahan yang dialami ini bersumber pada satu hal yaitu
pemahaman soal dan kegagalan dalam memilih rumus yang tepat untuk menyelesaikan soal. Terkait tahapan Newman Error Analysis subjek saat menyelesaikan lembar soal pemecahan
masalah matematika dapat dilihat pada lembar jawaban. Indikator pertama tahapan Newman Error
Analysis yaitu membaca soal dengan baik telah dipenuhi dan dilalui oleh semua subjek siswa
kemampuan tinggi (KTP1, KTP2, …, KTP8). Hal ini dapat dibuktikan dengan adanya usaha yang
ditulis subjek di lembar jawaban sesuai dengan semesta soal yang diberikan seperti pada Gambar 2
Selain itu dalam wawancara subjek juga menjelaskan bahwa tidak mengalami kesulitan saat membaca ewman Error Analysis Siswa Madrasah dalam Menyelesaikan Soal Cerita Matematika, Marni Zulyanty 385 soal. Sehingga subjek siswa kemampuan tinggi telah memenuhi indikator membaca soal dengan baik
saat menyelesaikan soal materi teorema Pythagoras. Gambar 2. Jawaban Subjek KTP1 untuk Soal No. 2 Gambar 2. Jawaban Subjek KTP1 untuk Soal No. 2 Gambar 2. Jawaban Subjek KTP1 untuk Soal No. 2 Selanjutnya untuk indikator kedua adalah identifikasi ciri masalah juga telah dilalui dan dipenuhi
oleh subjek KTP1, KTP2, …, KTP8 dengan baik. Nilai kebenaran dari identifikasi ciri masalah yang
dilalui subjek juga sudah benar. Hal ini terlihat dari lembar jawaban soal dan wawancara. Subjek dalam
melakukan identifikasi ciri masalah dapat menentukan informasi atau yang diketahui pada soal dan apa
yang ditanyakan pada soal. Dengan demikian subjek siswa kemampuan tinggi telah memenuhi indikator
identifikasi ciri masalah dalam menyelesaikan soal materi teorema Pythagoras. Indikator ketiga tahapan Newman Error Analysis yaitu merencanakan aktivitas pemecahan masalah
telah dilalui dan dipenuhi oleh subjek KTP1, KTP2, …, KTP8 dengan baik. Hal ini dapat dilihat dari
lembar jawaban subjek dan dari wawancara. Subjek dalam merencanakan aktivitas pemecahan masalah
ini telah dapat menemukan model matematika dari masalah teorema Pythagoras yang diberikan dan
menemukan rumus yang sesuai dengan model matematika yang dibuat walaupun model dan rumus yang
digunakan tersebut salah. Kesalahan Penyelesaian Soal Materi Teorema Pythagoras Kesalahan dalam menentukan model dan rumus untuk menyelesaikan soal ini
di alami subjek karena subjek gagal dalam mentransformasikan masalah ke dalam kajian matematika
sehingga terjadi kesalahan dalam memilih rumus untuk menyelesaikan soal. Subjek KTP1 salah karena dalam menyelesaikan soal menggunakan rumus luas segitiga. Subjek
KTP2, KTP4, KTP5, KTP6, KTP7, dan KTP8 salah karena menggunakan operasi penjumlahan dan
operasi pengurangan. KTP3 salah karena menggunakan operasi perkalian dan operasi pembagian. Dari
paparan diatas maka subjek siswa kemampuan tinggi telah memenuhi indikator merencanakan aktivitas
pemecahan masalah dalam menyelesaikan soal materi teorema Pythagoras. Berikutnya indikator memecahkan masalah juga telah dipenuhi dan dilalui subjek KTP1, KTP2, …,
KTP8 dengan baik. Hal ini terlihat pada lembar jawaban dan wawancara. Dari lembar jawaban terlihat
bahwa subjek telah memecahkan masalah teorema Pythagoras yang diberikan sesuai dengan model dan Jurnal Cendekia: Jurnal Pendidikan Matematika, Volume 03, No. 02, Agustus 2019, pp. 379-388 386 rumus yang didapat pada tahap sebelumnya. Saat memecahkan masalah ini subjek tidak mengalami
kesulitan dan langkah pengerjaan yang telah dilakukan juga sudah sesuai dengan model dan rumus yang
ditentukan sebelumnya walaupun rumus dan model yang digunakan bernilai salah. Berdasarkan uraian
diatas maka subjek siswa kemampuan tinggi telah memenuhi indikator memecahkan masalah dalam
menyelesaikan soal materi teorema Pythagoras. Indikator selanjutnya adalah kesimpulan proses pemecahan masalah, pada indikator ini terdapat
87,5% subjek gagal dalam menuliskan kesimpulan. Hanya 12,5% subjek telah melalui dan memenuhi
indikator kesimpulan proses pemecahan masalah ini yaitu KTP4. Kesimpulan yang dituliskan oleh
12,5% subjek ini sesuai dengan semesta masalah soal yang diberikan, walaupun kesimpulan yang
dituliskan bernilai salah. Sementara untuk 87,5% subjek yang gagal menuliskan kesimpulan ini
mengatakan bahwa subjek lupa untuk menuliskan kesimpulan karena subjek terbiasa menyelesaikan
soal cerita hanya sebatas penemuan nilai jawaban. Selain itu pada wawancara subjek mengatakan bahwa
tidak menuliskan kesimpulan karena belum yakin dengan jawaban yang ditemukan. Karena persentase
subjek yang tidak memenuhi tahap kesimpulan proses pemecahan masalah lebih besar daripada yang
memenuhi maka subjek siswa kemampuan tinggi belum memenuhi indikator kesimpulan proses
pemecahan masalah dalam menyelesaikan soal materi teorema Pythagoras. Berdasarkan uraian di atas, maka secara umum siswa kemampuan tinggi dalam menyelesaikan soal
cerita matematika masih mengalami kesalahan. Kesalahan ini terjadi terutama saat menyelesaikan soal
cerita terkait materi aljabar dan teorema Pythagoras. Ada beberapa bentuk kesalahan yang dialami siswa
kemampuan tinggi. Adapun kesalahan yang terjadi pada siswa kemampuan tinggi jika di analisis berdasarkan tahapan
Newman Error Analysis dikarenakan adanya kesalahan pada tahap memahami masalah
(comprehension). Kesalahan Penyelesaian Soal Materi Teorema Pythagoras Berdasarkan hasil pengerjaan siswa melalui lembar jawaban dan hasil wawancara,
pada tahap ini kesalahan ditandai dengan tidak mampunya siswa mengidentifikasi informasi yang
diketahui dan yang ditanyakan pada soal dengan benar. Hal ini yang menyebabkan siswa salah dalam
menyelesaikan soal atau tidak dapat menemukan solusi yang tepat. Hal ini sesuai dengan hasil observasi
Piaget (L.S. Shulman, 1970) yang menunjukkan bahwa kesalahan siswa menyebabkan tidak
ditemukannya solusi yang tepat. Selain kesalahan pada tahap memahami masalah (comprehension), kesalahan siswa kemampuan
tinggi dalam menyelesaikan soal cerita yang diberikan adalah kesalahan pada tahap transformasi
masalah (transformation). Pada tahap ini siswa mengalami kesalahan dalam menemukan hubungan
antar informasi pada soal dan gagal dalam menemukan rumus atau strategi yang tepat untuk
menyelesaikan soal. Hal ini sejalan dengan hasil penelitian Radatz, H (2013) yang menunjukkan bahwa
kebanyakan siswa salah dalam mengartikan bahasa matematika. Selain itu solusi dari masalah cerita
matematika lebih khusus adalah bagaimana mentranslasikan bahasa atau bentuk masalah ke dalam
bahasa matematika yang sesuai ( Aiken, 1972; Kane, Byme & Hater, 1974; Pippig, 1977). Hal ini juga
menyebabkan siswa salah dalam menyelesaikan soal atau tidak dapat menemukan solusi yang tepat. Newman Error Analysis Siswa Madrasah dalam Menyelesaikan Soal Cerita Matematika, Marni Zulyanty 387 KESIMPULAN Siswa madrasah dalam memecahkan masalah matematika dalam bentuk soal cerita masih
mengalami kesalahan terutama saat memecahkan masalah operasi aljabar dan teorema Pythagoras. Kesalahan ini dapat berupa kesalahan dalam hal memahami masalah, kesalahan dalam proses
penggunaan rumus, dan kesalahan pada solusi yang ditemukan. Sementara kesalahan siswa madrasah
berkemampuan tinggi dalam menyelesaikan soal cerita matematika materi operasi aljabar berdasarkan
Newman Error Analysis terjadi pada tahap comprehension. Kesalahan ini terjadi berupa kesalahan
dalam hal memaknai atau mengartikan soal cerita yang diberikan. Sementara kesalahan siswa madrasah
berkemampuan tinggi dalam menyelesaikan soal cerita matematika materi teorema Pythagoras
berdasarkan Newman Error Analysis terjadi pada tahap transformation. Kesalahan ini terjadi berupa
kesalahan dalam hal mentransformasikan soal cerita yang diberikan yang berujung pada kesalahan
penentuan rumus atau konsep dalam menyelesaikan soal. Tentunya kesalahan pada tahap
comprehension dan transformation ini menyebabkan kesalahan pada tahap selanjutnya sehingga
menyebabkan solusi atau jawaban dari soal cerita yang ditemukan bernilai salah. DAFTAR PUSTAKA Adebola & Sakiru. (2012). A Problem Solving Model as a Strategy for Improving Secondary School
Students’ Achievement and Retention in Further Mathematics. ARPN Journal of Science and
Technology, 2 (2), 122-130. Aiken, L. R. (1972). Language factors in learning mathematics. Review of Educational Research, 42,
359-385. A.L. White. (2005). Learning Mathematics in Classroom: Finding Out Why Children Make Mistakes-
and then Doing Something to Help Them. Sydney: Universitty of Western Sydney. A.L. White. (2010). Numeracy, Literacy, and Newman’s Error Analysis. Journal of Sciences and
Mathematics Education in Southeast Asia, 33 (2), 129-148. B, Sahar & AT, Rohani. (2010). Assessing Cognitive and Metacognitive Strategies during Algebra
Problem Solving Among University Students. Procedia Social and Behavioral Sciences, 8, 403-
410. Creswell. (2012). Educational Research “Planning, Conducting, and Evaluating Quantitative and
Qualitative Research. Amerika: Pearson. D, Setyono & S, Sutarni. (2013). Kesalahan Menyelesaikan Masalah Matematika dalam Bentuk Soal
Cerita Pokok Bahasan Aritmetika Sosial. Seminar Nasional Pendidikan Matematika, Surakarta. Kane, R. B., Byrne, M. A., & Hater, M. A. (1974). Helping children read mathematics. New York:
American Book. L.S. Shulman. (1970). Psychology and Mathematics Education. Chicago: University of Chicago Press
Chicago. Jurnal Cendekia: Jurnal Pendidikan Matematika, Volume 03, No. 02, Agustus 2019, pp. 379-388 388 NCTM. (2000). Principles and Standarts for School Mathematics. Reston: VA. Tello, E.A. (2010). Making Mathematics Word Problems Reliable Measures of Student Mathematics
Abilities. Journal of The Mathematics Education, 3 (1), 15-26. NCTM. (2000). Principles and Standarts for School Mathematics. Reston: VA. Pippig, G. (1977). Psychologische tIberlegungen zur Uiberwindung von denkfehlern. Mathematik in
der Schule, 15, 26-41. Radatz, H. (2013). Error Analysis in Mathematic Education. National Council of Teachers of
Mathematics. Journal for Research in Mathematics Education, 10 (3), 163-172. Sajadi M, Amiripour P, & Malkhalifeh M.R. (2013). The Examining Mathematical Word Problems
Solving Ability under Efficient Representation Aspec. Mathematics Education Trends and
Research, 1-11. Tello, E.A. (2010). Making Mathematics Word Problems Reliable Measures of Student Mathematics
Abilities. Journal of The Mathematics Education, 3 (1), 15-26.
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Meta-All: a system for managing metabolic pathway information
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Erschienen in: BMC Bioinformatics ; 7 (2006), 1. - 465. -
http://dx.doi.org/10.1186/1471-2105-7-465 Erschienen in: BMC Bioinformatics ; 7 (2006), 1. - 465. -
http://dx.doi.org/10.1186/1471-2105-7-465 Erschienen in: BMC Bioinformatics ; 7 (2006), 1. - 465. -
http://dx.doi.org/10.1186/1471-2105-7-465 BioMed Central BioMed Central Meta-All: a system for managing metabolic pathway information
Stephan Weise*1, Ivo Grosse1, Christian Klukas1, Dirk Koschützki1,
Uwe Scholz1, Falk Schreiber1 and Björn H Junker1,2 Email: Stephan Weise* - weise@ipk-gatersleben.de; Ivo Grosse - grosse@ipk-gatersleben.de; Christian Klukas - klukas@ipk-gatersleben.de;
Dirk Koschützki - koschuet@ipk-gatersleben.de; Uwe Scholz - scholz@ipk-gatersleben.de; Falk Schreiber - schreibe@ipk-gatersleben.de;
Björn H Junker - bjunker@bnl.gov * Corresponding author Received: 22 May 2006
Accepted: 23 October 2006 Received: 22 May 2006
Accepted: 23 October 2006 Published: 23 October 2006
BMC Bioinformatics 2006, 7:465
doi:10.1186/1471-2105-7-465
Received: 22 May 2006
Accepted: 23 October 2006
This article is available from: http://www.biomedcentral.com/1471-2105/7/465
© 2006 Weise et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativ MC Bioinformatics 2006, 7:465
doi:10.1186/1471-2105-7-465 This article is available from: http://www.biomedcentral.com/1471-2105/7/465 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. BMC Bioinformatics Open Access Background In an attempt to overcome these problems and to provide
the scientific community with a software system that
meets their requirements, we initiated the META-ALL
project. META-ALL is designed for management of
detailed information about metabolic pathways, includ-
ing reactions, translocations, substances, pathways, loca-
tions and kinetic parameters. The system contains a
versioning system and a Web interface for entering and
querying of data. g
Modern molecular biological research produces large
amounts of data. One main problem remains the elucida-
tion and representation of relationships between the com-
pounds of biological systems. A large number of
information systems holding data from molecular biol-
ogy has been developed by public and private research
projects [1]. A rapidly growing field is the collection of
data associated to metabolic pathways, available from
information systems such as KEGG [2], BRENDA [3], UM-
BBD [4] or Reactome [5]. Depending on the requirements
defined upon the initiation of these projects, the features
and implementations differ significantly among these sys-
tems. For example, BRENDA holds detailed kinetic infor-
mation about enzymes, whereas KEGG contains maps of
metabolic pathways and detailed descriptions about their
elements. Often, the existing systems are not well struc-
tured, because they have been grown over the years. Hence, they are not able to store all necessary information
[6]. For example, many of the systems are not capable of
describing the complexity of higher organisms. Especially
the differentiation between loci inside of organisms, the
consideration of developmental stages and stress factors,
and detailed representation of kinetics and regulation is
only partially possible. Additionally, there are errors
resulting from faulty text-mining procedures or genome-
based pathway predictions, which make the data only
conditionally useful. Hence, existing systems often repre-
sent collections of reference pathways. This is useful for
getting an idea of the metabolic processes as in the case of
the well-known Boehringer charts [7,8], however, in
many cases it is insufficient. Only rudimentary details are
stored about the real location of these processes, such as
the organism, tissue, cell type and compartment, while
comprising comprehensive information about pathway
structures in general. Also, only sparse information is
given about circumstances under which the data points
were determined, such as growth conditions or sampling
time. Hence, some of the represented pathways are non-
occurring in reality. http://www.biomedcentral.com/1471-2105/7/465 http://www.biomedcentral.com/1471-2105/7/465 Abstract Background: Many attempts are being made to understand biological subjects at a systems level. A major resource for these approaches are biological databases, storing manifold information about
DNA, RNA and protein sequences including their functional and structural motifs, molecular
markers, mRNA expression levels, metabolite concentrations, protein-protein interactions,
phenotypic traits or taxonomic relationships. The use of these databases is often hampered by the
fact that they are designed for special application areas and thus lack universality. Databases on
metabolic pathways, which provide an increasingly important foundation for many analyses of
biochemical processes at a systems level, are no exception from the rule. Data stored in central
databases such as KEGG, BRENDA or SABIO-RK is often limited to read-only access. If
experimentalists want to store their own data, possibly still under investigation, there are two
possibilities. They can either develop their own information system for managing that own data,
which is very time-consuming and costly, or they can try to store their data in existing systems,
which is often restricted. Hence, an out-of-the-box information system for managing metabolic
pathway data is needed. Results: We have designed META-ALL, an information system that allows the management of
metabolic pathways, including reaction kinetics, detailed locations, environmental factors and
taxonomic information. Data can be stored together with quality tags and in different parallel
versions. META-ALL uses Oracle DBMS and Oracle Application Express. We provide the META-
ALL information system for download and use. In this paper, we describe the database structure
and give information about the tools for submitting and accessing the data. As a first application of
META-ALL, we show how the information contained in a detailed kinetic model can be stored and
accessed. Conclusion: META-ALL is a system for managing information about metabolic pathways. It
facilitates the handling of pathway-related data and is designed to help biochemists and molecular
biologists in their daily research. It is available on the Web at http://bic-gh.de/meta-all and can be
downloaded free of charge and installed locally. Page 1 of 9
(page number not for citation purposes)
Konstanzer Online-Publikations-System (KOPS)
URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-2--wrl18rolr9yw1 Konstanzer Online-Publikations-System (KOPS)
URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-2--wrl18rolr9yw1 Konstanzer Online-Publikations-System (KOPS)
URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-2--wrl18rolr9yw1 Konstanzer Online-Publikations-System (KOPS)
URL: http://nbn-resolving.de/urn:nbn:de:bsz:352-2--wrl18rolr9yw1 Page 1 of 9
(page number not for citation purposes) BMC Bioinformatics 2006, 7:465 http://www.biomedcentral.com/1471-2105/7/465 Page 2 of 9
(page number not for citation purposes) Background As an exception the SABIO-RK system
[9] should be mentioned, which focuses on the storage of
fine-grained and high-quality data in a central instance. Meta-All is a software package that is initially distributed
with an example data-set only. It can be installed locally
to help biologists and biochemists in their daily research. The user can enter own experimental data as well as data
sets from other sources such as publications or databases. Once the user's instance of Meta-All is populated with suf-
ficient amounts of data, a variety of complex queries will
be possible due to the detailed structure of the database
schema. Examples for such queries are dependent on the
filling of the system, and could be: "What is the Km value
for enzyme x in compartment y of organ z of organism w?"
or "If a value for organism x is missing, what value was
measured in the closest relative (in taxonomical sense) of
that organism?" In this paper, at first we give an overview about the tech-
nical background of the system. In the second part we
describe the database schema, before we provide details
about the user-interface we developed for managing the
contents of the database. Finally, we discuss our system in
relation to other systems. In this paper, at first we give an overview about the tech-
nical background of the system. In the second part we
describe the database schema, before we provide details
about the user-interface we developed for managing the
contents of the database. Finally, we discuss our system in
relation to other systems. Locations and taxonomy y
Conversions take place at different locations inside an
organism depending on the developmental state and
environmental effects, and the database schema reflects as
many of these parameters as possible. We focused on the
use of existing ontologies in order to have a controlled
vocabulary allowing the comparison of data from differ-
ent sources. For our instance of META-ALL we decided to
use PlantOntology [15] terms to distinguish cytological
aspects of plants and also for developmental stages,
because the scientific focus of our institute is plant
research. The META-ALL system can be easily extended by
other ontologies or user-defined terms, respectively. To
determine the taxonomy of the organisms, we use the
NCBI taxonomy ID [16] enriched by established
attributes such as family, genus or species. Additionally,
META-ALL allows the storage of genetic distance matrices
of different organisms. The user can infer these matrices
for example by comparison of gene sequences from the
organisms stored in the database. Thus, in case a pathway
is poorly investigated in one organism, this feature allows
the recruitment of data from a phylogenetically closely
related species. Database schema The database schema is divided into several parts. The
main parts are conversions, substances, pathways, loca-
tions (taxonomy, developmental stage, cytology), refer-
ences and versioning. A simplified version reflecting all
objects storable in the database schema is shown in Fig. 1. The complete relational schema comprising 51 database
tables is available at the META-ALL project Web page. To
develop the database schema of META-ALL, we compared
several existing information systems storing data about
metabolic pathways and extensively discussed with exper-
imentalists about their needs and wishes. The schema was
improved in multiple cycles, according to the spiral model
[14] popular in software engineering. Conversions and substances Conversions and substances The central parts of the schema are conversions and sub-
stances. A conversion is a reaction or a translocation, both
of them either actively (enzyme-catalysed or transporter-
mediated, respectively) or passively (spontaneous or by
diffusion, respectively). All necessary information can be
stored with every conversion: name and synonym(s), for-
mula, type (reaction, translocation) and subtype (kinetic
reaction type for enzymes, type of transporter). Conver-
sions are categorised into pathways and pathways into
super-pathways. Furthermore, some specific information
can be included, e.g. for the layout of cycles (if there is a
clockwise or an anti-clockwise cycle, or if it is an open or
closed one). The substance is an umbrella term for trans-
porters, enzymes, metabolites and macromolecules, the
latter of which consists of one or more types of metabolite
units. Each substance plays a certain role in a conversion,
which can be catalyst in case the substance is an enzyme,
or substrate (= input substance) in case the substance is a
metabolite. Furthermore, a metabolite can be a modula-
tor (activator or inhibitor) of a reaction, the product (= http://www.biomedcentral.com/1471-2105/7/465 http://www.biomedcentral.com/1471-2105/7/465 BMC Bioinformatics 2006, 7:465 http://www.biomedcentral.com/1471-2105/7/465 META-ALL can be accessed in two ways. First, we provide
a demo instance of META-ALL on our server which is
accessible via our project Web page [11]. This instance is
intended to provide an overview about the abilities of
META-ALL. A guest user account with the username
"guest" and the password "meta-all" allows the user to see
a pathway from sucrose breakdown in potato as a test data
set [12]. The demo instance will be reset at regular inter-
vals. The second possibility is to download META-ALL
and to install it locally. That requires Oracle DBMS and
Oracle Application Express running. This can be either a
commercial version of Oracle software or an Oracle
Express Edition [13], which is a freely available light-
weight Oracle DBMS coming along with an integrated
Application Express installation. The META-ALL applica-
tion including the initial data set and the installation pro-
cedure is available for download free of charge at our
project Web page. output substance) or act as a coenzyme. Each of the role
information can be enriched by detailed information such
as Vmax values, affinity constants, etc. Each piece of infor-
mation is assigned to the location information and publi-
cations. p e
e tat o
Technical design of Meta-All META-ALL uses the Oracle database management system,
which is extensively used in academia and industry. Fur-
thermore, Oracle provides several useful tools for easy cre-
ation of user interfaces and a security concept for
protecting selected data. For the user-frontend, we use
Oracle Application Express [10]. It allows the wizard-
aided creation of Web-interfaces based on the underlying
database schema with the help of a set of components,
such as graphical and non-graphical reports, forms and
trees, which can be easily integrated into different areas
(regions) of Web pages. We use the user management pro-
vided by Oracle Application Express for allowing the cre-
ation of users with different rights such as read/write or
read-only access. Oracle Application Express addresses
also the concerns of multiple-user access with built-in
check constraints. Hence, it is possible for multiple users
to use META-ALL at the same time; it is ensured that two
users cannot edit the same data simultaneously and that
one user cannot overwrite the respective changes of the
other user. Another important problem biochemists and molecular
biologists are faced with is the management of their own
data. Currently, experimentalists can develop an own
information system for managing that own data, but this
is very time-consuming and costly. An alternative would
be to try to get their data into existing systems. Often, this
is not possible. In case that scientists have different opin-
ions about certain items, pathway data needs to be stored
in different parallel versions. However, this is not possible
with most of the existing systems. Page 2 of 9
(page number not for citation purposes) Page 2 of 9
(page number not for citation purposes) http://www.biomedcentral.com/1471-2105/7/465 Reference information As publications represent probably the most important
knowledge source, it is crucial to store reference informa-
tion with as many data points as possible. We decided to
use the widely accepted standard of PubMed IDs instead
of developing an integrated publication management. Hence, META-ALL stores the PubMed ID for each publica-
tion and a text field is provided to store necessary infor-
mation for yet unpublished data. The record has to be
updated manually once a PubMed ID is available. If using
the META-ALL Web-Interface described below, the user is
shown the PubMed ID and, if existing, the remark field. The NCBI Web page with the abstract of the publication
can be opened in a new window using a provided link. Page 3 of 9
(page number not for citation purposes) Quality tags and data versioning The data for which META-ALL is intended usually has dif-
ferent quality levels. Hence, the storage of additional
quality information and the possibility to store curated
data, as it was pointed out in [17], is required. In addition,
there is a strong need for a versioning system for a path-
way database like META-ALL, because biochemical data is
sometimes ambiguous and even long-established views
are subject to change. We need to be able to store different
revisions of pathways as scientists tend to have different Page 3 of 9
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(page number not for citation purposes) BMC Bioinformatics 2006, 7:465 http://www.biomedcentral.com/1471-2105/7/465 Simplified schema of the Meta-All database
Figure 1
Simplified schema of the Meta-All database. Simplified UML [30] structure diagram of the database schema. The com-
plete schema is available at the META-ALL project Web page. Rectangles symbolise classes (e.g. substance) without attributes
and methods. Lines between classes symbolise relationships between the classes, called associations. Small diamonds at one of
the ends of a line symbolise part-of relations. A black diamond means a composition (the single parts cannot exist on their
own), an open diamond means an aggregation (the single part can exist on its own). A line with a triangle at one of the ends
symbolises an inheritance relation (e.g. both metabolite class and enzyme class belong to the substance class). Numbers next to
lines specify the allowed cardinalities for an instance of that class, for example, the 1..* at the line between metabolite and mac-
romolecule means that a macromolecule consists of at least one (1) metabolite up to an unlimited (*) number of metabolites. Simplified schema of the Meta All database
Figure 1
Simplified schema of the Meta-All database. Simplified UML [30] structure diagram of the database schema. The com-
plete schema is available at the META-ALL project Web page. Rectangles symbolise classes (e.g. substance) without attributes
and methods. Lines between classes symbolise relationships between the classes, called associations. Small diamonds at one of
the ends of a line symbolise part-of relations. A black diamond means a composition (the single parts cannot exist on their
own), an open diamond means an aggregation (the single part can exist on its own). A line with a triangle at one of the ends
symbolises an inheritance relation (e.g. both metabolite class and enzyme class belong to the substance class). Quality tags and data versioning Numbers next to
lines specify the allowed cardinalities for an instance of that class, for example, the 1..* at the line between metabolite and mac-
romolecule means that a macromolecule consists of at least one (1) metabolite up to an unlimited (*) number of metabolites. able. The term "status" as used in META-ALL is a label
comprising the time-stamp at which a new version was
created. In addition, it contains information such as the
author of the version, the originality of the data (e.g. in-
house data, public database), the quality of the data (e.g. hand-curated, imported from external source, putative)
and the ID of the status record the newly generated status
(or version) is based on. The labelled data sets can be
improved gradually. Additionally, old pathways can be
kept for publication purposes (in case the pathway has
been improved in the meanwhile). opinions about certain issues. There are several possibili-
ties for data versioning [18,19]. Linear versioning could
be used, which means that each version has only one suc-
cessor. In contrast, as one of our requirements to the sys-
tem was to enable parallel instances of pathways, a
hierarchical versioning would be a benefit. Both linear
and hierarchical versioning can be subdivided into con-
tinuous versioning and discrete versioning. Continuous
versioning means that every simple change is a new ver-
sion. In contrast, if using discrete versioning, which was
used for META-ALL, a new version is created at a certain
interval. In the context of META-ALL, the decision to cre-
ate a new version is made by the user. All versioned values
are stored in the same table distinguished by the status. We do not work with shadow tables archiving older val-
ues, because then only one value would actually be avail- Results META-ALL is an information system for managing data
about metabolic pathways, which is available for down-
load and local installation, in order to help experimental-
ists in their daily research. We provide both the database
schema (together with an initial set of data from sucrose
breakdown in the potato tuber) and the user interface. It
is available from the META-ALL Web page. Additionally,
we provide a demo instance of the META-ALL system on
our server. An information system of high interest is SABIO-RK [9]. It
focuses on the curation of data about biochemical reac-
tions, including their kinetic properties. The limitations of
SABIO-RK are that data is stored in a central instance with
a read-only access and that there is no user interface for
inserting data. A system with similar goals as Meta-All is
aMAZE [26]. It consists of a WorkBench for storing path-
way data and a front-end (LightBench) for accessing this
data. Additional components, namely the Snow Work-
bench and a direct SQL access to the data, are available. To
the best of our knowledge, aMAZE cannot store informa-
tion about reaction kinetics, and is not able to write SBML
files. In the current version the aMAZE LightBench can be
used for querying data in the central installation in Bel-
gium only. The intension of META-ALL is to support
experimentalists in their daily work. While most of the
existing systems focus on storing and curating data in a
central instance, e. g. KEGG, and can just be accessed read- The Meta-All Web-Interface The META-ALL Web interface is the central user-interface
to the META-ALL database (Fig. 2). We used the Applica-
tion Express [10] technology from Oracle as described Page 4 of 9
(page number not for citation purposes) Page 4 of 9
(page number not for citation purposes) http://www.biomedcentral.com/1471-2105/7/465 BMC Bioinformatics 2006, 7:465 • The kinetic type of the reaction, together with the organ-
ism this was determined in. • The kinetic type of the reaction, together with the organ-
ism this was determined in. above (subsection Technical design of the system). META-
ALL comes along with two levels of navigation tabs reflect-
ing the certain parts of the database schema. Each part
(level one) consists of several pages (level two). Several
forms allow inserting new data, creating and assigning of
versions, publications, etc. For example, the user can
insert new substances (e.g. metabolites or enzymes). Con-
secutively, these substances can be assigned with conver-
sion processes, which can be assigned with pathways,
super-pathways and so on. All data can be visualised using
reports, and selective data can also be edited depending
on the authorisation of the user. Several reports are possi-
ble inside of META-ALL, e.g. graphical reports about con-
tents of the database as there are enzymes and
metabolites, or groupings of reactions regarding to the
pathways they belong to. Whole pathways can be selected
and then exported from the META-ALL Web-Interface in
SBML format [20]. META-ALL comes with the general rate
law for a number of standard kinetic types (e.g. Ping-Pong
bi bi) using MathML [21]. The user associates a conver-
sion with a kinetic type. When an SBML file is generated
for a pathway, META-ALL uses the necessary information
such as the binding order of the substrates, which are
stored together with the conversion, and fills the accord-
ing values into the rate law. The SBML file can be loaded
into a visualisation system, e. g. VANTED [22], as shown
in Fig. 3, or into a simulation system, e. g. COPASI [23]. • The name of each metabolite, the role it plays in which
reaction, and the binding- and dissociation order in these
reactions. • The kinetic parameters of each reaction such as binding
or inhibitory constants, maximal velocity and equilib-
rium constant; their value and unit; the organism, tissue,
cell, and compartment the constant was measured in. The Meta-All Web-Interface • For all data, the reference and the according PubMed ID
(if available) is given, together with a tag if this kinetic
type was measured or estimated. The structure of the metabolic network has been exported
from META-ALL as an SBML file and imported into the
data visualisation system VANTED for a visualisation of
the pathway. Figure 3 shows the result of the visualisation. While this procedure shows the potential of the system, it
should be pointed out that the connection of META-ALL
to VANTED is only one of numerous possibilities that
could be implemented depending on the preferences of
the user. Discussion Although there exist several information systems for stor-
ing metabolic pathway data, we decided to develop a new
one, because many of the existing systems are not capable
of meeting the requirements of experimentalists working
on higher organisms. We analysed a variety of existing
information systems concerning their advantages and dis-
advantages and made these information the fundament of
our requirement analysis. An exciting study in this field is
published in [25]. • The enzyme name, EC number and stoichiometric for-
mula. Page 5 of 9
(page number not for citation purposes) Case study
Th
h The pathway by which sucrose is transformed to starch in
developing potato tubers has been subject of numerous
studies over several decades (for a review see [24]). A
detailed kinetic model has been created [12] in an attempt
to better understand the dynamics of this pathway. The
model consists of 14 reactions with their according rate-
laws, which are further defined by a total of 74 constants. The data contained in the model represents a useful test
data set for META-ALL and was thus entered into the sys-
tem. It is possible to represent all data from the model in
META-ALL, including: • The enzyme name, EC number and stoichiometric for-
mula. Page 5 of 9
(page number not for citation purposes) Page 5 of 9
(page number not for citation purposes) http://www.biomedcentral.com/1471-2105/7/465 BMC Bioinformatics 2006, 7:465 Screenshot of the Meta-All Web-Interface
Figure 2
Screenshot of the Meta-All Web-Interface. The Web-Interface is divided into several parts, which correspond with the
main parts of the database schema as described in Fig. 1. There are tabs for substances, locations, conversions, pathways and
the management of versions and publications. These tabs open sub-pages each with several items belonging to the particular
part of the schema. a) shows an input form for inserting data about metabolites, and b) is a simple report listing enzymes. c) is
showing taxonomy information of the organisms stored in the demo instance of META-ALL, and d) shows a step from a wiz-
ard for browsing top-down through a pathway. On the left side, there is a select list for choosing one of the substances listed
in the report on the right side. These substances belong to a conversion selected in the previous step. By clicking on the
"next"-button, details of the chosen substance will be shown. Screenshot of the Meta All Web Interface
Figure 2 Screenshot
Figure 2 Screenshot of the Meta All Web Interface
Figure 2
Screenshot of the Meta-All Web-Interface. The Web-Interface is divided into several parts, which correspond with the
main parts of the database schema as described in Fig. 1. There are tabs for substances, locations, conversions, pathways and
the management of versions and publications. These tabs open sub-pages each with several items belonging to the particular
part of the schema. a) shows an input form for inserting data about metabolites, and b) is a simple report listing enzymes. c) is
showing taxonomy information of the organisms stored in the demo instance of META-ALL, and d) shows a step from a wiz-
ard for browsing top-down through a pathway. On the left side, there is a select list for choosing one of the substances listed
in the report on the right side. These substances belong to a conversion selected in the previous step. By clicking on the
"next"-button, details of the chosen substance will be shown. instruction containing database schema, user interface
and initial data set can be obtained from the META-ALL
project Web page. It can be used with either a commercial
Oracle license or the Oracle Database 10 g Express Edi-
tion, which is available free of charge from Oracle Corpo-
ration. only, META-ALL can be installed locally, and data can be
inserted and edited. Research groups or even whole insti-
tutes can manage their "fresh" data, or even data subject
to current investigations. The management is supported
by versioning and quality tagging. Furthermore, not only
reaction data, but also translocation data can be managed
with META-ALL. We prepared several reports for the META-ALL user inter-
face and used our system in conjunction with VANTED for
pathway visualisation. We plan to connect META-ALL to
the Systems Biology Modelling Environment (SYBME) META-ALL uses an Oracle database management system
for persistently storing metabolic pathway data and an
Oracle Application Express user interface. An installation Page 6 of 9
(page number not for citation purposes) Page 6 of 9
(page number not for citation purposes) http://www.biomedcentral.com/1471-2105/7/465 BMC Bioinformatics 2006, 7:465 ta from Meta-All visualised in Vanted
gure 3
ata from Meta-All visualised in Vanted. Visualisation of the sucrose breakdown pathway in the potato tuber [12]. hway data stored in META-ALL was exported into an SBML file using the SBML export filter. The file was subsequentl
ded into the network visualisation system VANTED [22]. Screenshot
Figure 2 As the SBML format does not specify a layout of the model,
omatic force directed layout was applied which served as a starting point for the manual refinement of the overall mo
out. Data from
Figure 3 g
Data from Meta-All visualised in Vanted. Visualisation of the sucrose breakdown pathway in the potato tuber [12]. The
pathway data stored in META-ALL was exported into an SBML file using the SBML export filter. The file was subsequently
loaded into the network visualisation system VANTED [22]. As the SBML format does not specify a layout of the model, an
automatic force directed layout was applied which served as a starting point for the manual refinement of the overall model
layout. Page 7 of 9
(page number not for citation purposes) Page 7 of 9
(page number not for citation purposes) http://www.biomedcentral.com/1471-2105/7/465 BMC Bioinformatics 2006, 7:465 Authors' contributions All authors participated in the design of the system. SW
implemented the system. SW and BHJ designed the data-
base schema and drafted the manuscript. All authors read
and approved the final version. • License: Apache License, Version 2.0 • License: Apache License, Version 2.0 Conclusion 4. Ellis LBM, Roe D, Wackett LP: The University of Minnesota Bio-
catalysis/Biodegradation Database: the first decade. Nucleic
Acids Research 2006:D517-D521. 4. Ellis LBM, Roe D, Wackett LP: The University of Minnesota Bio-
catalysis/Biodegradation Database: the first decade. Nucleic
Acids Research 2006:D517-D521. 5. Joshi-Tope G, Gillespie M, Vastrik I, D'Eustachio P, Schmidt E, de
Bono B, Jassal B, Gopinath GR, Wu GR, Matthews L, Lewis S, Birney
E, Stein L: Reactome: a knowledgebase of biological pathways. Nucleic Acids Res 2005:D428-432. In this paper, we presented META-ALL, a metabolic path-
way information system, which can be downloaded and
installed locally. It is intended to support biochemists and
molecular biologists in their daily research by providing a
platform for the management of detailed information
about metabolic pathways, including reactions, transloca-
tions, substances, pathways, locations and kinetic param-
eters. META-ALL contains a versioning system, quality tags
and a Web interface for entering and querying of data. Pathways can be exported into SBML files for use in visu-
alisation or simulation tools. 5. Joshi-Tope G, Gillespie M, Vastrik I, D'Eustachio P, Schmidt E, de
Bono B, Jassal B, Gopinath GR, Wu GR, Matthews L, Lewis S, Birney
E, Stein L: Reactome: a knowledgebase of biological pathways. Nucleic Acids Res 2005:D428-432. 5. Joshi-Tope G, Gillespie M, Vastrik I, D'Eustachio P, Schmidt E, de
Bono B, Jassal B, Gopinath GR, Wu GR, Matthews L, Lewis S, Birney
E, Stein L: Reactome: a knowledgebase of biological pathways. Nucleic Acids Res 2005:D428-432. 6. Philippi S, Köhler J: Addressing the problems with life-science
databases for traditional uses and systems biology. Nature
Reviews Genetics 2006, 7(6):482-488. 7. Michal G, (Ed): Biochemical Pathways (wall charts). Boehringer
Mannheim, Penzberg Third edition. 1993. 8. Michal G, (Ed): Biochemical Pathways – An Atlas of Biochemistry and
Molecular Biology A John Wiley & Sons, Inc. and Spektrum Akadem-
ischer Verlag Co-Publication; 1999. 7. Michal G, (Ed): Biochemical Pathways (wall charts). Boehringer
Mannheim, Penzberg Third edition. 1993. 8. Michal G, (Ed): Biochemical Pathways – An Atlas of Biochemistry and
Molecular Biology A John Wiley & Sons, Inc. and Spektrum Akadem-
ischer Verlag Co-Publication; 1999. 9. Wittig U, Golebiewski M, Kania R, Krebs O, Mir S, Weidemann A,
Anstein S, Saric J, Rojas I: SABIO-RK: Integration and Curation
of Reaction Kinetics Data. In Data Integration in the Life Sciences:
Third International Workshop, DILS 2006, Hinxton, UK, July 20–22, 2006. Acknowledgements We thank Sophia Biemelt, Frederik Börnke, Mohammad Hajirezaei, Hardy
Rolletschek and Uwe Sonnewald for valuable discussions, the anonymous
reviewers for valuable comments and the German Federal Ministry for Edu-
cation and Research (BMBF) for financial support. References 1. Galperin MY: The Molecular Biology Database Collection:
2006 update. Nucleic Acids Research 2006:D3-D5. 1. Galperin MY: The Molecular Biology Database Collection:
2006 update. Nucleic Acids Research 2006:D3-D5. For the daily work, the existing Web-Interface should be
appropriate. In the future, we plan to expand META-ALL
by an SBML importer allowing the import of larger
amounts of data at once. 2. Kanehisa M, Goto S, Hattori M, Aoki-Kinoshita KF, Itoh M,
Kawashima S, Katayama T, Araki M, Hirakawa M: From genomics
to chemical genomics: new developments in KEGG. Nucleic
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Leser U, Naumann F, Eckman B. Springer Berlin/Heidelberg;
2006:94-103. y
q
• Project name: META-ALL • Project home page: http://bic-gh.de/meta-all [27] to further support the user in the kinetic metabolic
modelling. Within SYBME, a user will be able to browse
through the information of metabolites and reactions
available in his/her META-ALL instance, may combine
this information into a kinetic model and can finally vis-
ualise and simulate the model with the two connected
simulators, GEPASI [28] and JARNAC [29]. • Operating system(s): Client: only a Web browser
required; Server: OS depending on the Oracle installation,
e. g. Microsoft Windows, Linux • Operating system(s): Client: only a Web browser
required; Server: OS depending on the Oracle installation,
e. g. Microsoft Windows, Linux • Operating system(s): Client: only a Web browser
required; Server: OS depending on the Oracle installation,
e. g. Microsoft Windows, Linux • Programming language: User-interface using the Oracle
Application Express technology and SQL, PL/SQL For the future, we plan to facilitate the following applica-
tion using META-ALL: the semi-automatic generation of
kinetic models of a certain pathway. The procedure for
this task will be as follows: the user enters the pathway
that should be modelled and defines the boundaries of
the model (substrates, products). An application of
META-ALL then queries the database for all information
about the enzymes present in this pathway at a specific
location in a given organism. If a certain kinetic parameter
is not available for an enzyme, it will be possible to take
the data from the taxonomically nearest neighbour spe-
cies for which the data is available with the help of the
genetic distance matrix included in META-ALL. This is a
common approach for constructing kinetic models, which
is normally done through tedious literature searches. The
data can then be presented to the user who can decide
which data to keep and which to change. Subsequently,
the model could be exported in standard formats such as
SBML. We are aware that this is a future application imply-
ing that the database is filled with a sufficient amount of
data, which is in the responsibility of the user. The user
could, for example, import large datasets from existing
sources and improve the data step by step. This procedure
is supported by the quality and versioning tagging
described in subsection Database schema. • Other requirements: Oracle DBMS 9i/10g (version
9.2.0.3 and higher) and Oracle Application Express 2.0
license if available, or Oracle Database 10g Express Edi-
tion (free of charge entry-level Oracle DBMS, including
Application Express) Availability and requirements y
q
• Project name: META-ALL Page 8 of 9
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(page number not for citation purposes) http://www.biomedcentral.com/1471-2105/7/465 http://www.biomedcentral.com/1471-2105/7/465 http://www.biomedcentral.com/1471-2105/7/465 BMC Bioinformatics 2006, 7:465 10. Oracle Application Express [http://www.oracle.com/technology/
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U, Novere NL, Loew LM, Lucio D, Mendes P, Minch E, Mjolsness ED,
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30. Fowler M, Scott K: UML Distilled: A Brief Guide to the Standard Object
Modeling Language: AND The Unified Process Explained 3rd edition. Add-
ison Wesley; 2004.
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https://www.frontiersin.org/articles/10.3389/fneur.2021.648958/pdf
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English
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Head-Down Tilt Bed Rest Studies as a Terrestrial Analog for Spaceflight Associated Neuro-Ocular Syndrome
|
Frontiers in neurology
| 2,021
|
cc-by
| 8,132
|
REVIEW REVIEW
published: 26 March 2021
doi: 10.3389/fneur.2021.648958 Edited by:
John Jing-Wei Chen,
Mayo Clinic, United States Reviewed by:
Thomas Mader,
Retired, Moab, United States
Michael S. Vaphiades,
University of Alabama at Birmingham,
United States
Ari Shinojima,
Keio University, Japan *Correspondence:
Heather E. Moss
hemoss@stanford.edu Specialty section:
This article was submitted to
Neuro-Ophthalmology,
a section of the journal
Frontiers in Neurology Keywords: spaceflight associated neuro-ocular syndrome, head-down tilt bed rest, astronaut, space medicine,
optic disc edema, microgravity, terrestrial analog, countermeasures Received: 03 January 2021
Accepted: 01 March 2021
Published: 26 March 2021 Keywords: spaceflight associated neuro-ocular syndrome, head-down tilt bed rest, astronaut, space medicine,
optic disc edema, microgravity, terrestrial analog, countermeasures Head-Down Tilt Bed Rest Studies as
a Terrestrial Analog for Spaceflight
Associated Neuro-Ocular Syndrome SANS is a disorder that is unique to spaceflight and has no terrestrial
equivalent. The prevalence of SANS increases with increasing spaceflight duration and
although there have been residual, structural, ocular changes noted, no irreversible or
permanent visual loss has occurred after SANS, with the longest spaceflight to date
being 14 months. These microgravity-induced findings are being actively investigated
by the United States’ National Aeronautics Space Administration (NASA) and SANS
is a potential obstacle to future longer duration, manned, deep space flight missions. The pathophysiology of SANS remains incompletely understood but continues to be a
subject of intense study by NASA and others. The study of SANS is of course partially
limited by the small sample size of humans undergoing spaceflight. Therefore, identifying
a terrestrial experimental model of SANS is imperative to facilitate its study and for testing
of preventative measures and treatments. Head-down tilt bed rest (HDTBR) on Earth
has emerged as one promising possibility. In this paper, we review the HDTBR as an
analog for SANS pathogenesis; the clinical and imaging overlap between SANS and
HDTBR studies; and potential SANS countermeasures that have been or could be tested
with HDTBR Head-Down Tilt Bed Rest Studies as
a Terrestrial Analog for Spaceflight
Associated Neuro-Ocular Syndrome Joshua Ong 1, Andrew G. Lee 2,3,4,5,6,7,8,9 and Heather E. Moss 10,11* 1 University of Pittsburgh School of Medicine, Pittsburgh, PA, United States, 2 Department of Ophthalmology, Houston
Methodist Hospital, Houston, TX, United States, 3 Baylor College of Medicine and the Center for Space Medicine, Houston,
TX, United States, 4 The Houston Methodist Research Institute, Houston Methodist Hospital, Houston, TX, United States,
5 Departments of Ophthalmology, Neurology, and Neurosurgery, Weill Cornell Medicine, New York, NY, United States,
6 Department of Ophthalmology, University of Texas Medical Branch, Galveston, TX, United States, 7 University of Texas MD
Anderson Cancer Center, Houston, TX, United States, 8 Texas A and M College of Medicine, Bryan, TX, United States,
9 Department of Ophthalmology, The University of Iowa Hospitals and Clinics, Iowa City, IA, United States, 10 Departments of
Ophthalmology, Stanford University, Palo Alto, CA, United States, 11 Departments of Neurology & Neurosciences, Stanford
University, Palo Alto, CA, United States Astronauts who undergo prolonged periods of spaceflight may develop a unique
constellation of neuro-ocular findings termed Spaceflight Associated Neuro-Ocular
Syndrome (SANS). SANS is a disorder that is unique to spaceflight and has no terrestrial
equivalent. The prevalence of SANS increases with increasing spaceflight duration and
although there have been residual, structural, ocular changes noted, no irreversible or
permanent visual loss has occurred after SANS, with the longest spaceflight to date
being 14 months. These microgravity-induced findings are being actively investigated
by the United States’ National Aeronautics Space Administration (NASA) and SANS
is a potential obstacle to future longer duration, manned, deep space flight missions. The pathophysiology of SANS remains incompletely understood but continues to be a
subject of intense study by NASA and others. The study of SANS is of course partially
limited by the small sample size of humans undergoing spaceflight. Therefore, identifying
a terrestrial experimental model of SANS is imperative to facilitate its study and for testing
of preventative measures and treatments. Head-down tilt bed rest (HDTBR) on Earth
has emerged as one promising possibility. In this paper, we review the HDTBR as an
analog for SANS pathogenesis; the clinical and imaging overlap between SANS and
HDTBR studies; and potential SANS countermeasures that have been or could be tested
with HDTBR. Astronauts who undergo prolonged periods of spaceflight may develop a unique
constellation of neuro-ocular findings termed Spaceflight Associated Neuro-Ocular
Syndrome (SANS). CLINICAL PRESENTATION OF SANS Spaceflight is associated with reduced visual acuity in ∼29%
of astronauts on short-duration (<6 months) missions and
60% of astronauts after LDSF (≥6 months) due to hyperopic
shift (4, 16). In 2011, the first report of hyperopic shift,
optic disc edema, cotton wool spots, choroidal folds, globe
flattening, and retina nerve fiber layer thickening were reported
in seven astronauts following a LDSF (6-month mission) on the
International Space Station (ISS) (17). In-flight orbital ultrasound
has revealed optic nerve sheath diameter dilation, and post-
flight lumbar punctures demonstrated slightly elevated but nearly
normal opening cerebrospinal fluid (CSF) pressures in some
individuals (4, 18). Intraocular pressure (IOP) increases initially
during spaceflight, documented with a 20–25% increase 44 min
into spaceflight (19). However, the Lifetime Surveillance of
Astronaut Health gathered in-flight IOP data on 15 astronauts
who underwent LDSF and found no significant changes between
baseline pre-flight IOP, 30th day in-flight IOP, 30 days prior to
returning to earth IOP, and post-flight IOP. Thus, suggesting
that the acute elevation in IOP during immediate exposure to
microgravity trends toward baseline shortly afterwards and stays
at baseline throughout the mission (4). Prospective studies of
SANS include the utilization of pre- and post-flight brain and
orbital magnetic resonance imaging (MRI), retina and optic
nerve optical coherence tomography (OCT), orbital ultrasound,
cycloplegic refraction, funduscopic examination, and lumbar
punctures (LP). Some of these measurements can be performed
in-flight but some (e.g., LP, MRI) are logistically not feasible on
the ISS (3). The exact pathophysiology of SANS continues to be an area
of investigation, but multiple hypotheses have emerged. The
first theory states that the ocular manifestations arise from
high ICP resulting from venous hypertension due to cephalad
fluid shifts that occur due to lack of gravity acting on the
intravascular fluids. Post-flight MRIs of astronauts have shown
findings similar to those seen in terrestrial high ICP states such
as idiopathic intracranial hypertension (IIH) including posterior
globe flattening and concavity of the pituitary dome (31). Jugular venous distension (JVD) occurs in microgravity studies
supporting a hypothesis of venous congestion in the microgravity
environment impeding cerebrospinal fluid absorption (3, 32, 33). Furthermore, optic disc edema seen in SANS is a hallmark
sign of terrestrial high ICP (3, 34). Citation: During
long-duration
spaceflight
(LDSF)
missions,
astronauts
undergo
a
number
of
microgravity-induced physiological changes such as skeletal muscle atrophy, decreased bone mass,
and height change (1–3). The neuro-ocular findings of LDSF include optic nerve head swelling,
choroidal folds, cotton wool patches, a hyperopic shift, and retinal nerve fiber layer thickening (3). This constellation of findings was initially termed Visual Impairment and Intracranial Pressure
(VIIP) syndrome based on the possibility of increased intracranial pressure (ICP) as the unifying During
long-duration
spaceflight
(LDSF)
missions,
astronauts
undergo
a
number
of
microgravity-induced physiological changes such as skeletal muscle atrophy, decreased bone mass,
and height change (1–3). The neuro-ocular findings of LDSF include optic nerve head swelling,
choroidal folds, cotton wool patches, a hyperopic shift, and retinal nerve fiber layer thickening (3). This constellation of findings was initially termed Visual Impairment and Intracranial Pressure
(VIIP) syndrome based on the possibility of increased intracranial pressure (ICP) as the unifying Ong J, Lee AG and Moss HE (2021)
Head-Down Tilt Bed Rest Studies as a
Terrestrial Analog for Spaceflight
Associated Neuro-Ocular Syndrome. Front. Neurol. 12:648958. doi: 10.3389/fneur.2021.648958 March 2021 | Volume 12 | Article 648958 Frontiers in Neurology | www.frontiersin.org HDTBR - SANS Terrestrial Analog Ong et al. mechanism. Over time however, the role of ICP alone in the
condition has evolved and the term Spaceflight Associated
Neuro-Ocular Syndrome (SANS) is likely more accurate (3). missions as a countermeasure for the microgravity-induced
hyperopic shifts (4, 20, 21). Although no permanent vision loss
has been reported, choroidal folds and posterior globe flattening
may persist years after LDSF (3, 17, 22). Several astronauts have
reported refractive changes that have yet to resolve (4). y
y
SANS has an elevated “Likelihood and Consequence” rating
from the NASA Human System Risk Board and accordingly
will require mitigation for manned deep space journey and
planetary missions of 1–3 years duration (4). In anticipation of
these future LDSF exploration missions including flights to Mars,
it is imperative to further study SANS, and develop strategies
to mitigate it. One potential challenge to studying SANS is
that it is uniquely associated with LDSF and the microgravity
environment, and therefore is difficult to study on the scale
required to understand the disease process. Thus, there is a need
for terrestrial analogs (i.e., methods to induce SANS-like findings
on earth) to accomplish this. SANS PATHOPHYSIOLOGY HYPOTHESES During LDSF, astronauts are exposed to a myriad of factors
that impose significant changes to the human mind and body. Microgravity, hypercarbia, and radiation must all be taken
into account when understanding the physiologic impact of
spaceflight. On Earth, there is a vertical hydrostatic pressure
gradient from gravitational downward force resulting in different
pressures throughout the body with relative increased pressure
in the lower extremities that are closer to the Earth’s center (23). A reduction in gravitational acceleration, such as that occurring
with travel away from earth, reduces the hydrostatic pressure
gradient and allows for a more uniform fluid redistribution in the
body with a net shift of fluid toward the cephalad region (3, 23–
25) (Figure 1). It is an important distinction that astronauts
onboard the ISS are still exposed to ∼90% of earth’s ground
gravitational pull (26). However, they are in a free fall state within
the ISS as they orbit at tremendous velocities that counterbalance
this gravitational pull, thus inducing the feeling of weightlessness
and the physiological effects of microgravity including cephalad
fluid migration (3, 23, 25–30). Citation: Head-Down Tilt Bed Rest (HDTBR)
is one such experimental model that has been used to simulate
the effects of microgravity to study multiple physiological systems
(5–14), and has become of interest as a terrestrial-based analog
for studying SANS and testing potential preventative strategies
and interventions, collectively referred to as countermeasures. SANS is characterized by an increased percentage of asymmetric
or unilateral disc edema and does not have similar patient
demographics found in terrestrial IIH (3, 15). Frontiers in Neurology | www.frontiersin.org CLINICAL PRESENTATION OF SANS Lastly, post-flight lumbar
puncture readings in astronauts with optic disc edema showed
an upper limit of high normal to slightly elevated post flight
opening pressures (21–28.5 cm H20), though these may not be
representative as they were collected 12–57 days post flight
(17). Interestingly, further studies of an astronaut with persistent
asymmetric optic disc swelling for 180 days demonstrated only
a mildly elevated opening pressure of 22 cm H20 6 days post-
flight and normal pressure of 16 cm H20 365 days post-flight (35). In addition, terrestrial IIH symptoms typically include headache
and pulsatile tinnitus, whereas the astronauts with optic disc
edema do not report these symptoms (36). Interestingly, optic
disc edema in astronauts may also persist up to 6 months after
spaceflight, long after the proposed cephalic fluid shift resolves
whereas the disc edema in IIH patients reduces relatively quickly
after reducing pressure in the optic nerve sheath (e.g., Optic Currently, astronauts utilize corrective plus sphere glasses
(in the past “Space Anticipation Glasses”), onboard spaceflight March 2021 | Volume 12 | Article 648958 Frontiers in Neurology | www.frontiersin.org 2 Ong et al. HDTBR - SANS Terrestrial Analog FIGURE 1 | Illustration of the initial cephalad fluid shift in the microgravity environment due to loss of hydrostatic pressure and cephalad fluid shift during terrestrial
HDTBR studies (not drawn to scale). FIGURE 1 | Illustration of the initial cephalad fluid shift in the microgravity environment due to loss of hydrostatic pressure an
HDTBR studies (not drawn to scale). FIGURE 1 | Illustration of the initial cephalad fluid shift in the microgravity environment due to loss of hydrostatic pressure and cephalad fluid shift during terrestrial
HDTBR studies (not drawn to scale). A third hypothesis is that SANS is not due to elevated
ICP but rather due to an upward shift of the brain during
microgravity (40). Post-flight MRIs in astronauts have shown
upward displacement of the optic chiasm (41). This observation
is postulated to be due to slight rotation of the brain in
microgravity, pulling the optic chiasm upwards and thereby
exerting tension on the optic nerve. Because the optic nerve
sheath (the dura that surrounds the optic nerve) is connected to
the periosteum of the orbital bone, this exerts a compressive force
on the optic nerve sheath, which in turn exerts a compressive
force on the posterior aspect of the eye. CLINICAL PRESENTATION OF SANS Together these cause
deformation of the eyeball (globe flattening) and expansion of
the optic nerve sheath without ICP elevation (40). Supporting
this hypothesis is a cohort study of twenty-two astronauts with
post-flight MRI scans demonstrating a 0.80 ± 0.74 mm (average
± SD) increase in optic nerve length from globe to chiasm
compared to pre-flight scans. This was associated with forward
displacement of the optic nerve head, which was related to
duration of spaceflight and clinical signs of SANS (42). nerve sheath fenestration) (35, 36). In addition, terrestrial IIH
commonly presents with bilateral optic disc edema in women
whereas SANS is characterized by an increased percentage of
asymmetric or unilateral disc edema more common in men (3,
4, 35, 36). The collection of these clinical findings eventually led
to the conclusion that venous hypertension leading to elevated
ICP may not be the sole reason for SANS. A second hypothesis postulates that in microgravity, CSF
pressure increases locally within the optic nerve sheath due to
a one-way valve-like mechanism allowing it to enter from the
cranial subarachnoid space, but not exit. A similar mechanism
of CSF accumulation has been theorized in the past with
terrestrial IIH patients who present with persistent optic disc
edema despite decreasing ICP (37). CSF biomarker studies have
characterized a difference in the CSF composition between the
subarachnoid spaces of the optic nerve and around the brain,
thus furthering the notion that CSF equilibration between the
spaces is incomplete (38). This theory of optic nerve sheath CSF
compartmentalization may account for the persistence of optic
disc edema in astronauts despite relatively normal to only slightly
elevated ICP pressures. In addition, astronauts with SANS exhibit
none of the typical terrestrial symptoms of increased ICP (e.g.,
headaches, diplopia, or tinnitus) (17, 36). Current study of the
pathophysiology that induces the compartmentalization of CSF
within the optic nerve sheath on Earth may give deeper insight
into this theory for SANS (39). While the pathogenesis behind SANS is not yet fully
understood, the development of these hypotheses allows for
proposal of potential experimental models of SANS on Earth
(terrestrial analogs). When high ICP was thought to be the main
cause, IIH was thought to be a close terrestrial analog but many
authors believe that IIH is a flawed model for SANS. Frontiers in Neurology | www.frontiersin.org HEAD-DOWN TILT BED REST STUDIES
AND CLINICAL FINDINGS Terrestrial-based analogs for LDSF have been of longstanding
interest in space medicine research as simulated microgravity is
an efficient platform to observe physiology in extreme situations. Analogs such as supine bed rest, head-down tilt bed rest
(HDTBR), wet immersion, dry immersion, and lower-extremity
limb suspension have been previously applied as earthbound
experimental models to further understand the human body
during spaceflight (24). Terrestrial-based analogs for LDSF have been of longstanding
interest in space medicine research as simulated microgravity is
an efficient platform to observe physiology in extreme situations. Analogs such as supine bed rest, head-down tilt bed rest
(HDTBR), wet immersion, dry immersion, and lower-extremity
limb suspension have been previously applied as earthbound
experimental models to further understand the human body
during spaceflight (24). HDTBR studies serve as a terrestrial analog by altering the
vector of gravitational force on the body to generate a cephalad
fluid shift similar to that seen in microgravity (43–45). Subjects
lay supine on a bed that has been tilted to lower the head at a
specific angle with the international standard angle established
at 6◦(46). Though other techniques such as parabolic flight
more closely simulate the actual microgravity condition, the brief
duration of exposure is not sufficient for manifestation of SANS-
like features. In contrast, long-duration HDTBR is feasible with
study durations of 370 days having been successfully completed
(24, 47). Furthermore, many outcomes analogous to those used
by NASA for study of astronauts (MRI, OCT, LP etc.) can be
measured in the HDT condition (Table 1). y
HDTBR studies have also been associated with short-term
ocular findings that may have implications for astronaut neuro-
ophthalmic health. A 7◦, 2 min head-down tilt was associated
with a decrease in choroidal pulsatile ocular blood flow,
suggesting retinal hypoperfusion (57). A 6◦, 12-h study HDTBR
observed no significant global minimum rim width thinning
compared to significant thinning seen in the seated position
for 12 h, although it was noted that Bruch’s membrane opening
height moved anteriorly during HDTBR. The study suggested
that this attenuation in neuroretinal rim thinning in postural
differences from seated to HDTBR may be due to translaminar
pressure difference, which has been proposed as a contributing
factor in glaucoma (58). A postural study on ambulatory
neurosurgical patients with continuous ICP monitoring observed
an increase in ICP during postural change from standing or
supine to 10◦& 20◦head-down tilt (4, 59). CLINICAL PRESENTATION OF SANS With these recent findings,
the role of the hypercapnic environment in SANS development
remains an area for further study. TABLE 1 | Spaceflight associated neuro-ocular syndrome (SANS) findings
demonstrated in head-down tilt bedrest studies. Spaceflight associated
neuro-ocular syndrome
(SANS) findings
Collective
head-down tilt
findings (up to
70 days)
Parameters
(angle and
duration)
References
Optic Nerve Sheath Distension
+
6◦, 60 min
(48)
Retinal Nerve Layer Thickening
+
6◦, 14, 30, & 70
days
(49, 50)
Optic Disc Edema
+
6◦, 30 days
(50, 51)
Choroidal Thickness Increase
+
6◦, 60 min
(48, 50)
(Possible) ICP Increase
+
10◦& 20◦, 5 min
(52)
Choroidal Folds
–
–
–
Hyperopic Shift
–
–
–
Cotton Wool Spots
–
–
– TABLE 1 | Spaceflight associated neuro-ocular syndrome (SANS) findings
demonstrated in head-down tilt bedrest studies. of other terrestrial analogs that may aid in understanding this
neuro-ophthalmic phenomenon (3). CLINICAL PRESENTATION OF SANS However,
further characterization of SANS has now led to consideration March 2021 | Volume 12 | Article 648958 Frontiers in Neurology | www.frontiersin.org 3 HDTBR - SANS Terrestrial Analog Ong et al. to 10 ± 2 mmHg when moving from a supine position without
pillow to with pillow, the research team hypothesized that use of
pillows and lifting the torso to eat meals may impact the cephalad
venous congestion induced by HDTBR and thus the HDTBR
protocol was modified with strict requirements prohibiting any
lifting of the head and torso, including raising elbows for support
to eat meals, or use of a standard pillow. The protocol was
also modified to more closely mimic ISS conditions with a mild
hypercapnic environment (PCO2 3.8 mmHg, 0.5%) compared to
earth’s surface (PCO2 0.6 mmHg, 0.04%), which was verified to
not impact arterial PCO2 levels (51, 53). A 6◦, 30-day HDTBR
study using this modified protocol was associated with Frisén
grade 1–2 optic disc edema observed in ∼45% of subjects (51). This provided strong support for strict HDTBR studies in a
hypercapnic environment as an experimental model to study the
pathophysiology and test countermeasures for SANS. The same
research group also compared healthy subjects undergoing strict
HDTBR with 20 astronauts during ∼30 days in spaceflight (50). In this study they found a larger increase in peripapillary total
retinal thickness in the strict HDTBR subjects than in the 20
astronauts with the average difference being 37 µm. Interestingly,
choroid thickness showed the opposite pattern; there was a
larger increase in astronauts compared to the strict HDTBR
subjects with the average difference being 27 µm. Another
study furthered this investigation of the mild hypercapnic
environment with HDBTR and observed that individuals who
developed SANS features from HDTBR demonstrated elevated
reliance on visuals cues when tested on cognitive performance
compared to non-SANS HDTBR individuals (54). The study
raised concerns that SANS in astronauts may influence inflight
performance for certain tasks or that SANS may be associated
with cognitive changes. However, a recent strict HDTBR study
in the mild hypercapnic environment of approximately 4 mmHg
PCO2 found no significant change in cerebrovascular reactivity,
hypercapnic ventilatory response, or arterialized PCO2 (55). Interestingly, observations of cerebral perfusion throughout
HDTBR demonstrated decrease in perfusion in all subjects with
higher perfusion in the subjects that developed SANS symptoms
compared to those who did not (56). Frontiers in Neurology | www.frontiersin.org HEAD-DOWN TILT BED REST STUDIES
AND CLINICAL FINDINGS 6◦, 60-min head-down One 6◦HDTBR study (14- & 70-days) observed a mild
increase in IOP with +1.42 and +1.79 mmHg from baseline,
respectively. This study also saw an increase in OCT peripapillary
retinal nerve thickening in the 70-day HDTBR compared to
the 14-day (+11.50 vs. +4.69 µm, superior peripapillary retinal
thickness), though overt optic disc edema was not apparent on
clinic exam (49). It was postulated that this RNFL thickening
represented early congestion of the optic nerve. Based on
the observation that subjects with Ommaya reservoirs, an
intraventricular catheter device that acts as a CSF conduit from
the ventricle to the scalp, had a reduction of ICP from 14 ± 2 March 2021 | Volume 12 | Article 648958 4 HDTBR - SANS Terrestrial Analog Ong et al. TABLE 2 | Intraocular pressure findings during spaceflight and head-down tilt
bedrest studies. IOP during
spaceflight
findings
Head-down tilt
findings
Parameters
(angle and
duration)
References
Acute IOP Increase
+
15◦, 21 min
(60)
Subsequent IOP
normalization to
baseline
Continued IOP
increase from
baseline
6◦, 14 & 70 days
(49) TABLE 2 | Intraocular pressure findings during spaceflight and head-down tilt
bedrest studies. devices that have been tested during spaceflight (65). Deflated
cuffs are typically placed around the thigh and inflated at specific
pressures (often ranging from 40 to 60 mmHg) to limit the
amount of fluid flow to the upper body (65). Interestingly, a 15◦
head-down tilt study utilizing bilateral thigh cuffs at 60 mmHg
for 10 min was not associated with significant differences in
peripapillary choroidal thickness or optic nerve sheath diameter
between cuffed individuals and controls (66). To mitigate the atrophying effect of microgravity on skeletal
muscle, astronauts undergo 2.5 h of intensive resistance and
aerobic exercise nearly every day onboard the ISS (67). However,
implementation of NASA’s integrated resistance and aerobic
training (iRAT) protocol during 70-day non-hypercarbic strict
6◦HDTBR was not associated with a significant difference in
retinal thickening or signs of optic disc edema compared to
a control HDTBR group who did not exercise, though IOP
was slightly higher (<1 mm Hg) in the exercise group (68). Interestingly, 15◦head down tilt for less than an hour was
associated with a decrease in IOP in subjects undergoing either
moderate-intensity aerobic, resistance, or high-intensity interval
aerobic exercise (69). These differences highlight how the impacts
of countermeasures are affected by the duration and angle of
HDTBR. HEAD-DOWN TILT BED REST STUDIES
AND CLINICAL FINDINGS Integration of results from different HDTBR models is
likely necessary to understand the short- and long-term effects. tilt was associated with optic nerve sheath distension on orbital
ultrasound, similar to that seen in astronauts after 1 month
in-flight (4, 18, 48), as well as increased subfoveal choroidal
thickness on OCT (48). Certain SANS findings such as refractive
shift, cotton wool spots, and choroidal folds have not yet been
observed in individuals in HDTBR studies. Some studies have identified possible risk factors for SANS. 15◦, 21-min HDT was associated with higher peak IOP in
moderate myopes (19.8 mmHg) compared to emmetropes and
low myopes (18.6 & 18.7 mmHg, respectively) (60) (Table 2). In another study, single-nucleotide polymorphisms involved
in vitamin B9 and B12 metabolism (5-methyltetrahydrofolate-
homocysteine methyltransferase reductase (MTRR) 66 G and
serine hydroxymethyltransferase 1 (SHMT1) 1420C alleles) were
associated with the magnitude of optic disc edema during
strict HDTBR (61). These studies highlight the potential utility
of HDTBR to generate hypotheses regarding SANS etiology,
identify screening tests, as well as a possible role to empirically
screen astronaut candidates. HDTBR has been leveraged to develop devices for monitoring
SANS features that are feasible for inflight use. For example,
monitoring ICP during spaceflight is a topic of interest, with
invasive measurements such as lumbar puncture currently not
feasible inflight. Therefore, deployment of non-invasive devices
to measure ICP is desirable (70, 71). Otoacoustic Emission
(OAE) phase change is a candidate non-invasive method to
monitor ICP. Changes to the positioning and tension of the
middle ear caused by ICP changes are detected by a OAE probe
placed at the opening of the ear canal. The OAE technique has
been tested in head-down tilt and helps guide interpretation
of OAE measurements taken aboard the ISS (72). Ocular
vestibular evoked myogenic potentials (oVEMPs), which record
extraocular muscle activity during vestibular stimulation, were
observed to be associated with head-down tilt magnitude,
supporting
oVEMPs
as
a
non-invasive
ICP
monitoring
tool (73). Frontiers in Neurology | www.frontiersin.org LIMITATIONS OF HDTBR Although
HDTBR
is
a
promising
terrestrial
analog
for
investigating SANS and for testing the efficacy of any potential
countermeasures, certain limitations must be taken into account. HDTBR results support genetic traits such as MTRR and
SHMT1 as risk factors. HDTBR itself may be a practical
way to screen for risk of SANS development in individuals. HDTBR results also support the use of lower body negative
pressure as a countermeasure for SANS. Other countermeasures
may include artificial gravity, dietary supplementation, varying
training regiments, topical or oral medication, non-invasive
monitoring, and external device utilization during or before
flight/HDTBR. HDTBR may be helpful to identify the best in-
flight measurements with which to diagnose and monitor SANS. One notable limitation is the small sample size of HDTBR
studies, reflecting their time and resource intensive nature. The commitment of subjects to participate for months while
maintaining a strict position that may be uncomfortable and
induce headaches, which may make recruitment challenging
(76). Generalizing HDTBR observations to LDSF and SANS
may be limited by differences between terrestrial subjects and
astronauts. Two years before a spaceflight mission, astronauts
undergo intensive pre-flight strength and aerobic conditioning
(77). Matching HDTBR subjects to a similar training regiments
prior to HDTBR would be ideal but impractical. Screening
protocols, such as a modified Air Force Class III physical exam
and clearance by NASA Test Subject Screening facility have been
applied to address this and select subjects similar to astronauts in
terms of age, height, weight and physical fitness (48). Beyond government-funded space exploration, private space
companies (e.g., SpaceX, Blue Origin) strive to increase the
accessibility to spaceflight for civilian populations as space
tourism (78–80). Short-term HDTBR may be a practical
way to screen civilians for susceptibility to consequences of
cephalad shifts. Furthermore, a collaborative effort between
private space companies and HDTBR researchers may provide
novel understandings to short-term spaceflight in the civilian
population when comparing pre/post-flight and HDTBR data. As we head into a new era of spaceflight, HDTBR emerges
as a promising terrestrial analog to understand how to
optimally protect the neuro-ophthalmic health of civilians
and astronauts. The variability in conditions for various studies also makes
it challenging to integrate the findings of HDTBR with SANS. While a uniform, international standard 6◦angle has been
established, the strictness of HDT positioning is not standardized
and reports of studies often do not include sufficient detail to
permit replication. COUNTERMEASURE TESTING AND
MONITORING DEVELOPMENT IN
HEAD-DOWN TILT BED REST While advancing our understanding of SANS is an important
application of HDTBR, the study model is also useful for
testing possible countermeasures which may mitigate SANS. Lower body negative pressure (LBNP) device is a non-invasive
technique that surrounds the pelvic area and legs and simulates
the effects of gravity by inducing fluid redistribution from
the cephalad region toward the lower body (52). LBNP
has been studied during spaceflight as a countermeasure for
cardiovascular responses toward microgravity-induced fluid
shifts and orthostatic intolerance upon returning to earth’s gravity
(62). LBNP of −20 mmHg during a 5-h HDTBR was associated
with less increase in optic nerve sheath diameter as well as
orbital and intracranial cerebrospinal fluid volume measured
with MRI compared to the control group (63). Another HDTBR
countermeasure study found a 40% decrease in increased choroid
volume in association with use of −20 mmHg LBNP for 8
h/day during 3 days of strict 6◦HDTBR. Although the choroid
volume was still increased during this HDTBR study, this
significant attenuation suggests that LBNP may be an effective
countermeasure for SANS (64). Thigh cuffs are also non-invasive For years during spaceflight, OCT has been crucial in
quantifying the retinal nerve fiber layer (RNFL) changes to
supplement the clinical observations of SANS. In December
2018, OCT angiography (OCTA) became available on the ISS
(3). OCTA is a non-invasive, high resolution ocular imaging
technique that measures blood flow information and provides
angiographic data that corresponds to retinal and choroidal
vessels (74). Compared to invasive, contrast enhanced, fundus
fluorescein angiography and indocyanine green angiography,
OCTA is a non-invasive approach to blood flow visualization in
the retina and choroid and may play an increasingly important
role in evaluating the ocular vasculature in SANS (3, 75). This
technology onboard the ISS will offer a clearer understanding of
the volumetric shifts during spaceflight, particularly in the setting
of the SANS cephalad fluid theory. Utilizing OCTA with HDTBR
studies and comparing these results to ISS OCTA will provide March 2021 | Volume 12 | Article 648958 Frontiers in Neurology | www.frontiersin.org 5 HDTBR - SANS Terrestrial Analog Ong et al. completed on Earth, are an important tool with which to achieve
this and HDTBR is a promising one as, with the right parameters,
it is associated with many of the ophthalmic findings seen in
SANS. COUNTERMEASURE TESTING AND
MONITORING DEVELOPMENT IN
HEAD-DOWN TILT BED REST Further HDTBR studies may provide useful information
by applying advances in ophthalmic and neuro imaging and
image analysis. novel information on HDTBR’s ability to mimic spaceflight-
induced fluid shifts within the retina vasculature. AUTHOR CONTRIBUTIONS JO performed the primary literature review and drafted the initial
manuscript, tables, and figure, and edited the manuscript. HM
and AL provided feedback and edited the manuscript. All authors
contributed to the article and approved the submitted version. LIMITATIONS OF HDTBR There are conditions induced by HDTBR that
are inconsistent with spaceflight, such as subjects having their
posterior side in contact with the bed at all times. It is not known
how these features impact results and the homology between
HDTBR and SANS. FUNDING This work was supported by NIH p30 026877, Research to
Prevent Blindness unrestricted grant to Stanford Department
of Ophthalmology. DISCUSSION AND FUTURE DIRECTIONS A planned manned mission to Mars will require even longer
duration of spaceflight than the longest ISS flights to date. Understanding the pathogenesis; identifying those at increased
risk; and mitigating the effects of SANS are of critical importance
so that astronauts can complete LDSF missions safely. Terrestrial
analogs, that is, experimental models of SANS that can be 4. Stenger MB, Tarver WJ, Brunstetter T, Gibson CR, Laurie SS, Lee S, et al.
Evidence Report: Risk of Spaceflight Associated Neuro-Ocular Syndrome
(SANS)
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Research
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spaceflight. Nature. (2020) 582. doi: 10.1038/d41586-020-01554-8 80. Witze A. SpaceX to launch astronauts - and a new era of private human
spaceflight. Nature. (2020) 582. doi: 10.1038/d41586-020-01554-8 Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest. Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest. 64. Lawley JS, Babu G, Janssen S, Petersen LG, Hearon CM Jr, Dias KA,
et al. Daily generation of a footward fluid shift attenuates ocular changes
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31. doi: 10.1152/japplphysiol.00250.2020 The Handling Editor declared a past co-authorship with two of the authors,
AL and HM. 65. Arbeille P, Fomina G, Achaibou F, Pottier J, Kotovskaya A. Cardiac
and vascular adaptation to 0g with and without thigh cuffs (Antares 14
and Altair 21 day Mir spaceflights). Acta Astronaut. (1995) 36:753–62. doi: 10.1016/0094-5765(95)00166-2 Copyright © 2021 Ong, Lee and Moss. This is an open-access article distributed
under the terms of the Creative Commons Attribution License (CC BY). The use,
distribution or reproduction in other forums is permitted, provided the original
author(s) and the copyright owner(s) are credited and that the original publication
in this journal is cited, in accordance with accepted academic practice. No use,
distribution or reproduction is permitted which does not comply with these terms. 66. Macias B, Balasubramanian S, Huang AS, Liu JH, Lee SM, Laurie S. Fluid
Shift Induced Alterations of the Optic Nerve Head and Peripapillary Choroid
Assessed using Optical Coherence Tomography Investigative Ophthalmology
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Ophthalmology & Visual Science (2017). REFERENCES March 2021 | Volume 12 | Article 648958 Frontiers in Neurology | www.frontiersin.org
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2023 MART – psixologik kompetentlik–pedagogik jarayonda sog’lom psixologik muhitni yarata
olish, talabalar va ta’lim jarayonining boshqa ishtirokchilari bilan ijobiy muloqotni tashkil
etish, turli salbiy psixologik ziddiyatlarni o’z vaqtida anglay olish va bartaraf eta olish[15]; – metodik kompetentlik– pedagogik jarayonni metodik jihatdan oqilona tashkil etish,
ta’lim yoki tarbiyaviy faoliyat shakllarini to’g’ri belgilash, metod va vositalarni maqsadga
muvofiq tanlay olish, metodlarni samarali qo’llay olish, vositalarni muvaffaqiyatli qo’llash;
informasion kompetentlik – axborot muhitida zarur, muhim, kerakli, foydali ma’lumotlarni
izlash, yig’ish, saralash, qayta ishlash va ulardan maqsadli, o’rinli, samarali foydalanish[7]; – metodik kompetentlik– pedagogik jarayonni metodik jihatdan oqilona tashkil etish,
ta’lim yoki tarbiyaviy faoliyat shakllarini to’g’ri belgilash, metod va vositalarni maqsadga
muvofiq tanlay olish, metodlarni samarali qo’llay olish, vositalarni muvaffaqiyatli qo’llash;
informasion kompetentlik – axborot muhitida zarur, muhim, kerakli, foydali ma’lumotlarni
izlash, yig’ish, saralash, qayta ishlash va ulardan maqsadli, o’rinli, samarali foydalanish[7]; – kreativ kompetentlik – pedagogik faoliyatga tanqidiy, ijodiy yondashish, o’zining
ijodkorlik malakalariga egaligini namoyish eta olish; – innovasion kompetentlik–pedagogik jarayonni takomillashtirish, ta’lim sifatini
yaxshilash, tarbiya jarayonining samaradorligini oshirishga doir yangi g’oyalarni ilgari
surish, ularni amaliyotga samarali tatbiq etish[14]; – kommunikativ kompetentlik – ta’lim jarayonining barcha ishtirokchilari, jumladan,
talabalar bilan samimiy muloqotda bo’lish, ularni tinglay bilish, ularga ijobiy ta’sir ko’rsata
olish[8]. – Shaxsiy kompetentlik – izchil ravishda kasbiy o’sishga erishish, malaka darajasini
oshirib borish, kasbiy faoliyatda o’z ichki imkoniyatlarini namoyon qilish. – Shaxsiy kompetentlik – izchil ravishda kasbiy o’sishga erishish, malaka darajasini
oshirib borish, kasbiy faoliyatda o’z ichki imkoniyatlarini namoyon qilish. – Texnologik
kompetentlik
–
kasbiy-pedagogik
BKMni
boyitadigan
ilg’or
texnologiyalarni o’zlashtirish, zamonaviy vosita, texnika va texnologiyalardan foydalana
olish. – Ekstremal kompetentlik – favqulotda vaziyatlar (tabiiy ofatlar, texnologik jarayon
ishdan chiqqan)da, pedagogik nizolar yuzaga kelganda oqilona qaror qabul qilish, to’g’ri
harakatlanish malakasiga egalik[9]. Kasbiy-pedagogik kompetentlikka ega bo’lishda o’z ustida ishlash, o’zo’zini rivojlantirish
muhim ahamiyatga ega[16]. O’z-o’zini rivojlantirish vazifalari o’zini o’zi tahlil qilish va
o’zini o’zi baholash orqali aniqlanadi. O’z ustida ishlash – pedagogning izchil ravishda
o’zining kasbiy BKM va shaxsiy sifatlarini rivojlantirib borish yo’lida amaliy harakatlarni
tashkil etishi[10]. O’z ustida ishlash quyidagilarda ko’rinadi: O’z ustida ishlash quyidagilarda ko’rinadi: - kasbiy BKMni takomillashtirib borish; - faoliyatga tanqidiy va ijodiy yondashish; - kasbiy va ijodiy hamkorlikka erishish;- ishchanlik qobiliyatini rivojlantiri - kasbiy va ijodiy hamkorlikka erishish;- ishchanlik qobiliyatini rivojlantirish;
- salbiy odatlarni bartaraf etib borish; ijobiy sifatlarni o’zlashtirish. salbiy odatlarni bartaraf etib borish; ijobiy sifatlarni o’zlashtirish. PEDAGOGNING KASBIY KOMPETENTLIGI –TA’LIM SIFATINING GAROVI Maqsuda Norbosheva,
Termiz davlat universiteti
Maktabgacha ta’lim kafedrasi mudiri Yangiboyeva Gavhar,
Jarqo’rg’on biznes va yengil-sanoat texnikumi
O'quv tarbiyaviy ishlar bo'yicha direktor o'rinbosari Annotatsiya: Tarbiyachi-pedagog yosh avlodni xalqimizning munosib farzandlari
qilib tayyorlashdek muhim, faxrli ish bilan birga mas’uliyatli vazifani bajaradi. Tarbiyachi-
pedagogning siyosiy yetukligi bolalarni tarbiyalash sifati uchun xalq hamda jamiyat
oldidagi o’z mas’uliyatini anglashga, ta’lim-tarbiya vazifalarini ijodiy yondashishga, o’z
mahoratini doimo takomillashtirib borish va ishdagi o’rtoqlarini o’sishiga yordam beradi. Kalit so`z: maktabgacha, ta’lim, tarbiya, jarayon, shaxs, bola. Demak, tarbiyachi-pedagog-pedagog avvalo, bilimli bo’lishi, o’zi yashab turgan
ulkan hayotini bilishi, tabiat va jamiyatning qonuniyatlarini tushunishi, ijtimoy faol bo’lishi,
umumiy va maktabgacha tarbiya pedagogikasini, bolalar ruhiyati va fiziologiyasini
egallashi va bolalarning yosh xususiyatlarini bilishi kerak. Shuningdek, pedagogik
tarbiyachi-pedagogning hodisalarni tahlil qilishga ilmiy nuqtai nazardan yondashuvi bolani
har tomonlama rivojlantirish muvaffaqiyatini amalga oshirishga imkon beradi[1]. “Kompetentlik” tushunchasi ta’lim sohasiga psixologik izlanishlar natijasida kirib
kelgan. Shu sababli kompetentlik “noan’anaviy vaziyatlar, kutilmagan hollarda
mutaxassisning o’zini qanday tutishi, muloqotga kirishishi, raqiblar bilan o’zaro
munosabatlarda yangi yo’l tutishi, noaniq vazifalarni bajarishda, ziddiyatlarga to’la
ma’lumotlardan foydalanishda, izchil rivojlanib boruvchi va murakkab jarayonlarda
harakatlanish rejasiga egalik”ni anglatadi[5]. Kasbiy kompetentlik – mutaxassis tomonidan kasbiy faoliyatni amalga oshirish uchun
zarur bo’lgan bilim, ko’nikma va malakalarning egallanishi va ularni amalda yuqori
darajada qo’llay olinishidir. Ijtimoiy kompetentlik – ijtimoiy munosabatlarda faollik ko’rsatish ko’nikma,
malakalariga egalik, kasbiy faoliyatda subyektlar bilan muloqotga kirisha olish. Maxsus
kompetentlik–kasbiy-pedagogik faoliyatni tashkil etishga tayyorlanish, kasbiy-pedagogik
vazifalarni oqilona hal qilish, faoliyati natijalarini real baholash, BKMni izchil rivojlantirib
borish bo’lib, ushbu kompetentlik negizida psixologik, metodik, informasion, kreativ,
innovasion va kommunikativ kompetentlik ko’zga tashlanadi. Ular o’zida quyidagi
mazmunni ifodalaydi[6]: 74 ADABIYOTLAR: 1. Farberman B. Ilgor pedagogik texnologiyalar. – T.: «Fan», 2000 y. 3. Tolipova J.O. Pedagogik texnologiyalar-do’stona muhit yaratish omili. -T.: YUNISEF, 2005 y. 4. Tolipov U.Q, Usmonboyeva M. Pedagogik texnologiyalarning tatbiqiy asoslari (o’quv qo’llanma). nashriyoti, 2006 y. y
dukarimovich, Y. T. (2023, January). SPECIFICITY OF PEDAGOGICAL AND PSYCHOLOGICAL 5. Abdukarimovich, Y. T. (2023, January). SPECIFICITY OF PEDAGOGICAL AND PSYCHOLOGICAL 5. Abdukarimovich, Y. T. (2023, January). SPECIFICITY OF PEDAGOGICAL AND PSYCHOLOGICAL
PREPARATION OF YOUNG PEOPLE FOR THE FAMILY. In Conference Zone (pp. 147-149). 6. Yo‘ldoshev Tojiddin Abdukarimovich. (2023). OILADA BOLALARNI TO‘G‘RI TARBIYALASHNING ASOSIY PREPARATION OF YOUNG PEOPLE FOR THE FAMILY. In Conference Zone (pp. 147-149). 6. Yo‘ldoshev Tojiddin Abdukarimovich. (2023). OILADA BOLALARNI TO‘G‘RI TARBIYALASHNING ASOSIY (pp
)
6. Yo‘ldoshev Tojiddin Abdukarimovich. (2023). OILADA BOLALARNI TO‘G‘RI TARBIYALASHNING ASOSIY 6. Yo‘ldoshev Tojiddin Abdukarimovich. (2023). OILADA BOLALARNI TO‘G‘RI TARBIYALASHNING ASOSIY
SHARTLARI VA MUAMMOLARI. SCHOLAR, 1(1), 139–143. ,
,
,
,
,
(
)
RELATIONSHIP OF RESPONSIBILITY TO THE FAMILY IN YOUNG PEOPLE. (
)
RELATIONSHIP OF RESPONSIBILITY TO THE FAMILY IN YOUNG PEOPLE. 8. Abdukarimovich, Y. T. (2022). BOSHLANG ‘ICH SINF O ‘QUVCHILARNI TARBIYALASHDA MA ‘NAVIY- 8. Abdukarimovich, Y. T. (2022). BOSHLANG ‘ICH SINF O ‘QUVCHILARNI TARBIYALASHDA MA ‘NAVIY-
AXLOQIY XUSUSIYATLARNI SHAKLLANTIRISH. Uzbek Scholar Journal, 11, 114-116. 8. Abdukarimovich, Y. T. (2022). BOSHLANG ‘ICH SINF O ‘QUVCHILARNI TARBIYALASHDA MA ‘NAVIY-
AXLOQIY XUSUSIYATLARNI SHAKLLANTIRISH. Uzbek Scholar Journal, 11, 114-116. ,
(
)
Q
AXLOQIY XUSUSIYATLARNI SHAKLLANTIRISH. Uzbek Scholar Journal, 11, 114-116. 9
Abd k i
i h Y T N
t
i h K A & K kh
S S (2022) C i i
f th A
f M t
it
Th AXLOQIY XUSUSIYATLARNI SHAKLLANTIRISH. Uzbek Scholar Journal, 11, 114-116. 9. Abdukarimovich, Y. T., Nurmamatovich, K. A., & Kakhramonovna, S. S. (2022). Crisis of the Age of Maturity, The
Formation of The Image of “I”. Global Scientific Review, 9, 21-25. g
, ,
10. Abdukarimovich, Y. T., & Melikmurodovich, S. U. B. (2022). Art Therapy in Eliminating Aggressiveness in the
Individual. Global Scientific Review, 9, 1-4. Individual. Global Scientific Review, 9, 1-4. 11. Isakovich, U. F., & Abdukarimovich, Y. T. (2022). THE PECULIARITIES OF THE FORMATION OF SPIRITUAL , ,
11. Isakovich, U. F., & Abdukarimovich, Y. T. (2022). THE PECULIARITIES OF THE FORMATION OF SPIRITUAL 11. Isakovich, U. F., & Abdukarimovich, Y. T. (2022). THE PECULIARITIES OF THE FORMATION OF SPIRITUAL EDUCATION IN CHILDREN IN THE FAMILY. 2023 MART -faoliyatiga fan-texnikaning so’nggi yangiliklarini samarali tatbiq etish; - kasbiy va malakalarini takomillashtirish; - kasbiy va malakalarini takomillashtirish; - ko’nikma salbiy pedagogik nizolarning oldini olish, bartaraf etish choralarini izlash yo’lida
olib boradigan amaliy harakati uning o’z ustida ishlashini ifodalaydi. - ko’nikma salbiy pedagogik nizolarning oldini olish, bartaraf etish choralarini izlash yo’lida
olib boradigan amaliy harakati uning o’z ustida ishlashini ifodalaydi. Pedagoglarning o’z ustilarida izchil, samarali ishlashlarida faoliyatga loyihali yondasha
olishlari qo’l keladi. Ularning loyihali yondashuv asosida quyidagi modelni shakllantira
olishlari maqsadga muvofiqdir[11]. Pedagogning kasbiy kompetentlikka ega bo’lishida o’zini tahlil qila olishi ham
ahamiyatli sanaladi. O’zini o’zi tahlil qilish pedagog tomonidan kasbiy faoliyatda tashkil
etayotgan o’z amaliy harakatlari mohiyatining o’rganilishidir. Xulosa o’rnida shuni aytish mumkinki, tarbiyachi-pedagog-maxsus pedagogik, psixologik
va mutaxassisligi bo’yicha yetuk bilimli va yuksak fazilatli shaxs sanaladi. Shuning uchun
zamonaviy tarbiyachi-pedagog-murabbiy qiyofasida ko’plab fazilatlarni namoyon eta olishi
kerak[12]. Zamonaviy murabbiy o’zini mutaxassisligi bo’yicha chuqur puxta bilimga ega
bo’lish, o’z ustida tinimsiz ishlashi kerak. U pedagogika va psixologiya, fiziologiya
fanlarining asoslarini bilish, ta’lim-tarbiya jarayonida bolalarning yoshi va psixologik
xususiyatlarini inobatga olgan holda faoliyatni tashkil etishi kerak. Pedagogning kasbiy kompetentlikka ega bo’lishida o’zini tahlil qila olishi ham
ahamiyatli sanaladi. O’zini o’zi tahlil qilish pedagog tomonidan kasbiy faoliyatda tashkil
etayotgan o’z amaliy harakatlari mohiyatining o’rganilishidir. 2023 MART Pedagogning mutaxassis sifatida[13]: - aniq maqsad, intilish asosida pedagogik jarayonni takomillashtirish;-pedagogik jarayon
samaradorligini, o’zining ishchanlik faolligini oshirish; - aniq maqsad, intilish asosida pedagogik jarayonni takomillashtirish;-pedagogik jarayon
samaradorligini, o’zining ishchanlik faolligini oshirish; -izchil ravishda yangilanib borayotgan pedagogik bilimlarni o’zlashtirish;-ilg’or
texnologiya, metod hamda vositalardan xabardor bo’lish; -izchil ravishda yangilanib borayotgan pedagogik bilimlarni o’zlashtirish;-ilg’or
texnologiya, metod hamda vositalardan xabardor bo’lish; 75 ADABIYOTLAR: European Journal of Interdisciplinary Research and Development, 9, 1-4. 12. Yuldashev, T. (2022, February). Measures to eliminate or prevent the manifestation of personality aggression. In
Conference Zone (pp. 227-229). EDUCATION IN CHILDREN IN THE FAMILY. European Journal of Interdisciplinary Research and Development, 9, 1-4. 12. Yuldashev, T. (2022, February). Measures to eliminate or prevent the manifestation of personality aggression. In
Conference Zone (pp. 227-229). (pp
)
13. Abdukarimovich, Y. T., & Melikboboyevich, S. U. (2022). FORMING A RESPONSIBLE ATTITUDE TO THE
FAMILY IN YOUNG PEOPLE. Galaxy International Interdisciplinary Research Journal, 10(1), 653-657. 14
Abd k i
i h Y T (2021) P d
i
l di
ti
ti
b t
f
il
d d
ti
l
i
ti 13. Abdukarimovich, Y. T., & Melikboboyevich, S. U. (2022). FORMING A RESPONSIBLE ATTITUDE TO THE
FAMILY IN YOUNG PEOPLE. Galaxy International Interdisciplinary Research Journal, 10(1), 653-657. 14
Abd k i
i h
(2021)
d
i
l di
i
i
b
f
il
d d
i
l
i
i FAMILY IN YOUNG PEOPLE. Galaxy International Interdisciplinary Research Journal, 10(1), 653-657. (
)
g g
p
y
g
15. Yo’Ldoshev, T. (2021). KELAJAK AVLODNI TARBIYALASHNING PSIXOLOGIK VA PEDAGOGIK
XUSUSIYATLARI. Academic research in educational sciences, 2(NUU Conference 1), 333-336. (
)
g g
p
y
g
15. Yo’Ldoshev, T. (2021). KELAJAK AVLODNI TARBIYALASHNING PSIXOLOGIK VA PEDAGOGIK
XUSUSIYATLARI. Academic research in educational sciences, 2(NUU Conference 1), 333-336. 16. Йулдошев, Т. А. (2018). ЦЕННОСТНЫЕ ОРИЕНТАЦИИ В ПРОФЕССИОНАЛЬНОЙ ДЕЯТЕЛЬНОСТИ
БУДУЩИХ ПЕДАГОГОВ Ж
б
П
б XUSUSIYATLARI. Academic research in educational sciences, 2(NUU Conference 1), 333 336. 16. Йулдошев, Т. А. (2018). ЦЕННОСТНЫЕ ОРИЕНТАЦИИ В ПРОФЕССИОНАЛЬНОЙ ДЕЯТЕЛЬНОСТИ
БУДУЩИХ ПЕДАГОГОВ. Журнал выпускается ежемесячно, публикует статьи по гуманитарным наукам. Подробнее
на, 85. 76 76
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Scorpions of Sri Lanka (Scorpiones: Buthidae, Chaerilidae, Scorpionidae) with description of four new species of the genera Charmus Karsch, 1879 and Reddyanus Vachon, 1972, stat. n
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Scorpions of Sri Lanka (Arachnida, Scorpiones: Buthidae,
Chaerilidae, Scorpionidae) with Description of Four New
Species of the Genera Charmus Karsch, 1879 and Reddyanus
Vachon, 1972, stat. n.
František Kovařík, Graeme Lowe, Kithsiri B. Ranawana, David Hoferek,
V. A. Sanjeewa Jayarathne, Jana Plíšková & František Šťáhlavský
March 2016 – No. 220 Scorpions of Sri Lanka (Arachnida, Scorpiones: Buthidae,
Chaerilidae, Scorpionidae) with Description of Four New
Species of the Genera Charmus Karsch, 1879 and Reddyanus
Vachon, 1972, stat. n. František Kovařík, Graeme Lowe, Kithsiri B. Ranawana, David Hoferek,
V. A. Sanjeewa Jayarathne, Jana Plíšková & František Šťáhlavský Scorpions of Sri Lanka (Arachnida, Scorpiones: Buthidae,
Chaerilidae, Scorpionidae) with Description of Four New
Species of the Genera Charmus Karsch, 1879 and Reddyanus
Vachon, 1972, stat. n. March 2016 – No. 220 March 2016 – No. 220 March 2016 – No. 220 Derivatio Nominis The name Euscorpius Thorell, 1876 refers to the most common genus of scorpions in the
Mediterranean region and southern Europe (family Euscorpiidae). Euscorpius is located at: http://www.science.marshall.edu/fet/Euscorpius (Marshall University, Huntington, West Virginia 25755-2510, USA) (Marshall University, Huntington, West Virginia 25755-2510, USA) Euscorpius
Occasional Publications in Scorpiology
EDITOR: Victor Fet, Marshall University, ‘fet@marshall.edu’
ASSOCIATE EDITOR: Michael E. Soleglad, ‘soleglad@znet.com’ Euscorpius is the first research publication completely devoted to scorpions (Arachnida:
Scorpiones). Euscorpius takes advantage of the rapidly evolving medium of quick online
publication, at the same time maintaining high research standards for the burgeoning field of
scorpion science (scorpiology). Euscorpius is an expedient and viable medium for the publication
of serious papers in scorpiology, including (but not limited to): systematics, evolution, ecology,
biogeography, and general biology of scorpions. Review papers, descriptions of new taxa, faunistic
surveys, lists of museum collections, and book reviews are welcome. Publication date: 28 March 2016
http://www.zoobank.org/urn:lsid:zoobank.org:pub:DD0DF45D-F63A-4AA2-8EFF-03CF99E297EF Summary Data about all 18 known scorpion species of Sri Lanka are summarized. All previously known species were newly
collected and four new species discovered during a scorpiological expedition in 2015. Information is provided
about their taxonomy, distribution, ecology, and reproductive biology, fully complemented with color photos of
live and preserved specimens, as well as their habitat. Subgenus Isometrus (Reddyanus) Vachon, 1972 is elevated
to genus level, Reddyanus stat. n., supported by new characters of setation on leg tarsomere II and hemi-
spermatophore morphology. Charmus saradieli sp. n., Reddyanus ceylonensis sp. n., R. jayarathnei sp. n., and R. ranawanai sp. n. are described, compared with other species and fully illustrated. Heterometrus serratus (Pocock,
1900) is restored and differentiated from H. indus (Geer, 1778). Charmus minor Lourenço, 2002 is synonymized
with Charmus laneus Karsch, 1879; Isometrus garyi Lourenço et Huber, 2002 is synonymized with Reddyanus
loebli (Vachon, 1982) comb. n., and Heterometrus spinifer solitarius Couzijn, 1981 is synonymized with
Heterometrus indus (Geer, 1778). A key and distribution maps for all 18 Sri Lankan scorpion species are
presented. Hemispermatophores of Buthoscorpio sarasinorum (Karsch, 1892), Charmus laneus Karsch, 1879,
Isometrus thwaitesi Pocock, 1897, Lychas srilankensis Lourenço, 1997, Reddyanus basilicus (Karsch, 1879)
comb. n., R. ceylonensis sp. n., R. loebli comb. n. and Heterometrus gravimanus (Pocock, 1894) are illustrated
and described for the first time. In additional to morphological analysis, we also describe the karyotype of
Buthoscorpio sarasinorum (2n=14); Charmus laneus (2n=9); Isometrus thwaitesi (2n=8); Lychas srilankensis
(2n=16); Reddyanus basilicus comb. n. (2n=15–16); R. ceylonensis sp. n. (2n=16); and R. loebli comb. n. (2n=17). The significance of cytogenetics and hemispermatophore morphology in buthid taxonomy are discussed. other described species, with type localities cited as
"India or Ceylon" (Heterocharmus cinctipes Pocock,
1892 = Charmus laneus Karsch, 1879), and "India"
(Heterometrus indus (De Geer, 1778)) are actually Sri
Lankan endemics. Euscorpius — Occasional Publications in Scorpiology. 2016, No. 220
Scorpions of Sri Lanka (Scorpiones: Buthidae, Chaerilidae,
Scorpionidae) with description of four new species of the
genera Charmus Karsch, 1879 and Reddyanus Vachon,
1972, stat. n.
František Kovařík 1, 4, Graeme Lowe 2, Kithsiri B. Ranawana 3, David Hoferek 1, V. A.
Sanjeewa Jayarathne 3, Jana Plíšková 4 & František Šťáhlavský 4
1 P.O. Box 27, CZ-145 01 Praha 45, Czech Republic, www.scorpio.cz
2 Monell Chemical Senses Center, 3500 Market St., Philadelphia, PA 19104-3308, USA
3 Department of Zoology, Faculty of Science, University of Peradeniya, Sri Lanka
4 Department of Zoology, Charles University, Viničná 7, CZ-128 44 Praha 2,
Czech Republic http://www.zoobank.org/urn:lsid:zoobank.org:pub:DD0DF45D-F63A-4AA2-8EFF-03CF99E297EF http://www.zoobank.org/urn:lsid:zoobank.org:pub:DD0DF45D-F63A-4AA2-8EFF-03CF99E297EF ICZN COMPLIANCE OF ELECTRONIC PUBLICATIONS: Electronic (“e-only”) publications are fully compliant with ICZN (International Code of Zoological Nomenclature)
(i.e. for the purposes of new names and new nomenclatural acts) when properly archived and registered. All
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2001 2012 Between 2000 and 2013, ICZN did not accept online texts as "published work" (Article 9.8). At this time,
Euscorpius was produced in two identical versions: online (ISSN 1536-9307) and CD-ROM (ISSN 1536-9293) (laser
disk) in archive-quality, read-only format. Both versions had the identical date of publication, as well as identical
page and figure numbers. Only copies distributed on a CD-ROM from Euscorpius in 2001-2012 represent
published work in compliance with the ICZN, i.e. for the purposes of new names and new nomenclatural acts. disk) in archive quality, read only format. Both versions had the identical date of publication, as well as identical
page and figure numbers. Only copies distributed on a CD-ROM from Euscorpius in 2001-2012 represent
published work in compliance with the ICZN, i.e. for the purposes of new names and new nomenclatural acts. In September 2012, ICZN Article 8. What constitutes published work, has been amended and allowed for electronic
publications, disallowing publication on optical discs. From January 2013, Euscorpius discontinued CD-ROM
production; only online electronic version (ISSN 1536-9307) is published. For further details on the new ICZN
amendment, see http://www.pensoft.net/journals/zookeys/article/3944/. Introduction Early authors (1879–1913) cited scorpion records
from Sri Lanka mostly in the course of broader works on
Indian or worldwide scorpions, and these records were
based on several often solitary specimens without exact
localities. They described six Sri Lankan species, with
type localities cited only as "Ceylon", of which five are
still valid (i.e. Charmus laneus Karsch, 1879; Isometrus
thwaitesi Pocock, 1897; Reddyanus basilicus (Karsch,
1879) comb. n.; Heterometrus gravimanus (Pocock,
1894); Scorpio ceylonicus Herbst, 1800 = Heterometrus
indus; Heterometrus serratus (Pocock, 1900)). Two Until now, the most comprehensive study on Sri
Lankan scorpions was authored by Vachon (1982) who
analyzed material collected by entomologists Claude
Besuchet and Ivan Löbl from MHNG. Vachon also cited
older records and created the first distribution map and
key for 11 species or subspecies (Vachon, 1982: 96, fig. 50, 102–110). Two of the subspecies that he listed are
now placed in synonymy. A second paper on Sri Lankan Euscorpius — 2016, No. 220 2 Figures 1–4: Figure 1. Sri Lankan scorpion expedition 2015 research team, the official photo. Figure 2. Collecting in the
locality 15CA, within Peradeniya Univesity land. Figure 3. The main building of the Faculty of Science, Univesity of Pera-
deniya. Figure 4. F. Kovařík taking photo of Heterometrus swammerdami in the locality 15CB. Figures 1–4: Figure 1. Sri Lankan scorpion expedition 2015 research team, the official photo. Figure 2. Collecting in the
locality 15CA, within Peradeniya Univesity land. Figure 3. The main building of the Faculty of Science, Univesity of Pera-
deniya. Figure 4. F. Kovařík taking photo of Heterometrus swammerdami in the locality 15CB. Figures 1–4: Figure 1. Sri Lankan scorpion expedition 2015 research team, the official photo. Figure 2. Collecting in the
locality 15CA, within Peradeniya Univesity land. Figure 3. The main building of the Faculty of Science, Univesity of Pera-
deniya. Figure 4. F. Kovařík taking photo of Heterometrus swammerdami in the locality 15CB. Figures 1–4: Figure 1. Sri Lankan scorpion expedition 2015 research team, the official photo. Figure 2. Collecting in the
locality 15CA, within Peradeniya Univesity land. Figure 3. The main building of the Faculty of Science, Univesity of Pera-
deniya. Figure 4. F. Kovařík taking photo of Heterometrus swammerdami in the locality 15CB. Kovařík et al.: Scorpions of Sri Lanka 3 p
Figures 5–11: Figure 5. The reserarch team at a field station in the locality 15CB. Figure 6. Tea time in Girithale Wild
Training Center close to the locality 15CD. From left P. Devasurendra, F. Kovařík and K. B. Ranawana. Figure 7. P. Rajkum
(Environmental Research, Jaffna), F. Kovařík and local people in the locality 15CK video recording Hottentotta tamulus. Figu
8. S. Jayarathne taking a picture of a dangerous snake Hypnale hypnale in the locality 15CD. Figure 9. Herpetologist
Goonewardene and F. Kovařík in the locality 15CN. Figure 10. Meeting chief monk, in Monaragala "Sri Sambodhi Viharay
who has helped to research scorpions in the locality 15CQ. Figure 11. Profesor K. B. Ranawana in his office at University
Peradeniya. Figures 5–11: Figure 5. The reserarch team at a field station in the locality 15CB. Figure 6. Tea time in Girithale Wildlife
Training Center close to the locality 15CD. From left P. Devasurendra, F. Kovařík and K. B. Ranawana. Figure 7. P. Rajkumar
(Environmental Research, Jaffna), F. Kovařík and local people in the locality 15CK video recording Hottentotta tamulus. Figure
8. S. Jayarathne taking a picture of a dangerous snake Hypnale hypnale in the locality 15CD. Figure 9. Herpetologist S. Goonewardene and F. Kovařík in the locality 15CN. Figure 10. Meeting chief monk, in Monaragala "Sri Sambodhi Viharaya",
who has helped to research scorpions in the locality 15CQ. Figure 11. Profesor K. B. Euscorpius — 2016, No. 220 pre-Cambrian crystalline rocks, while sedimentary
deposits of Miocene limestone are confined to the
northwest, including the Jaffna Peninsula (Senanayake,
1990). )
Parts of the island have, through subsidence,
elevation, erosion and, faulting, produced three pene-
plains or erosion levels. They represent the lowlands, the
uplands and the highlands of the country. The first
peneplain represents the coasts and lowlands up to an
elevation of 305 m a.s.l., with a few scattered inselbergs. The slope in the coastal belt ranges up to 15°. The first
peneplain covers ca. 40,000 km2 of land area (about 75%
of the island). The lowlands are most extensive in the
northern and eastern parts, and are very narrow in the
southwestern, southern and southeastern regions, where
their width is less than 4 km. The second peneplain is an
upland belt encircling the third peneplain covering about
15,000 km2. It is composed of ridge and valley top-
ography with highly dissected plateaus. The mean slope
ranges from 10° to 30° along the upland ridges. The
upland belt is considerably narrower in the northern and
south-eastern sectors than elsewhere. The third pene-
plain covers an area of ca. 10,000 km2 of the south
central mountains. It is a complex of well-defined high
plains and plateaus rimmed with montane peaks and
ridges. The highlands are not continuous, but separated
from intervening dissected lower plateaus and uplands
by steep escarpments and valleys. We have continued this important task by con-
ducting a scorpiological expedition during the period of
19 April – 8 May 2015 to survey 20 carefully selected
localities. Sri Lankan arachnologists from Peradenyia
University also visited several other localities before and
after our expedition. Our collection efforts yielded fresh
material which, after analysis was found to represent a
total of 18 valid species. We report here our results
which provide the most complete and up-to-date account
of the Sri Lankan scorpiofauna yet available, with pre-
cise locality data and detailed descriptions using modern
methods. Thus, the south central mountains are arranged in
the shape of an anchor with two arms arching from the
southernmost region of a central ridge. In addition to the
south-central complex of major highlands, there are
several isolated mountain highland massifs, the two
most noteworthy of these being the Knuckles (Dumbara)
and the Deniyaya-Rakwana ranges. Euscorpius — 2016, No. 220 4 scorpions of more limited scope was published by
Lourenço & Huber (2002). They listed 7 species or sub-
species, based on specimens in MHNG collected in
1970–1979, a juvenile Chaerilus ceylonensis collected in
1992, and 7 specimens of Reddyanus besucheti comb. n. collected illegally in 2000. Two of their described taxa,
Isometrus (Reddyanus) garyi (see below) and Isometrus
thwaitesi pallidus (c.f. Kovařík & Ojanguren, 2013), and
one that was elevated from a subspecies, Heterometrus
titanicus (c.f. Kovařík, 2004), are now placed in
synonymy. Lourenço (2002) also described Charmus
minor based on specimens which are probably topotypes
of Charmus laneus Karsch, 1879 (see below). In
taxonomy, knowledge of the true distributions of species
is of critical importance. Historically, wrong assump-
tions or incomplete data about distributions was a major
factor causing the above documented errors in taxonomy
of Sri Lankan scorpions. This indicates the need for
comprehensive and rigorous field surveys of the Sri
Lankan scorpion fauna. Recent works have begun to
address this need. Ranawana et. al. (2013) and Veronika
et. al. (2013) surveyed scorpions of the Jaffna Peninsula
and accurately recorded distributions of 3 species: Hot-
tentotta tamulus, Isometrus maculatus and Heterometrus
swammerdami. scorpions of more limited scope was published by
Lourenço & Huber (2002). They listed 7 species or sub-
species, based on specimens in MHNG collected in
1970–1979, a juvenile Chaerilus ceylonensis collected in
1992, and 7 specimens of Reddyanus besucheti comb. n. collected illegally in 2000. Two of their described taxa,
Isometrus (Reddyanus) garyi (see below) and Isometrus
thwaitesi pallidus (c.f. Kovařík & Ojanguren, 2013), and
one that was elevated from a subspecies, Heterometrus
titanicus (c.f. Kovařík, 2004), are now placed in
synonymy. Lourenço (2002) also described Charmus
minor based on specimens which are probably topotypes
of Charmus laneus Karsch, 1879 (see below). In
taxonomy, knowledge of the true distributions of species
is of critical importance. Historically, wrong assump-
tions or incomplete data about distributions was a major
factor causing the above documented errors in taxonomy
of Sri Lankan scorpions. This indicates the need for
comprehensive and rigorous field surveys of the Sri
Lankan scorpion fauna. Recent works have begun to
address this need. Ranawana et. al. (2013) and Veronika
et. al. (2013) surveyed scorpions of the Jaffna Peninsula
and accurately recorded distributions of 3 species: Hot-
tentotta tamulus, Isometrus maculatus and Heterometrus
swammerdami. Euscorpius — 2016, No. 220 The Knuckles massif
has a maximum elevation of 2035 m a.s.l., is situated
northeast of Kandy, and is separated from the central
highland mass by deeply eroded valleys carved out by
the Mahaweli River. The Deniyaya-Rakwana Massif
reaches up to an elevation of 1488 m a.s.l., and is sep-
arated from the central highland proper by the valleys
formed as a result of erosion caused by the head streams
of the Kalu Ganga. Ranawana in his office at University of
Peradeniya. Figures 5–11: Figure 5. The reserarch team at a field station in the locality 15CB. Figure 6. Tea time in Girithale Wildlife
Training Center close to the locality 15CD. From left P. Devasurendra, F. Kovařík and K. B. Ranawana. Figure 7. P. Rajkumar
(Environmental Research, Jaffna), F. Kovařík and local people in the locality 15CK video recording Hottentotta tamulus. Figure
8. S. Jayarathne taking a picture of a dangerous snake Hypnale hypnale in the locality 15CD. Figure 9. Herpetologist S. Goonewardene and F. Kovařík in the locality 15CN. Figure 10. Meeting chief monk, in Monaragala "Sri Sambodhi Viharaya",
who has helped to research scorpions in the locality 15CQ. Figure 11. Profesor K. B. Ranawana in his office at University of
Peradeniya. Euscorpius — 2016, No. 220 6 inter-monsoons. The duration of inter-monsoons is less
than those of the monsoons but their contribution to the
total annual precipitation is remarkable and more out-
standing than that of the monsoons. On the basis of the
time of occurrence and the direction of the cloud
movements, each of the two rainy seasons can be further
subdivided into two periods, thus yielding four seasons: only collected samples needed for taxonomic revisions
of each species. For conservation purposes, additional
samples of species that were encountered at various sites
were not taken, but were recorded photographically in
the field. Nomenclature and measurements herein follow
Stahnke (1971), Kovařík (2009), and Kovařík & Ojan-
guren Affilastro (2013), except for trichobothriotaxy
(Vachon, 1974), metasomal carinae (Francke, 1977),
hemispermatophore (see below), and sternum (Soleglad
& Fet, 2003). Unless otherwise noted, all diagnostic
morphological characters mentioned in the text refer to
adults of both sexes. First Inter-monsoon: March – Mid-May
Southwest monsoon: Mid-May – September
Second Inter-monsoon: October – November
Northeast monsoon: December – February Rainfall and its seasonality is the main determinant
of the distribution of natural vegetation in Sri Lanka. This climatic regime is the basis for dividing the island
into three major climatic zones viz. wet, intermediate
and dry (Figs. 12–15). Tropical lowland wet evergreen
forests or lowland rain forests which are restricted to the
wet southwest of the country up to an elevation of 900 m
a.s.l. is the climax vegetation of the wet zone of the
island (Gunatilleke et al, 2008). Wet evergreen montane
forests are restricted to the uppermost elevations (> 1500
m a.s.l.) of the country while the lower montane forests
are confined to middle elevations (900–1500 m a.s.l.) of
the Peak Wilderness, Knuckles (Dumbara Hills), Namu-
nukula, and the Rakwana-Deniyaya ranges. Tropical
moist semi-evergreen forests are the characteristic veg-
etation type in the intermediate zone which includes
seasonally dry northern and eastern plains. Tropical dry
mixed evergreen forests are found in the dry zone. The
tropical dry, mixed evergreen forests represent 54% of
the island’s natural forest cover and 16% of the total
land area. The arid zones of the north-western and south-
eastern extremities of the island are covered with
tropical thorn forests. Hemispermatophore terminology. We use 'dorsal',
'internal', 'ventral' and 'external' to refer to axes of the
scorpion mesosoma with hemispermatophores in situ. Euscorpius — 2016, No. 220 For flagelliform buthid hemispermatophore structures,
we use the following terms: f, flagellum; pr, pars recta of
flagellum; prf, pars reflecta of flagellum; t, trunk (c.f. Lamoral, 1979; Sissom, 1990; Vachon, 1952); bl, basal
lobe (Maury, 1970; Ojanguren-Affilastro, 2005; Stock-
well, 1989; Vachon, 1952); ml, median lobe (Vachon,
1952) with median lobe carina (mlc). For lamelliform
scorpionid hemispermatophore structures, we use the
folowing terms: dl, distal lamina; h, hook; t, trunk
(Lamoral, 1979; Sissom, 1990); bl, basal lobe (Couzijn,
1981; Lamoral, 1979); dtdl, dorsal trough of distal
lamina (Lamoral, 1979); il, inner lobe (Couzijn, 1981;
Lamoral, 1979); ml, median lobe (Lamoral, 1979;
Vachon, 1952); mtt, median transverse trough, (Lamoral,
1979); pl, proximal lobe (Koch, 1977); tf, truncal flexure
(Bastawade, 1994; Stockwell, 1989). Note that the 'basal
lobe' and 'median lobe' of lamelliform hemisperma-
tophores are not necessarily homologous to structures
bearing the same names in flagelliform hemisper-
matophores. Specimens cited and/or studied herein are preserved
in ethanol 80% and deposited in the following
collections: Specimens cited and/or studied herein are preserved
in ethanol 80% and deposited in the following Geology and environment of Sri Lanka Sri Lanka (05°54'–09°52'N and 79°39'–81°53'E) is
an island lying off the south-eastern corner of the Indian
subcontinent. The extent of the continental shelf and the
nature of rock formations in both India and Sri Lanka
provide evidence in support of the hypothesis that Sri
Lanka was once a part of India and separated to become
an island in the late Miocene. The islands south western
part appears to have been the first to separate from India,
with alternate shallow flooding and elevation at various
times thereafter (Cruz, 1986). Sri Lanka was inter-
mittently connected to mainland India during the last
Pleistocene glaciations until sea level rise resulted in the
present separation ca. 11,000 y BP (Vaz, 2000). The
65,610 km2 island was, in the distant geological past, a
part of Gondwana, which was never fully submerged by
the sea. Nearly 90% of the island landmass comprises Sri Lanka receives 40–60% of its rain from the
monsoons while the rest is inter-monsoonal. The spatial
differences in the rain climate of Sri Lanka are a clear
manifestation of an orographic effect of the south-central
highlands. Although the Sri Lankan mountains have no
great vertical extensions by global standards, their ele-
vational range has a considerable influence on the
climate, given the small land area involved (Pemadasa,
1996). The annual rainfall period of Sri Lanka extends
from March to February. On the basis of the relative
dominance of the causative factors, two general cate-
gories of rainy seasons can be recognized. These are
regarded by climatologists as the monsoons and the 5 Kovařík et al.: Scorpions of Sri Lanka Kovařík et al.: Scorpions of Sri Lanka Figures 12–15: Maps showing the known geographical distribution of the Sri Lankan species. Figures 12–15: Maps showing the known geographical distribution of the Sri Lankan species. Methods and Material Specimens collected during the scorpiological
expedition between 19 April and 8 May 2015 are cited
as "leg. Kovařík et al." These scorpions were collected
by a research team consisting of the following members:
František Kovařík and David Hoferek (Czech Republic);
Kithsiri B. Ranawana, Sanjeewa Jayarathne, Sanjaya
Karunarathne, A. N. Thudugala, Sewwandi Wijesooriya,
Dilan Chathuranga, Chamalka Halyala, and Ajith Hema-
chandra (University of Peradeniya, Sri Lanka) (Fig. 1). At localities 15CJ and 15CK, we were ably assisted by
P. Rajkumar (Sri Lanka, Environmental Research,
Jaffna) and local volunteers (Fig. 7). At locality 15CN (a
private garden) we were assisted by herpetologist Suraj
Goonewardene (Fig. 9). BMNH (The Natural History Museum, London, United
Kingdom); FKCP (František Kovařík, private collection, Prague,
Czech Republic); MHNG (Museum d`Histoire naturelle of Geneve, Geneva, Switzerland); MNHN (Muséum National d´Histoire Naturelle, Paris,
France); NHRS (Naturhistoriska Riksmuseet, Stockholm,
Sweden); NMPC (National Museum of Natural History, Prague,
Czech Republic); Czech Republic); UPSL (Faculty of Science, University of Peradeniya,
Peradeniya, Sri Lanka); Specimens examined here were collected and
exported legally, via permit No. WL/3/2/79/14 issued by
the Department of Wildlife Conservation, Sri Lanka. We ZMHB (Museum für Naturkunde der Humboldt-
Universität, Berlin, Germany); Kovařík et al.: Scorpions of Sri Lanka Kovařík et al.: Scorpions of Sri Lanka 7 Figures 16–19: Buthoscorpio sarasinorum. Figures 16–17. Male from locality 15CF in dorsal (16) and ventral (17) views. Figures 18–19. Female from locality 15CG in dorsal (18) and ventral (19) views. Figures 16–19: Buthoscorpio sarasinorum. Figures 16–17. Male from locality 15CF in dorsal (16) and ventral (17) views. Figures 18–19. Female from locality 15CG in dorsal (18) and ventral (19) views. Euscorpius — 2016, No. 220 8 gures 20–23: Buthoscorpio sarasinorum. Figures 20, 22. Male from locality 15CF, chelicerae, carapace and tergites I–
) and sternopectinal region and sternite III (22). Figures 21, 23. Female from locality 15CG, chelicerae, carapace and tergit
II (21) and sternopectinal region and sternite III (23). Figures 20–23: Buthoscorpio sarasinorum. Figures 20, 22. Male from locality 15CF, chelicerae, carapace and tergites I–I
20) and sternopectinal region and sternite III (22). Figures 21, 23. Female from locality 15CG, chelicerae, carapace and tergit
–III (21) and sternopectinal region and sternite III (23). Figures 20–23: Buthoscorpio sarasinorum. Figures 20, 22. Male from locality 15CF, chelicerae, carapace and tergites I–III
(20) and sternopectinal region and sternite III (22). Figures 21, 23. Methods and Material Female from locality 15CG, chelicerae, carapace and tergites
I–III (21) and sternopectinal region and sternite III (23). Kovařík et al.: Scorpions of Sri Lanka 9 Figures 24–29: Buthoscorpio sarasinorum. Figures 24–26. Male from locality 15CF, metasoma and telson, lateral (24), ventral
(25), and dorsal (26) views. Figures 27–29. Female from locality 15CG, metasoma and telson, lateral (27), ventral (28), and
dorsal (29) views. Figures 24–29: Buthoscorpio sarasinorum. Figures 24–26. Male from locality 15CF, metasoma and telson, lateral (24), ventral
(25), and dorsal (26) views. Figures 27–29. Female from locality 15CG, metasoma and telson, lateral (27), ventral (28), and
dorsal (29) views. Hottentotta tamulus (Fabricius, 1798)
Isometrus maculatus (De Geer, 1778)
Isometrus thwaitesi Pocock, 1897 *
= Isometrus thwaitesi pallidus Lourenço et Huber,
2002
Lychas srilankensis Lourenço, 1997 *
= Lychas ceylonensis Lourenço et Huber, 1999
Reddyanus basilicus (Karsch, 1879) comb. n. *
Reddyanus besucheti (Vachon, 1982) comb. n. *
Reddyanus ceylonensis sp. n. *
Reddyanus jayarathnei sp. n. *
Reddyanus loebli (Vachon, 1982) comb. n. *
= Isometrus garyi Lourenço et Huber, 2002 Syn. n. ZMUH (Zoologisches Institut und Zoologisches Mus-
eum, Universität Hamburg, Germany). ZMUH (Zoologisches Institut und Zoologisches Mus-
eum, Universität Hamburg, Germany). ZMUH (Zoologisches Institut und Zoologisches Mus-
eum, Universität Hamburg, Germany). ZMUH (Zoologisches Institut und Zoologisches Mus-
eum, Universität Hamburg, Germany). For explanation of locality codes 15CA–15CT see
legends of Figs. 575–598. Checklist of Scorpions of Sri Lanka
Family Buthidae C. L. Koch, 1837
Buthoscorpio sarasinorum (Karsch, 1892) *
Charmus laneus Karsch, 1879 *
= Heterocharmus cinctipes Pocock, 1892
= Charmus minor Lourenço, 2002 Syn. n. Charmus saradieli sp. n. *
Hottentotta tamulus (Fabricius, 1798)
Isometrus maculatus (De Geer, 1778)
Isometrus thwaitesi Pocock, 1897 *
= Isometrus thwaitesi pallidus Lourenço et Huber,
2002
Lychas srilankensis Lourenço, 1997 *
= Lychas ceylonensis Lourenço et Huber, 1999
Reddyanus basilicus (Karsch, 1879) comb. n. *
Reddyanus besucheti (Vachon, 1982) comb. n. *
Reddyanus ceylonensis sp. n. *
Reddyanus jayarathnei sp. n. *
Reddyanus loebli (Vachon, 1982) comb. n. *
= Isometrus garyi Lourenço et Huber, 2002 Syn. n. For explanation of locality codes 15CA–15CT see
legends of Figs. 575–598. Checklist of Scorpions of Sri Lanka Family Buthidae C. L. Koch, 1837
Buthoscorpio sarasinorum (Karsch, 1892) *
Charmus laneus Karsch, 1879 *
= Heterocharmus cinctipes Pocock, 1892
= Charmus minor Lourenço, 2002 Syn. n. Charmus saradieli sp. n. * Reddyanus besucheti (Vachon, 1982) comb. n. *
* Reddyanus ceylonensis sp. n. *
* Reddyanus jayarathnei sp. n. y
j y
p
Reddyanus loebli (Vachon, 1982) comb. n. * 10 Euscorpius — 2016, No. 220 Figures 30–33: Capsule region and flagellum of left hemispermatophore of Buthoscorpio sarasinorum from locality 15CF. External (30), dorsal (31), internal (32) and ventral (33) views. Note: damaged section of pars reflecta of flagellum is interpolated
as a dotted outline. Scale bar: 500 µm. Abbreviations: bl, basal lobe; f, flagellum; ml, median lobe; mlc, median lobe carina; pr,
pars recta of flagellum; prf, pars reflecta of flagellum; t, trunk. Figures 30–33: Capsule region and flagellum of left hemispermatophore of Buthoscorpio sarasinorum from locality 15CF. External (30), dorsal (31), internal (32) and ventral (33) views. Note: damaged section of pars reflecta of flagellum is interpolated
as a dotted outline. Scale bar: 500 µm. Abbreviations: bl, basal lobe; f, flagellum; ml, median lobe; mlc, median lobe carina; pr,
pars recta of flagellum; prf, pars reflecta of flagellum; t, trunk. Heterometrus serratus (Pocock, 1900) * Heterometrus serratus (Pocock, 1900) * Heterometrus swammerdami Simon, 1872 Reddyanus ranawanai sp. n. * Reddyanus ranawanai sp. n. Family Chaerilidae Pocock, 1893
Chaerilus ceylonensis Pocock, 1894 *
Family Scorpionidae Latreille, 1802
Heterometrus gravimanus (Pocock, 1894)
Heterometrus indus (De Geer, 1778) *
= Scorpio ceylonicus Herbst, 1800
= Heterometrus (Heterometrus) spinifer solitarius
Couzijn, 1981 Syn. n. Heterometrus serratus (Pocock, 1900) *
Heterometrus swammerdami Simon, 1872
= Heterometrus (Gigantometrus) swammerdami
titanicus Couzijn, 1981 Family Buthidae C. L. Koch, 1837
Genus Buthoscorpio Werner, 1936
(Figs. 12, 16–40, 193, 421–422, 547) = Stenochirus Karsch, 1892: 305–306, fig. 30, Pl. XXII;
a junior homonym of Stenochirus Oppel, 1862
(Crustacea); Kraepelin, 1899: 39; Pocock, 1900:
32–33; Kraepelin, 1913: 131–132; Werner, 1934:
271; Vachon, 1982: 83–84, figs. 12–18, 84–85;
Tikader & Bastawade, 1983: 152–153. = Scorpio ceylonicus Herbst, 1800 = Heterometrus (Heterometrus) spinifer solitarius
Couzijn, 1981 Syn. n. Heterometrus swammerdami Simon, 1872 = Heterometrus (Gigantometrus) swammerdami
titanicus Couzijn, 1981 = Pocockius Francke, 1985: 13, 16 (syn. by Fet, 1997:
246). Buthoscorpio Werner, 1936: 191; Fet, 1997: 246; Fet &
Lowe, 2000: 90; Kovařík, 2009: 31; Javed et al., Buthoscorpio Werner, 1936: 191; Fet, 1997: 246; Fet &
Lowe, 2000: 90; Kovařík, 2009: 31; Javed et al., 2010: 5–10, figs. 1–8; Aswathi et al., 2015: 213–
218, figs. 1–5.
DISTRIBUTION. India, Sri Lanka. 2010: 5–10, figs. 1–8; Aswathi et al., 2015: 213–
218, figs. 1–5. DISTRIBUTION. India, Sri Lanka. Buthoscorpio sarasinorum (Karsch, 1892)
(Figs. 12, 16–40, 47, 193, 421–422, 547) * Sri Lankan endemics * Sri Lankan endemics Kovařík et al.: Scorpions of Sri Lanka 11 Figures 34–38: Buthoscorpio sarasinorum. Figures 34. Male at locality 15CF. Figures 35. Female at locality 15CS under UV
Figures 36–38. Female from locality 15CF (36) with newborns (37), and with juveniles after first ecdysis (38). Figures 34–38: Buthoscorpio sarasinorum. Figures 34. Male at locality 15CF. Figures 35. Female at locality 15CS under UV. Figures 36–38. Female from locality 15CF (36) with newborns (37), and with juveniles after first ecdysis (38). Euscorpius — 2016, No. 220
12
Figures 39–46: Chela of pedipalps (39–45) and movable finger of chela (46). Figures 39–40. Buthoscorpio sarasinorum, male
from locality 15CF (39) and female from locality 15CG (40). Figures 41–43. Charmus laneus, male (41) and female (42) from
locality 15CO, and female holotype (43). Figure 44. Charmus saradieli, sp. n., female holotype. Figures 45–46. Hottentotta
tamulus, male from locality 15CK. Euscorpius — 2016, No. 220 12 Figures 39–46: Chela of pedipalps (39–45) and movable finger of chela (46). Figures 39–40. Buthoscorpio sarasinorum, male
from locality 15CF (39) and female from locality 15CG (40). Figures 41–43. Charmus laneus, male (41) and female (42) from
locality 15CO, and female holotype (43). Figure 44. Charmus saradieli, sp. n., female holotype. Figures 45–46. Hottentotta
tamulus, male from locality 15CK. TYPE SPECIES. Stenochirus sarasinorum Karsch, 1892 DIAGNOSIS. Total length 30–52 mm. Sternum type 1,
subpentagonal or subtriangular, roughly as wide as long,
exhibiting horizontal compression. Pedipalps with tri-
chobothrial pattern Aα; femur trichobothrium d2 located
dorsally, patella d3 external to dorsomedian carina; chela
with 3 Eb trichobothria on manus. Cheliceral fixed
finger with two ventral accessory denticles. Movable
finger of pedipalp chela longer than manus. Pectines
with fulcra. Dentate margin of pedipalp chela movable
finger with distinct granules divided into 10–11 linear
rows, apical rows of 3–4 granules, and 3 terminal gran-
ules. Tergites I–VI smooth or finely granular, with one
carina. Carapace smooth to finely granular without cari-
nae, anterior edge with epistome present medially. Meta-
somal segments punctate, without stridulatory areas,
without carinae except for dorsolateral carinae which
may be developed. Telson vesicle punctate, subaculear
tooth absent or weakly developed. Pedipalps, metasoma
and telson glabrous. Legs III and IV with well de-
veloped, long tibial spurs; ventral aspect of tarsomere II
with two rows of spiniform setae. Stenochirus sarasinorum Karsch, 1892: 305–306, fig. 30, Pl. XXII; Kraepelin, 1899: 39–40; Pocock,
1900: 33–34; Moritz & Fischer, 1980: 323; Vachon,
1982: 83–84, figs. 12–18, 84–85. Buthoscorpio sarasinorum: Fet, 1997: 246; Fet & Lowe,
2000: 90 (? in part); Lourenço, 2012: 188–189, figs. 6–12. TYPE LOCALITY AND TYPE REPOSITORY. Ceylon (now Sri
Lanka), Peradeniya, ZMHB No. 6806. MATERIAL
EXAMINED. Sri Lanka, North Central
Province, Polonnaruwa District, near Kaudulla National
Park, 08°08'40.6"N 080°51'04"E, 101 m a.s.l. (Locality
15CF, Fig. 581), 23.IV.2015, 1♂ (Figs. 16–17, 20, 22,
24–26, 30–34, 193, 421, 547), FKCP, 1♀ (Figs. 36–38)
UPSL, leg. Kovařík et al.; Central Province, Matale
District, Habarana, Wananiwahana Resort, 07°59'25.8"N
080°43'24.6"E, 280 m a.s.l. (Locality 15CG, Fig. 584),
23. –24.IV.2015, 1♀ (Figs. 18–19, 21, 23, 27–29, 422),
FKCP, leg. Kovařík et al.; Eastern Province, Ampara 13 Kovařík et al.: Scorpions of Sri Lanka Figures 47–53: Figure 47. Left hemispermatophore of Buthoscorpio sarasinorum from locality 15CF. Figures 48–53. Charmus laneus from locality 15CO. Figure 48. Left hemispermatophore. Figures 49–53. Capsule region and flagellum. External (49), dorsal (50), internal (51) and ventral (52) views of left hemispermatophore, and externo-dorsal (53) view of right
hemispermatophore presented as mirror image for comparison. Scale bars: 1 mm for 47, 500 µm for 48, 250 µm for 49–52, and
200 µm for 53. District, Ampara env., 07°20'01.3"N 081°41'57.1"E, 56
m a.s.l. (Locality 15CS, Fig. 597), 4.V.2015, 1♂, UPSL,
♀ (Fig. 35), FKCP, leg. Kovařík et al.; Central Pro-
1
vince, Kandy District, Tree Centre Wildlife Trust Sri
Lanka “Rantambe”, 07°12'22.1"N 080°57'20.7"E, 171 m TYPE SPECIES. Stenochirus sarasinorum Karsch, 1892 Abbreviations: bl, basal lobe; f, flagellum; ml, median lobe; mlc, median lobe carina; pr, pars recta of flagellum;
prf, pars reflecta of flagellum; t, trunk. Figures 47–53: Figure 47. Left hemispermatophore of Buthoscorpio sarasinorum from locality 15CF. Figures 48–53. Charmus laneus from locality 15CO. Figure 48. Left hemispermatophore. Figures 49–53. Capsule region and flagellum. External (49), dorsal (50), internal (51) and ventral (52) views of left hemispermatophore, and externo-dorsal (53) view of right
hemispermatophore presented as mirror image for comparison. Scale bars: 1 mm for 47, 500 µm for 48, 250 µm for 49–52, and
200 µm for 53. Abbreviations: bl, basal lobe; f, flagellum; ml, median lobe; mlc, median lobe carina; pr, pars recta of flagellum;
prf, pars reflecta of flagellum; t, trunk. Euscorpius — 2016, No. 220 14 Euscorpius — 2016, No. 220
14
Figures 54–59: Charmus laneus from locality 15CO. Figures 54–55. Male 14.7 mm long in dorsal (54) and ventral (55) views. Figures 56–57. Juvenile male 7.5 mm long in dorsal (56) and ventral (57) views. Figures 58–59. Female 20.1 mm long in dorsal
(58) and ventral (59) views. District, Ampara env., 07°20'01.3"N 081°41'57.1"E, 56
a.s.l. (Locality 15CT, Fig. 598), 5.V.2015, 1♂, FKCP, Figures 54–59: Charmus laneus from locality 15CO. Figures 54–55. Male 14.7 mm long in dorsal (54) and ventral (55) views. Figures 56–57. Juvenile male 7.5 mm long in dorsal (56) and ventral (57) views. Figures 58–59. Female 20.1 mm long in dorsal
(58) and ventral (59) views. a.s.l. (Locality 15CT, Fig. 598), 5.V.2015, 1♂, FKCP,
1♂, UPSL leg. Kovařík et al. District, Ampara env., 07°20'01.3"N 081°41'57.1"E, 56
m a.s.l. (Locality 15CS, Fig. 597), 4.V.2015, 1♂, UPSL,
♀ (Fig. 35), FKCP, leg. Kovařík et al.; Central Pro-
1
vince, Kandy District, Tree Centre Wildlife Trust Sri
Lanka “Rantambe”, 07°12'22.1"N 080°57'20.7"E, 171 m a.s.l. (Locality 15CT, Fig. 598), 5.V.2015, 1♂, FKCP,
1♂, UPSL leg. Kovařík et al. DIAGNOSIS. Total length 25–52 mm. Dentate margin of
pedipalp chela movable finger with distinct granules Kovařík et al.: Scorpions of Sri Lanka 15 Figures 60, 62. Male, chelicerae, carapace and tergites I–III (60) and
sternopectinal region and sternites III–VII (62). Figures 61, 63. Female, chelicerae, carapace and tergites I–III (61) and
sternopectinal region and sternite III (63). Figures 60–63: Charmus laneus from locality 15CO. Figures 60, 62. Male, chelicerae, carapace and tergites I–III (60) and
sternopectinal region and sternites III–VII (62). Figures 61, 63. TYPE SPECIES. Stenochirus sarasinorum Karsch, 1892 Dorsal (
(white light + UV), and ventroexternal (67b) aspect (UV) showing 2 denticles on ventral surface of
b
200 Kovařík et al.: Scorpions of Sri Lanka 17 17 Figures 66–67: Left chelicera of male Charmus laneus from locality 15CO. Figures 66–67. Dorsal (66) and ventral (67a) view
(white light + UV), and ventroexternal (67b) aspect (UV) showing 2 denticles on ventral surface of fixed finger (d1, d2). Scale
bar: 200 µm. Genus Charmus Karsch, 1879
(Figs. 12, 41–43, 47–119, 194, 423–426, 548, Tables 1–
2) one clearly visible carina. Carapace granular without
carinae, anterior edge with epistome present medially. Metasomal segments IV–V punctate without developed
carinae. Telson vesicle punctate, without subaculear
tooth. Pedipalps, metasoma and telson densely hirsute. Legs III and IV with well developed long tibial spurs,
first and second tarsomeres with ventral setae. Genus Charmus Karsch, 1879
(Figs. 12, 41–43, 47–119, 194, 423–426, 548, Tables 1–
2) Charmus Karsch, 1879: 104; Kraepelin, 1899: 39;
Pocock, 1900: 31–32; Kraepelin, 1913: 131;
Vachon, 1982: 79, 81; Tikader & Bastawade, 1983:
140–152, figs. 382–416; Sissom, 1990: 101;
Kovařík, 1998: 120; Lourenço, 2000: 295; Kovařík,
Soleglad & Fet, 2007: 201; Kovařík, 2009: 31. NOTE. A remarkable feature of the metasoma and telson
–73). All segments bear an abundance of fine
setae of various lengths emerging from pits containing
h intense pinpoint fluorescence at the tip (Figs. 8–70). Comparing these setae to similar kinds of setae
915 and C. singhagadensis
ikader et Bastawade, 1983 (Sreenivasa Reddy, 1966:
of Charmus laneus is the extremely dense pubescence
(Figs. 71
sockets or perforations in the thickened cuticle. These
setae can be divided into at least two types: (1) straight
or uniformly curved, non-fluorescent golden setae; and
(2) terminally curved, brightly fluorescent, translucent
setae wit
6
found in other scorpions, we suggest that type 1 setae
may be mechanoreceptive and tactile, and type 2 setae
may be chemoreceptive in function. Putative chem-
otactic microsetae in other scorpions are typically also
fluorescent and exhibit a similar, apically curved shape,
but are usually quite short compared to the long
fluorescent setae seen here. A similar densely hirsute
metasoma is also present in C. saradieli sp. n. and was
also described in the other two known members of the
genus, C. indicus Hirst, 1
T monotypy Heterocharmus cinctipes Pocock, 1892
g
= Heterocharmus Pocock, 1892: 46–47, type species by
(= Charmus laneus Karsch, 1879) (syn. TYPE SPECIES. Stenochirus sarasinorum Karsch, 1892 Female, chelicerae, carapace and tergites I–III (61) and
sternopectinal region and sternite III (63). Figures 60–63: Charmus laneus from locality 15CO. Euscorpius — 2016, No. 220 16 Figures 64–65: Charmus laneus from locality 15CO, showing granulation and color pattern of male. Chelicerae, carapace and
tergites (64). Coxosternal region and sternites (65). Scale bar: 500 µm. Figures 64–65: Charmus laneus from locality 15CO, showing granulation and color pattern of male. Chelicerae, carapace and
tergites (64). Coxosternal region and sternites (65). Scale bar: 500 µm. dorsal carina. Basal lobe strongly developed as a prom-
inent, blunt, rounded scoop arising dorsally from base of
median lobe carina. External surface of capsule region
roughly sculptured with series of transverse corru-
gations. divided into 10 linear rows, apical rows of 3 granules,
and 3 terminal granules. Tergites and carapace smooth to
finely granular (more so in males). Median eyes located
anteriorly in the ratio ca 1:2.5. Metasomal segments III–
IV with well developed dorsolateral carinae. Dorsal
surface of metasomal segments mesially granulated. Metasomal segments I–V granulated. Pectinal teeth
number 14–17 in both sexes. Telson without subaculear
tooth. COMMENTS. Buthoscorpio sarasinorum is probably en-
demic to Sri Lanka. Tikader & Bastawade (1983: 158–
163, figs. 430–452) and Thulsi Rao et al. (2005: 7) cited
B. sarasinorum from India but according to the char-
acters they cited, their records probably represent a
different species. Aswathi et al. (2015: 218) cited B. sarasinorum only from Sri Lanka (Aswathi et al., 2015:
217, fig. 1) but in their table (Aswathi et al., 2015: 218,
tab. 2) they listed variable or incorrect diagnostic char-
acters for B. sarasinorum. HEMISPERMATOPHORE (Figs. 30–33, 47). Trunk and cap-
sule region very narrow, elongate (Fig. 47). Flagellum
cylindriform, short, pars recta with broad base; pars
flecta thickened, about the same length as capsule
re
region. Median lobe narrow with prominent, curved Kovařík et al.: Scorpions of Sri Lanka
17
Figures 66–67: Left chelicera of male Charmus laneus from locality 15CO. Figures 66–67. Dorsal (66) and ventral (67a) view
(white light + UV), and ventroexternal (67b) aspect (UV) showing 2 denticles on ventral surface of fixed finger (d1, d2). Scale
bar: 200 µm. Kovařík et al.: Scorpions of Sri Lanka
Figures 66–67: Left chelicera of male Charmus laneus from locality 15CO. Figures 66–67. TYPE SPECIES. Stenochirus sarasinorum Karsch, 1892 by
Kraepelin, 1899: 39; Pocock, 1900: 31). TYPE SPECIES. Charmus laneus Karsch, 1879 TYPE SPECIES. Charmus laneus Karsch, 1879 2
dorsally, patella d3 dorsal of dorsomedian carina;
ela with 3 Eb trichobothria on manus. Movable finger
ventral surface (Fig. 67a). Tergites I–VI granular, with
DIAGNOSIS. Small buthids, adults 12 mm (male) – 23.5
mm (female). Sternum type 1, subpentagonal, roughly as
wide as long, exhibiting horizontal compression. Ped-
ipalps trichobothrial pattern Aα; femur trichobothrium d
cated
lo
ch
of pedipalp longer than manus. Pectines with or without
fulcra. Dentate margin of pedipalp chela movable finger
with distinct granules divided into 8–9 linear rows,
apical rows of 4–6 granules, and 3 terminal granules. Cheliceral fixed finger armed with two denticles on Euscorpius — 2016, No. 220
18
n
Figures 68–70: Charmus laneus, male from locality 15CO. granulation, punctuation and setation in dorsal (68), lateral (69) a
247–256; Tikader & Bastawade, 1983: 140–152). A
similar, probably homologous development of dense
setation is also observed in the closely related genus
Thaicharmus (Kovařík, 1995, 2013; Mirza et. al., 2016). Metasoma V and telson under UV fluorescence, showing
d ventral (70) views. Scale bar: 500 µm. This massive concentration of multimodal sensory input
indicates that Charmus is another example of the evo-
lution of the metasoma into a specialized sensory organ. As noted previously, this has apparently occurred in- Euscorpius — 2016, No. 220 18 n
Figures 68–70: Charmus laneus, male from locality 15CO. granulation, punctuation and setation in dorsal (68), lateral (69) a
Metasoma V and telson under UV fluorescence, showing
d ventral (70) views. Scale bar: 500 µm. 247–256; Tikader & Bastawade, 1983: 140–152). A
similar, probably homologous development of dense
setation is also observed in the closely related genus
Thaicharmus (Kovařík, 1995, 2013; Mirza et. al., 2016). This massive concentration of multimodal sensory input
indicates that Charmus is another example of the evo-
lution of the metasoma into a specialized sensory organ. As noted previously, this has apparently occurred in- 247–256; Tikader & Bastawade, 1983: 140–152). A
similar, probably homologous development of dense
setation is also observed in the closely related genus
Thaicharmus (Kovařík, 1995, 2013; Mirza et. al., 2016). 247–256; Tikader & Bastawade, 1983: 140–152). A
similar, probably homologous development of dense
setation is also observed in the closely related genus
Thaicharmus (Kovařík, 1995, 2013; Mirza et. al., 2016). This massive concentration of multimodal sensory input
indicates that Charmus is another example of the evo-
lution of the metasoma into a specialized sensory organ. DISTRIBUTION. India, Sri Lanka. DISTRIBUTION. India, Sri Lanka. TYPE SPECIES. Charmus laneus Karsch, 1879 As noted previously, this has apparently occurred in- 19 Kovařík et al.: Scorpions of Sri Lanka s
Figures 71–73: Metasoma and telson of Charmus laneus male from locality 15CO, showing granulation, punctuation, setation
and color pattern in dorsal (71), lateral (72) and ventral (73) view . Scale bar: 1 mm. s
Figures 71–73: Metasoma and telson of Charmus laneus male from locality 15CO, showing granulation, punctuation, setation
and color pattern in dorsal (71), lateral (72) and ventral (73) view . Scale bar: 1 mm. Euscorpius — 2016, No. 220 20 Euscorpius — 2016, No. 220 Figures 74–79: Charmus laneus from locality 15CO. Figures 74–76. Male, metasoma and telson, lateral (74), ventral (75), and
dorsal (76) views. Figures 77–79. Female, metasoma and telson, lateral (77), ventral (78), and dorsal (79) views. Figures 74–79: Charmus laneus from locality 15CO. Figures 74–76. Male, metasoma and telson, lateral (74), ventral (75), and
dorsal (76) views. Figures 77–79. Female, metasoma and telson, lateral (77), ventral (78), and dorsal (79) views. dependently in several different buthid lineages, e.g. Butheoloides Hirst, 1925; Isometroides Keyserling,
1885; Karasbergia Hewitt, 1914; Microbuthus Krae-
pelin, 1898; Orthochirus Karsch, 1892; etc. (E. Fet et al.,
2003; Lourenço, 2001, 2003; Lowe, 2010; Prendini,
2004). = Heterocharmus cinctipes Pocock, 1892: 47–48, pl. IIIB, fig. 2, 2a–b (TL: India or Sri Lanka; BMNH). Syn. by Kraepelin, 1899: 39. = Charmus minor Lourenço, 2002: 19–24, figs. 1–14
(TL: Sri Lanka, Mannar District, Wilpattu National
Park, 0.5 miles NE Cockmuttai; ZMUH). Syn. n. p
(
2003; Lourenço, 2001, 2003; Lowe, 2010; Prendini,
2004). DISTRIBUTION. India, Sri Lanka. Charmus laneus Karsch, 1879
(Figs. 12, 41–43, 47–83, 85–86, 96–98, 118–119, 194,
423–424, 548, Tables 1–2) TYPE LOCALITY AND TYPE REPOSITORY. Ceylon; ZMHB. Charmus laneus Karsch, 1879
(Figs. 12, 41–43, 47–83, 85–86, 96–98, 118–119, 194,
423–424, 548, Tables 1–2) TYPE MATERIAL EXAMINED. Ceylon (now Sri Lanka),
leg. Hoffmeister, 1♀ holotype (Figs. 43, 80–82, 96–98),
ZMHB No. 3051. TYPE MATERIAL EXAMINED. Ceylon (now Sri Lanka),
leg. Hoffmeister, 1♀ holotype (Figs. 43, 80–82, 96–98),
ZMHB No. 3051. Charmus laneus Karsch, 1879: 104–105; Kraepelin,
1899: 39; Pocock, 1900: 32; Kraepelin, 1913: 131;
Sreenivasa-Reddy, 1966: 253–254; Moritz & Fis-
cher, 1980: 317; Kovařík, 1998: 108; Fet & Lowe,
2000: 123 (in part). OTHER MATERIAL EXAMINED. Sri Lanka, North Central
Province, Puttalam District, Eluwankulam, 08°12'35.1
"N 079°51'32"E, 52 m a.s.l. (Locality 15CN, Fig. 591),
28.IV.2015, 1♂, FKCP, leg. Kovařík et al.; North Cen-
tral Province, Puttalam District, Eluwankulam, 08°17' OTHER MATERIAL EXAMINED. Sri Lanka, North Central
Province, Puttalam District, Eluwankulam, 08°12'35.1
"N 079°51'32"E, 52 m a.s.l. (Locality 15CN, Fig. 591),
28.IV.2015, 1♂, FKCP, leg. Kovařík et al.; North Cen-
tral Province, Puttalam District, Eluwankulam, 08°17' Kovařík et al.: Scorpions of Sri Lanka 21 Kovařík et al.: Scorpions of Sri Lanka
21
Figures 80–84: Comparing Sri Lankan Charmus female me
metasoma and telson, lateral (80), ventral (81), and dorsal (82
15CO, metasoma and telson dorsal. Figures 84. Female holotyp
dorsal. tasoma
ale holotype of Charmus laneus,
)
e of
(L
li
15CO Fi
d l
s. Figures 80–82. Fem
views. Figures 83. Female of Charmus laneus from locality
Charmus saradieli sp. n. (MHNG) metasoma and telson
15"N 079°50'38 7"E 38
l
bl
k
di
l
f
l
i l
bl
k
i h Figures 80–84: Comparing Sri Lankan Charmus female me
metasoma and telson, lateral (80), ventral (81), and dorsal (82
15CO, metasoma and telson dorsal. Figures 84. Female holotyp
dorsal. tasoma
ale holotype of Charmus laneus,
)
e of
s. Figures 80–82. Fem
views. Figures 83. Female of Charmus laneus from locality
Charmus saradieli sp. n. (MHNG) metasoma and telson (Locality 15CO, Fig. 92), 28.IV.2015, 1♂ (Figs. 41, 48–55, 60, 62, 74–76,
18, 194, 42
, 63, 77–79,
8
(Figs. 64–73) 1
. 15"N 079°50'38.7"E, 38 m a.s.l. 5
1
3, 548) 1♀ (Figs. 42, 58–59, 61
3, 85, 119, 424) 1juv. (Figs. 56–57, 86), FKCP, 2♂
♀, UPSL, leg. Kovařík et al (Locality 15CO, Fig. 92), 28.IV.2015, 1♂ (Figs. 41, 48–55, 60, 62, 74–76,
18, 194, 42
, 63, 77–79,
8
(Figs. 64–73) 1
. 15"N 079°50'38.7"E, 38 m a.s.l. 5
1
3, 548) 1♀ (Figs. TYPE LOCALITY AND TYPE REPOSITORY. Ceylon; ZMHB. 42, 58–59, 61
3, 85, 119, 424) 1juv. (Figs. 56–57, 86), FKCP, 2♂
♀, UPSL, leg. Kovařík et al adul
seve
sh
ith
s;
ranular without carinae, anterior edge with epistome
V punctate without developed cari-
ts black; pedipalp femur almost entirely black with
ral small yellow spots; pedipalp patella yellowi
several black spots; legs yellow with black spot
w
chelicerae brown, with black reticulation. Carapace
g
present medially. Tergites I–VI granular, obviously with
one carina. Sternites without carinae. Metasomal seg-
ments IV–V or III– mal
DIAGNOSIS. Total length 14 mm (male) – 21.3 mm (fe-
e). Mesosoma, carapace, metasoma and telson of Euscorpius — 2016, No. 220 22 p
,
l (86) of Charmus laneus from locality 15CO. Figure 87. Male
paratype of Charmus saradieli sp. n. (UPSL). Figures 85–87: Charmus. Figures 85–86. Male (85) and fema e l (86) of Charmus laneus from locality 15CO. Figure 87. Male
paratype of Charmus saradieli sp. n. (UPSL). Figures 85–87: Charmus. Figures 85–86. Male (85) and fema e 23 Kovařík et al.: Scorpions of Sri Lanka Kovařík et al.: Scorpions of Sri Lanka Kovařík et al.: Scorpions of Sri Lanka Figures 88–91: Charmus saradieli sp. n. Figures 88–89. Male paratype (MHNG) in dorsal (88) and ventral (89) views, and
original labels. Figures 90–91. Female holotype in dorsal (90) and ventral (91) views, and original labels. Scale bar: 5 mm. Figures 88–91: Charmus saradieli sp. n. Figures 88–89. Male paratype (MHNG) in dorsal (88) and ventral (89) views, and
original labels. Figures 90–91. Female holotype in dorsal (90) and ventral (91) views, and original labels. Scale bar: 5 mm. Euscorpius — 2016, No. 220 24 Figures 92–100: Charmus. Figures 92–95, 99–100. Charmus saradieli sp. n., male paratype (MHNG), chelicerae, carapace
and tergites I–IV (92), sternopectinal region and sternites III–IV (94), and telson lateral (99); female holotype, chelicerae,
carapace and tergites I–III (93), sternopectinal region and sternite III (95), and telson lateral (100). Figures 96–98. Charmus
laneus, female holotype, chelicera and carapace (96), original label (97), and sternopectinal region and sternites III–IV (98). ae. Fifth metasomal segment length / width ratio
l
h i
female 1.692–1.791. Movable and fixed fingers of pedi-
l
d 3
i
l
l
h
f
l
n
1.288–1.425 in female. Pectines with or without fulcra. R i
f
di
l
h l
l
h / fi
d fi
palps bearing 8 rows of granules, apical rows of 4–6 Figures 92–100: Charmus. Figures 92–95, 99–100. Charmus saradieli sp. n., male paratype (MHNG), chelicerae, carapace
and tergites I–IV (92), sternopectinal region and sternites III–IV (94), and telson lateral (99); female holotype, chelicerae,
carapace and tergites I–III (93), sternopectinal region and sternite III (95), and telson lateral (100). Figures 96–98. Charmus
laneus, female holotype, chelicera and carapace (96), original label (97), and sternopectinal region and sternites III–IV (98). female 1.692–1.791. Movable and fixed fingers of pedi-
granules, and 3 terminal granules; each row of granules
palps bearing 8 rows of granules, apical rows of 4–6 ae. Fifth metasomal segment length / width ratio
nger length in
n
1.288–1.425 in female. Pectines with or without fulcra. Ratio of pedipalp chela length / fixed fi Kovařík et al.: Scorpions of Sri Lanka
25
Figures 101–106: Charmus saradieli sp. n. Figures 101–103. Male paratype (MHNG), metasoma and telson, lateral (101)
ventral (102), and dorsal (103) views. Figures 104–106. Female holotype, metasoma and telson, lateral (104), ventral (105), and
dorsal (106) views. Kovařík et al.: Scorpions of Sri Lanka 25 Figures 101–106: Charmus saradieli sp. n. Figures 101–103. Kovařík et al.: Scorpions of Sri Lanka Male paratype (MHNG), metasoma and telson, lateral (101),
ventral (102), and dorsal (103) views. Figures 104–106. Female holotype, metasoma and telson, lateral (104), ventral (105), and
dorsal (106) views. (except last) with one internal and two external acces-
sory granules. Pectinal teeth number 16–18 in both
sexes. Telson vesicle punctate, rather bulbous in male. aspect of manus and fixed finger with dense brush of
microsetae, most if not all appearing fluorescent with
intense terminal pinpoint fluorescence (i.e. putative
chemoreceptive setae). Microsetae also present but
sparse on ventral aspect of movable finger. Fingers with
typical buthid dentition (Vachon, 1963). Fixed finger
with distal and subdistal denticles, and 2 basal denticles
fused into bicusp. Dorsal margin of movable finger with
5 denticles: 1 large distal and 1 large medial, 1 smaller
subdistal, and 2 small partially fused basal denticles. Ventral margin of movable finger with 3 denticles: 1
large distal and 2 smaller medial denticles. Ventral
icro-
setae (Fig. 67b). surface of fixed finger armed medially and basally with
2 small denticles concealed by dense brush of m HEMISPERMATOPHORE (Figs. 48–53). Trunk very nar-
row, elongate, capsule region short (Fig. 48). Flagellum
cylindriform, relatively short, robust, coiled. Median
lobe broad, distally truncate, with straight dorsal carina
near internal margin. Basal lobe well developed, a prom-
inent, blunt, bilobate scoop arising dorsally near base of
median lobe carina. C
(Figs. 66–67). Manus with dark reticulated
ith 2 dorsal macrosetae on anterior half. Ventrointernal
HELICERA
patterns on dorsal and ventral surfaces. Dorsal manus
ith 9 macrosetae near anterior margin. Movable finger
w
w 26 Euscorpius — 2016, No. 220 Figures 107–119: Charmus. Figures 107–114. Charmus saradieli sp. n., female holotype. Pedipalp movable finger (107). Pedipalp chela, dorsal (108), external (109), and ventral (110) views. Pedipalp patella, dorsal (111) and external (112) views. edipalp femur and trochanter, internal (113) and dorsal (114) views. The trichobothrial pattern is indicated in Figures 1
P
1
08a–
14a. Figures 115–117. Charmus saradieli sp. n., male paratype (MHNG), chelicerae (115), pedipalp dorsal (116), and fourth
leg dorsal (117). Figures 118–119. Charmus laneus from locality 15CO. Pedipalp dorsal of male (118) and female (119). Figures 107–119: Charmus. Figures 107–114. Charmus saradieli sp. n., female holotype. Pedipalp movable finger (107). Pedipalp chela, dorsal (108), external (109), and ventral (110) views. Pedipalp patella, dorsal (111) and external (112) views. edipalp femur and trochanter, internal (113) and dorsal (114) views. Kovařík et al.: Scorpions of Sri Lanka The trichobothrial pattern is indicated in Figures 1
P
1
08a–
14a. Figures 115–117. Charmus saradieli sp. n., male paratype (MHNG), chelicerae (115), pedipalp dorsal (116), and fourth
leg dorsal (117). Figures 118–119. Charmus laneus from locality 15CO. Pedipalp dorsal of male (118) and female (119). Kovařík et al.: Scorpions of Sri Lanka
27
Charmus laneus . Charmus saradieli sp. n. Dimensions (mm)
♀ holotype
♀ 15CO
♀ holotype
♂ paratype
Carapace
L / W
2.125 / 2.425
2.250 / 2.900
2.625 / 2.900
1.450 / 1.575
Mesosoma
L
6.100
6.900
8.800
3.800
Tergite VII
L / W
1.250 / 2.050
1.550 / 2.600
1.600 / 3.000
0.825 / 1.375
Metasoma
& telson
L
10.275
10.900
12.070
6.925
Segment I
L / W / D 1.175 / 1.475 / 1.260
1.150 / 1.625 / 1.425
1.375 / 1.700 / 1.500
0.800 / 0.900 / 0.825
Segment II
L / W / D 1.350 / 1.450 / 1.375
1.500 / 1.680 / 1.480
1.575 / 1.600 / 1.525
0.875 / 0.825 / 0.825
Segment III
L / W / D 1.600 / 1.550 / 1.425
1.625 / 1.825 / 1.600
1.600 / 1.650 / 1.610
0.925 / 0.850 / 0.825
Segment IV
L / W / D 1.750 / 1.600 / 1.425
1.875 / 1.875 / 1.675
2.100 / 1.580 / 1.630
1.250 / 0.825 / 0.775
Segment V
L / W / D 2.300 / 1.625 / 1.375
2.480 / 1.925 / 1.575
2.900 / 1.610 / 1.600
1.675 / 0.800 / 0.738
Telson
L / W / D 2.100 / 0.910 / 0.850
2.275 / 1.125 / 0.950
2.520 / 1.020 / 1.000
1.400 / 0.525 / 0.475
Pedipalp
L
7.050
7.520
9.200
5.200
Femur
L / W
1.800 / 0.530
1.900 / 0.630
2.050 / 0.700
1.275 / 0.425
Patela
L / W
2.150 / 0.750
2.325 / 0.900
2.900 / 0.980
1.625 / 0.525
Chela
L
3.000
3.300
4.250
2.300
Manus
L / W / D 1.050 / 0.600 / 0.575
1.025 / 0.725 / 0.710
1.300 / 0.800 / 0.790
0.625 / 0.475 / 0.475
Fixed finger
L
1.675
1.950
2.925
1.400
Movable finger
L
2.050
2.275
2.950
1.675
Total length
18.50
20.10
23.50
12.18
Table 1: Comparative measurements of adults of Charmus laneus and C. saradieli sp. n. Kovařík et al.: Scorpions of Sri Lanka Abbreviations: length (L), width (W,
in carapace it corresponds to posterior width) depth (D) Kovařík et al.: Scorpions of Sri Lanka 27 Charmus saradieli sp. n. Table 1: Comparative measurements of adults of Charmus laneus and C. saradieli sp. n. Abbreviations: length (L), width (W,
in carapace it corresponds to posterior width), depth (D). Table 1: Comparative measurements of adults of Charmus laneus and C. saradieli sp. n. Abbreviations: length (L), width (W,
in carapace it corresponds to posterior width), depth (D). Ratios of adult females
C. laneus
(n = 4)
C. saradieli sp. n. (n = 1)
Metasomal segment I (L/W)
0.707–0.796
0.808
Metasomal segment II (L/W)
0.892–0.931
0.984
Metasomal segment IV (L/W)
1.000–1.093
1.329
Metasomal segment IV (L/D)
1.119–1.228
1.288
Metasomal segment V (L/W)
1.288–1.425
1.801
Metasomal segment V (L/D)
1.574–1.672
1.812
Telson (L/D)
2.394–2.470
2.520
Pedipalp chela (L/W)
4.551–5.000
5.312
Pedipalp chela (L) / fixed finger (L)
1.692–1.791
1.452
Pedipalp chela (L) / movable finger (L)
1.450–1.512
1.440
Total (L)
18.50–21.30
23.50
Table 2: Comparison among Sri Lankan Charmus species (specimens), based upon selected morphometric ratios of adult
males. Abbreviations: length (L), width (W), depth (D). fe Table 2: Comparison among Sri Lankan Charmus species (specimens), based upon selected morphometric ratios of adult
males. Abbreviations: length (L), width (W), depth (D). fe us in spite of the fact that he cited six characters for
is own figure (Lourenço, 2002: 18,
1) shows that the male "paratype" of C. minor has
COMMENTS. Lourenço did not study any specimens of C. lane
distinguishing C. laneus from C. minor (Lourenço,
2002: 23). Several of these 'character differences' lie
within the range of intraspecific variation (e.g. fulcra of
pectines), and others are not valid. Lourenço stated that
the movable finger of C. laneus bears 7–8 rows of
granules, while that of C. minor bears 9 rows of
granules. However, h
fig. only 8 rows of granules. Another problem is that
according to the type material section (Lourenço, 2002:
19) there exist only two types of C. minor – the male
holotype and a juvenile (second instar) female paratype. g to Vachon’s opinion they all belong
Neither a male paratype, nor an adult female paratype
were listed. Thus, it is surprising that Lourenço claimed
that the female of C. minor has a differently flattened
sternum than the male (Lourenço, 2002: 23). Kovařík et al.: Scorpions of Sri Lanka If this was
a reference to the second instar juvenile female paratype,
the diagnostic character needs to specified for adult
females. Under "Ecological observations" Lourenço
(2002: 23) wrote that "The specimens of C. laneus
studied by Vachon (1982) were all collected in the
central-south region of Sri Lanka which is characterised
by high altitudes reaching to more than 1000 m...". In
fact, the three specimens studied by Vachon (1982: 81)
were collected at altitudes of 250 m, 600 m, and 1350 m
a.s.l. and accordin g to Vachon’s opinion they all belong
Neither a male paratype, nor an adult female paratype
were listed. Thus, it is surprising that Lourenço claimed
that the female of C. minor has a differently flattened
sternum than the male (Lourenço, 2002: 23). If this was
a reference to the second instar juvenile female paratype,
the diagnostic character needs to specified for adult
females. Under "Ecological observations" Lourenço
(2002: 23) wrote that "The specimens of C. laneus
studied by Vachon (1982) were all collected in the
central-south region of Sri Lanka which is characterised
by high altitudes reaching to more than 1000 m...". In
fact, the three specimens studied by Vachon (1982: 81)
were collected at altitudes of 250 m, 600 m, and 1350 m
a.s.l. and accordin 28 Euscorpius — 2016, No. 220 to the same species, 'C. laneus'. Neither Vachon (1982)
nor Lourenço (2002) studied the holotype of C. laneus,
and as we show here, these three Charmus specimens
from central Sri Lanka actually belong to a different
species. These three specimens are herein designated as
types of C. saradieli sp. n., and C. minor Lourenço,
2002 is synonymized with C. laneus Karsch, 1879 as
there are no significant differences between them at the
species level. to the same species, 'C. laneus'. Neither Vachon (1982)
nor Lourenço (2002) studied the holotype of C. laneus,
and as we show here, these three Charmus specimens
from central Sri Lanka actually belong to a different
species. These three specimens are herein designated as
types of C. saradieli sp. n., and C. minor Lourenço,
2002 is synonymized with C. laneus Karsch, 1879 as
there are no significant differences between them at the
species level. Metasomal segments IV–V punctate without developed
carinae. Fifth metasomal segment length/ width ratio 1.8
in female. Pectines with or without fulcra. Movable and
fixed finger of pedipalps long, ratio of pedipalp chela
length/ fixed finger length in female 1.452. Movable and
fixed fingers of pedipalps bear 8 rows of granules, apical
rows of 4–6 granules, and 3 terminal granules; each row
of granules (except most proximal) with one internal and
two external accessory granules. Pectinal teeth number
12–17 in males, 16 in female. Telson vesicle punctate,
rather elongate in male. DISTRIBUTION. Sri Lanka. DISTRIBUTION. Sri Lanka. http://www.zoobank.org/urn:lsid:zoobank.org:act:C
CA71154-E551-49C4-B328-E84B86656252 p
g
g
CA71154-E551-49C4-B328-E84B86656252 Charmus laneus (misidentification): Vachon, 1982: 79–
83, figs. 1–11, 82–83; Lourenço, 2000: 297, figs. 2–
3; Lourenço, 2002: 24; Fet & Lowe, 2000: 123 (in
part in references). Coloration (Figs. 87–91). Mesosoma and the carapace
yellow with black ornamentation, to almost entirely
black, metasoma yellow to reddish black with black orn-
amentation, and pedipalp femur and patella mainly black
with yellow spots, pedipalp chela yellow with black spots
on manus, pedipalp fingers yellow without spots. Telson
yellow to reddish black. Legs yellow with black spots. Chelicerae yellow, with black reticulation. TYPE LOCALITY AND TYPE REPOSITORY. Sri Lanka,
Hasalaka; MHNG. TYPE LOCALITY AND TYPE REPOSITORY. Sri Lanka,
Hasalaka; MHNG. TYPE MATERIAL EXAMINED. Sri Lanka, Hasalaka (Loc. No. 9), 250 m a.s.l., 18.I.1970, 1♀ (holotype, Figs. 44,
80–82, 90–91, 93, 95, 100, 104–114, 426), MHNG, leg. Y. Löbl et C. Besuchet; Haputale (Loc. No. 19), 1350 m
a.s.l., 23.I.1970, 1♂ (paratype, Figs. 88–89, 92, 94, 99,
101–103, 115–117, 425), MHNG, leg. Y. Löbl, Kandy
(Loc. No. 18), 600 m a.s.l., 22.I.1970, 1juv. (paratype),
MHNG, leg. Y. Löbl et C. Besuchet; Eastern Province,
Padiyatalawa (ca 7°25'N 81°15'E), IV.1994, 1♂ (para-
type), FKCP, leg. P. Senft; Central Province, Kandy
District, Gannoruwa village, 07°17'10" 080°35'35", 10. IX.2015, 1♂ (paratype, Fig. 87), UPSL, leg. S. Jaya-
rathne. Carapace (Figs. 92–93). Granular without carinae, an-
terior edge straight with epistome present medially. Granulation stronger in female. Median furrow present,
more distinct in male. Three well developed and two
reduced or absent lateral eyes. Mesosoma (Figs. 92–95). Tergites with one carina,
densely granulate in the female. In the males granules
are larger and sparse. Pectinal tooth count 16 in female,
12–17 in males. Marginal tips of pectines extending to
3/4 quarters of sternite III in female, to end of sternite III
in the males. Pectines with 3 marginal lamellae and 7
middle lamellae, fulcra either present (female) or absent
(male). Sternites III–VII smooth, without carinae. Stigmata short, ovoid. ETYMOLOGY. Named after the popular Sri Lankan folk
hero Deekirikevage Saradiel whose activities between
Colombo and Kandy in the 1850s–1860s were com-
parable to the world famous legend of Robin Hood. His
forest sanctuary was on the summit of Mt. Utuwan-
kanda. Metasoma and telson (Figs. 101–106, 425–426). Metasoma relatively narrow, segment V length/ width
ratio 1.8 in female. Segments I–III strongly granulated,
more so in female, with 8–10 granulated carinae. Segments IV–V may have two smooth, poorly deve-
loped dorsal carinae; segments IV–V and telson punctate
except for dorsal surface. Telson rather elongate, es-
pecially in smaller males. Entire metasoma and the
telson densely hirsute. Vesicle of telson somewhat elon-
tae on
ventral surface, fewer on other surfaces; bristle combs
bsent. gate with long, strongly curved aculeus. Legs (Figs. 117). Long tibial spur present on the third
and fourth legs. Tarsus hirsute, more densely so on
ventral surface. Tarsomeres bearing numerous se
a Metasoma and telson (Figs. 101–106, 425–426). Metasoma relatively narrow, segment V length/ width
ratio 1.8 in female. DISTRIBUTION. Sri Lanka. Charmus saradieli Kovařík, Lowe, Ranawana, Hoferek
et Jayarathne, sp. n. (Figs. 12, 44, 84, 87–95, 99–117, 425–426, Tables 1–2)
http://www.zoobank.org/urn:lsid:zoobank.org:act:C
CA71154-E551-49C4-B328-E84B86656252 Charmus saradieli Kovařík, Lowe, Ranawana, Hoferek
et Jayarathne, sp. n. DESCRIPTION. The adult male paratypes are 12.18–18.42
mm long, the adult female holotype is 23.5 mm long. For habitus see Figs. 88–91. For position and dis-
tribution of trichobothria of pedipalps see Figs. 108–114. For measurements and ratios see Tables 1–2. Sexual di-
morphism is manifested mainly in total length. Other
sexual differences are noted below. (Figs. 12, 44, 84, 87–95, 99–117, 425–426, Tables 1–2) (
g
,
,
,
,
,
,
)
http://www.zoobank.org/urn:lsid:zoobank.org:act:C http://www.zoobank.org/urn:lsid:zoobank.org:act:C
CA71154-E551-49C4-B328-E84B86656252 TYPE LOCALITY AND TYPE REPOSITORY. Sri Lanka,
Hasalaka; MHNG. Segments I–III strongly granulated,
more so in female, with 8–10 granulated carinae. Segments IV–V may have two smooth, poorly deve-
loped dorsal carinae; segments IV–V and telson punctate
except for dorsal surface. Telson rather elongate, es-
pecially in smaller males. Entire metasoma and the
telson densely hirsute. Vesicle of telson somewhat elon-
gate with long, strongly curved aculeus. DIAGNOSIS. Total length 12.18 mm (male) – 23.5 mm
(female). Sexual dimorphism manifested mainly in total
length. Mesosoma and carapace yellow with black
ornamentation, to almost entirely black; pedipalp femur
ith epistome present medially. Tergites I–VI granular,
ith one clearly visible carina. Sternites without carinae. and patella almost entirely black with several small
yellow spots; telson yellow to reddish black; legs yellow
with black spots; chelicerae yellow, with black retic-
ulation. Carapace granular without carinae, anterior edge
w
w DIAGNOSIS. Total length 12.18 mm (male) – 23.5 mm
(female). Sexual dimorphism manifested mainly in total
length. Mesosoma and carapace yellow with black
ornamentation, to almost entirely black; pedipalp femur
ith epistome present medially. Tergites I–VI granular,
ith one clearly visible carina. Sternites without carinae. and patella almost entirely black with several small
yellow spots; telson yellow to reddish black; legs yellow
with black spots; chelicerae yellow, with black retic-
ulation. Carapace granular without carinae, anterior edge
w
w Kovařík et al.: Scorpions of Sri Lanka Kovařík et al.: Scorpions of Sri Lanka 29 Kovařík et al.: Scorpions of Sri Lanka
29
Figures 120–124: Hottentotta tamulus, male from locality 15CK, dorsal (120) and ventral (121) views, chelicerae, carapace
and tergites I–III (122), sternopectinal region and sternites III–IV (123), and telson lateral (124). Figures 120–124: Hottentotta tamulus, male from locality 15CK, dorsal (120) and ventral (121) views, chelicerae, carapace
and tergites I–III (122), sternopectinal region and sternites III–IV (123), and telson lateral (124). Pedipalps (Figs. 107–114, 116). Femur with 4 granulated
carinae, surface finely granulated. patella smooth without
developed carinae, only internal surface with several
larger granules. Chela smooth, lacking carinae. Pedipalps
hirsute on all segments. Movable and fixed fingers of
pedipalp bear 8 rows of granules, each row (except most
proximal) with one internal and two external accessory
granules; fingers also with apical rows of 4–6 granules
and 3 terminal granules. butions (Fig. 12), these two species can be separated
from each other by: 1) the color of the patella of
pedipalps, which is yellowish with several black spots in
C. laneus (Figs. TYPE SPECIES. Scorpio hottentotta Fabricius, 1787. DIAGNOSIS. Medium to large buthids, adults 30–130
mm. Sternum type 1, triangular in shape. Pedipalps
orthobothriotaxic, type Aβ, femur trichobothrium d2
dorsal, patella d3 dorsal of dorsomedian carina. Chelal
trichobothrium db usually located between est and et,
but may be level with trichobothrium est, and rarely
between est and esb. Trichobothrium eb clearly posi-
tioned on fixed finger of pedipalp. Pectines with fulcra. Dentate margin of pedipalp chela movable finger with
distinct granules divided into 11–16 linear rows and (4)
5–7 terminal granules. Chelicerae with typical buthid
dentition, fixed finger armed with two denticles on
ventral surface. Tergites I–VI granular, with three
carinae, tergite VII with 5 carinae. Carapace with dis-
tinct carinae, entire dorsal surface nearly planar. First
sternite with two granulated lateral stridulatory areas,
which may be reduced in some species. Metasoma elon-
gate, segment I with 10 carinae, segments II-IV with 8–
10 carinae. Ventrolateral carinae of fifth metasomal seg-
ment with all granules more or less equal in size, never
lobate. Telson bulbous, lumpy and granulated, without
subaculear tooth. Legs III and IV with well developed
tibial spurs, first and second tarsomeres with paired
ventral setae. DIAGNOSIS. Total length 50–90 mm. Trichobothrium db
on fixed figer of pedipalp chela situated between tri-
chobothria et and est, but may be level with est. Males
with proximal margins of pedipalp fingers flexed, manus
of pedipalps wider than female. Pectinal teeth number
30–39 in males, 27–34 in females. Chelicerae yellow,
reticulated. Pedipalps densely hirsute, legs and meta-
soma sparsely hirsute. Setae on patella of pedipalps
short. Color uniformly yellow to reddish, mesosoma
dark. Ventral carinae on metasomal segments usually
black. Pedipalp femur with 5 carinae, patella with two or
4 carinae on internal surface, no other carinae. Chela
lacking carinae. Movable fingers of pedipalps with 13–
15 rows of granules and 5 or 6 terminal granules. Seventh sternite with 4 well marked black carinae. First
to third metasomal segments with 10 carinae; fourth
with 10 or rarely 8 carinae; fifth with 5 or 7 carinae. Metasoma sparsely to densely granulated between cari-
nae. Dorsal surface densely and very finely granulated,
often bearing two short, inconspicuous marginal carinae. Telson granulated. Dorsal carinae of metasomal seg-
ments with posterior terminal granules of size approx-
imately equal to preceding granules. First metasomal
segments of adult female wider than long (in male
usually as long as wide), second metasomal segment
about 1.1. Kovařík et al.: Scorpions of Sri Lanka 31 TYPE LOCALITY AND TYPE REPOSITORY. “India ori-
entalis”; original type lost. Neotype from India, Maha-
rashtra State, Bombay env., designated by Kovařík,
2007: 76; NMPC. Buthus (Hottentotta) Birula, 1908: 141. Hottentotta: Fet & Lowe, 2000: 133–145 (complete
reference list until 1998); Kovařík & Ojanguren
Affilastro, 2013: 159–180, figs. 942–1250 (refer-
ence list until 2013). SRI LANKAN MATERIAL EXAMINED. Sri Lanka, Northern
Province, Jaffna District, Palali, 09°44'16.83"N 080°
05'2.88"E, 2012, 1♂, FKCP, col. K. B. Ranawana;
Northern Province, Jaffna District, 09°49'15.4"N 080°
08'41.6"E, 19 m a.s.l. (Locality 15CK, Fig. 588), 27. IV.2015, 1♂ (45–46, 120–124, 126, 195, 427)
1♀im.1juv., FKCP, 1♀ (Fig. 125) 1juv., UPSL, leg. Kovařík et al. Mesobuthus (in part): Fet & Lowe, 2000: 169–180. TYPE LOCALITY AND TYPE REPOSITORY. Sri Lanka,
Hasalaka; MHNG. 118–119) and black with several little
yellow spots in C. saradieli sp. n. (Figs. 111 and 116);
2) the shape of the metasoma, as the metasomal segment
V length/ width ratio is 1.288–1.425 in female of C. laneus (Figs. 80–83, Tables 1–2) and 1.800 in female of
C. saradieli sp. n. (Fig. 84, Tables 1–2); and 3) the
shape of pedipalp chela, as the chela length/ fixed finger
length ratio is 1.692–1.791 in female of C. laneus (Figs. 42–43, Tables 1–2) and 1.452 in female of C. saradieli
sp. n. (Fig. 44, Tables 1–2). AFFINITIES. Charmus saradieli sp. n. and C. laneus
Karsch, 1879 are the only two members the genus
known from Sri Lanka. Apart from their disjunct distri- Euscorpius — 2016, No. 220
30
. Figures 125–126: Hottentotta tamulus from locality 15CK, female (125) and male (126) Euscorpius — 2016, No. 220 Euscorpius — 2016, No. 220 30 . Figures 125–126: Hottentotta tamulus from locality 15CK, female (125) and male (126) Scorpio tamulus Fabricius, 1798: 294. Hottentotta tumulus [sic]: Veronika & al., 2013: 70–71,
fig. 1, 9–14, tab. 1. Kovařík et al.: Scorpions of Sri Lanka
Genus Hottentotta Birula, 1908
(Figs. 12, 45–46, 195, 427) Kovařík et al.: Scorpions of Sri Lanka
Genus Hottentotta Birula, 1908
(Figs. 12, 45–46, 195, 427) TYPE SPECIES. Scorpio hottentotta Fabricius, 1787. Length to width
ratio of fourth metasomal segment about 1.5. Telson
longer than wide in both sexes. Second to fourth meta-
somal segments width ratio
bulbous, especially in large females. Telson granulated. Dorsal carinae of metasomal seg-
ments with posterior terminal granules of size approx-
imately equal to preceding granules. First metasomal
segments of adult female wider than long (in male
usually as long as wide), second metasomal segment
about 1.1. Length to width
ratio of fourth metasomal segment about 1.5. Telson
longer than wide in both sexes. Second to fourth meta-
somal segments width ratio
bulbous, especially in large females. ISTRIBUTION. Hottentotta is one of the most widely
sia to Pakistan and India. D
distributed genera of the family Buthidae, with species
resent throughout Africa, the Arabian Peninsula, and in
p
A Hottentotta tamulus (Fabricius, 1798)
(Figs. 12, 45–46, 120–126, 195, 427) DISTRIBUTION. India (Andhra Pradesh, Bihar, Gujarat,
Jharkhand, Madhya Pradesh, Maharashtra, Pondicherry,
Rajasthan, Tamil Nadu and West Bengal States), Pak-
istan, Sri Lanka. Scorpio tamulus Fabricius, 1798: 294. Mesobuthus tamulus: Fet & Lowe, 2000: 179–180
(complete reference list until 1998). Isometrus Ehrenberg, 1828
(Figs. 13, 127–151, 196–197, 221–224, 237–244, 252–
253, 403–406, 549–555, 557–560) 5–1008, 1173 (reference list
until 2013); Ranawana et al., 2013: 3–7, figs.1–12;
Hottentotta tamulus: Kovařík & Ojanguren Affilastro,
2013: 176, figs. 100
Veronika & al., 2013: 73–75, figs. 1, 9–14, tab. 1. Buthus (Isometrus) Ehrenberg in Hemprich & Ehren-
berg, 1828: pl. 1, fig. 3; Hemprich & Ehrenberg,
1829: 351. Buthus (Isometrus) Ehrenberg in Hemprich & Ehren-
berg, 1828: pl. 1, fig. 3; Hemprich & Ehrenberg,
1829: 351. Hottentotta tumulus [sic]: Veronika & al., 2013: 70–71,
fig. 1, 9–14, tab. 1. 32 Euscorpius — 2016, No. 220 igures 127–130: Isometrus maculatus. Figures 127–128. Male from locality 15CP in dorsal (127) and ventral (128) views
F
Figures 129–130. Female from locality 15CI in dorsal (129) and ventral (130) views. igures 127–130: Isometrus maculatus. Figures 127–128. Male from locality 15CP in dorsal (127) and ventral (128) views. F
Figures 129–130. Female from locality 15CI in dorsal (129) and ventral (130) views. Kovařík et al.: Scorpions of Sri Lanka
33
Figures 131–134: Isometrus maculatus. Figures 131, 133. M
(131) and sternopectinal region and sternite III (133). Figures
tergites I–III (132) and sternopectinal region and sternite III (134
ale from locality 15CP, chelicerae, carapace and tergites I–III
32, 134. Female from locality 15CI, chelicerae, carapace and
. TYPE SPECIES. Scorpio hottentotta Fabricius, 1787. 1
) Kovařík et al.: Scorpions of Sri Lanka 33 Figures 131–134: Isometrus maculatus. Figures 131, 133. M
(131) and sternopectinal region and sternite III (133). Figures
tergites I–III (132) and sternopectinal region and sternite III (134
ale from locality 15CP, chelicerae, carapace and tergites I–III
32, 134. Female from locality 15CI, chelicerae, carapace and
. 1
) Isometrus (Isometrus): Vachon, 1972: 169–180, figs. 1–
13, 15, 17, 19; Vachon, 1982: 86–90, 100–101, 108,
figs. 28–39, 64–65; Fet & Lowe, 2000: 146–150
(complete reference list until 1998); Kovařík, 2003:
2; Kovařík & Ojanguren, 2013: 182–184, 347, 353,
355–357, figs. 1251–1264, 1354–1357, 1368–1375,
1376–1385. meres of legs I–III with setae not arranged into bristle
combs on dorsal surfaces. Tarsomeres II of leg IV with
two rows of more than 30 dense setae. Mesosoma
dorsally with one median carina. Telson with subaculear
tooth pointed (except for Isometrus formosus Pocock,
1894). Pedipalp and metasomal segments longer in
males than females. figs. 771–840. Isometrus (Raddyanus [sic]): Tikader & Bastawade,
1983: 254–311 (in part), COMMENTS. The taxonomic position of Isometrus for-
mosus Pocock, 1894 from Indonesia is unclear. This
ecies is probably not a member of the genus
in remarks on the Indian species I. oni Pocock, 1893, wrote: "There are also two
specimens in
led Ceylon". H
een reco
hat I. sp
Isometrus, but comprises a separate genus. Two species
of Isometrus, I. maculatus (De Geer, 1778) and I. thwaitesi Pocock, 1897 are known from Sri Lanka. Po-
cock, 1900: 48,
thurst
the British Museum label
owever, as no other specimen of I. thurstoni has ever
rded from Sri Lanka, we believe t
b
thurstoni probably does not occur there. ikader & Bastawade, 1983:
311–
, 1994:
201)
= Isometrus (Closotrichus) T
316, figs. 896–910 (syn. by Kovařík
. TYPE SPECIES. Scorpio macu tus De Geer, 1778.
la TYPE SPECIES. Scorpio macu tus De Geer, 1778. la Ster
dipalps ortho-
betw
Tibi
fing
l
Cheliceral fixed finger with a single ventral denticle. hird and fourth legs with tibial spurs. Tibia and tarso-
DIAGNOSIS. Medium sized buthids, adults 30–75 mm. num type 1, triangular in shape. Pe
bothriotaxic, type Aβ. Chelal trichobothrium db located
een dt and et. Three to five pairs of lateral eyes. al spurs absent on all legs. Movable and fixed
ers of pedipalps with six rows of granules, severa
accessory granules and external and internal granules. T DIST
riental region from India to Melanesia
ee comments for I. maculatus). RIBUTION. O
(s Euscorpius — 2016, No. 220
34
Figures 135–137: Isometrus maculatus, female from locality 15CH (135) with newborns before first ecdysis (136), and with
juveniles after first ecdysis (137). Euscorpius — 2016, No. 220 Euscorpius — 2016, No. 220 34 Figures 135–137: Isometrus maculatus, female from locality 15CH (135) w
juveniles after first ecdysis (137). Figures 135–137: Isometrus maculatus, female from locality 15CH (135) with newborns before first ecdysis (136), and w
juveniles after first ecdysis (137). Figures 135–137: Isometrus maculatus, female from locality 15CH (135) with newborns before first ecdysis (136), and with
juveniles after first ecdysis (137). Kovařík et al.: Scorpions of Sri Lanka 35 Kovařík et al.: Scorpions of Sri Lanka
35
139. Mal
igures 140–141. Female from locality 15CO in dorsal (140) and
94–207 566–
Figures 138–141: Isometrus thwaitesi. Figures 138–
e from locality 15CH in dorsal (138) and ventral (139) views. ventral (141) views. Isometrus (Isometrus) maculatus: Vachon, 1972: 169–
180, figs. 1–13, 15, 17, 19; Fet & Lowe, 2000: 147;
Lourenço & Huber, 2002: 266; Teruel & Kovařík,
2012: 88–91 figs 4 27 36–37 46 1
F
Isometrus maculatus (De Geer, 1778)
(Figs. 13, 127–137, 196, 221–222, 237–238, 242, 252,
403–404, 555, 557, 559) 139. Mal
igures 140–141. Female from locality 15CO in dorsal (140) and
Figures 138–141: Isometrus thwaitesi. Figures 138–
e from locality 15CH in dorsal (138) and ventral (139) views. ventral (141) views. F Isometrus maculatus (De Geer, 1778)
(Figs. 13, 127–137, 196, 221–222, 237–238, 242, 252,
403–404, 555, 557, 559) 94–207, 566–
569; Kovařík & Ojanguren, 2013: 182–184, 347,
355–356,
Isometrus (Isometrus) maculatus: Vachon, 1972: 169–
180, figs. 1–13, 15, 17, 19; Fet & Lowe, 2000: 147;
Lourenço & Huber, 2002: 266; Teruel & Kovařík,
2012: 88–91, figs. 4, 27, 36–37, 46, 1
figs. TYPE SPECIES. Scorpio macu tus De Geer, 1778.
la 1251–1255, 1264, 1368–1375, 1376– 94–207, 566–
569; Kovařík & Ojanguren, 2013: 182–184, 347,
355–356,
Isometrus (Isometrus) maculatus: Vachon, 1972: 169–
180, figs. 1–13, 15, 17, 19; Fet & Lowe, 2000: 147;
Lourenço & Huber, 2002: 266; Teruel & Kovařík,
2012: 88–91, figs. 4, 27, 36–37, 46, 1
figs. 1251–1255, 1264, 1368–1375, 1376– Isometrus maculatus (De Geer, 1778)
(Figs. 13, 127–137, 196, 221–222, 237–238, 242, 252,
403–404, 555, 557, 559)
Scorpio maculatus De Geer, 1778: 346. Isometrus maculatus: Kraepelin, 1899: 66. 403–404, 555, 557, 559)
Scorpio maculatus De Geer, 1778: 346. Isometrus maculatus: Kraepelin, 1899: 66. Scorpio maculatus De Geer, 1778: 346. Isometrus maculatus: Kraepelin, 1899: 66. Euscorpius — 2016, No. 220 36 al region and sternite III (145). Figures 142–145: Isometrus thwaitesi. Figures 142, 144. Male from locality 15CH, chelicerae, carapace and tergites I–II
(142) and sternopectinal region and sternite III (144). Figures 143, 145. Female from locality 15CO, chelicerae, carapace an
tergites I–III (143) and sternopectin al region and sternite III (145). Figures 142–145: Isometrus thwaitesi. Figures 142, 144. Male from locality 15CH, chelicerae, carapace and tergites I–III
(142) and sternopectinal region and sternite III (144). Figures 143, 145. Female from locality 15CO, chelicerae, carapace and
tergites I–III (143) and sternopectin Kovařík et al.: Scorpions of Sri Lanka Kovařík et al.: Scorpions of Sri Lanka 37 Figures 146–149: Left hemispermatophore of Isometrus thwaitesi from locality 15CH. External (146), dorsal (147), internal
(148) and ventral (149) views. Note: distal part of flagellum and basal part of trunk (foot) were damaged during dissection. Scale
bar: 500 µm. Abbreviations: bl, basal lobe; f, flagellum; ml, median lobe; mlc, median lobe carina; pr, pars recta of flagellum; prf,
pars reflecta of flagellum; t, trunk. Figures 146–149: Left hemispermatophore of Isometrus thwaitesi from locality 15CH. External (146), dorsal (147), internal
(148) and ventral (149) views. Note: distal part of flagellum and basal part of trunk (foot) were damaged during dissection. Scale
bar: 500 µm. Abbreviations: bl, basal lobe; f, flagellum; ml, median lobe; mlc, median lobe carina; pr, pars recta of flagellum; prf,
pars reflecta of flagellum; t, trunk. Veronika & al., 2013: 75, figs. 1, 15–20, tab. 1. 1383 (reference and synonymes list until 2013); Marichchukkaddi env, border of Wilpattu National Park,
08°33'32.3"N 079°56'51"E, 7 m a.s.l. (Locality 15CI,
134,
al.;
villa
calit
127
59), FKCP, leg. Kovařík et al.; Eastern Province, Am-
Kovařík et al.; Northern Province, Mannar District,
Fig. 585), 25. –26.IV.2015, 1♂1♀ (Figs. 129–130, 132,
196, 222, 238, 404) 1 juv., FKCP, leg. Kovařík et
Southern Province, Matara District, Kekanadura
ge, 05°58'28.2"N 080°36'20.5"E, 40 m a.s.l. (Lo-
y 15CP, Figs. 593–594), 30.IV.2015, 1♂ (Figs. –128, 131, 133, 221, 237, 242, 252, 403, 555, 557,
5 TYPE LO
e and
P
CALITY AND TYPE REPOSITORY. “Surinam
ennsylvania”; NHRS. RI LANKAN MATERIAL EXAMINED. Sri Lanka, Northern
, 08°48'26.3"N
ig. 584), 24. –25.IV.2015, 1♂1♀, FKCP, 1♀2juvs., UPSL, leg. S
Province, Mannar District, Madhu Road
080°10'26"E, 90 m a.s.l. (Locality 15CH, F Euscorpius — 2016, No. 220 38 Figures 150–151: Isometrus thwaitesi from locality 15CF, male (150) and female (151). Figures 150–151: Isometrus thwaitesi from locality 15CF, male (150) and female (151). Kovařík et al.: Scorpions of Sri Lanka Kovařík et al.: Scorpions of Sri Lanka 39 Kovařík et al.: Scorpions of Sri Lanka Figures 152–155: Lychas srilankensis from locality 15CJ. Figures 152–153. Male in dorsal (152) and ventral (153) views. Figures 154–155. Female in dorsal (154) and ventral (155) views. Figures 152–155: Lychas srilankensis from locality 15CJ. Figures 152–153. Male in dorsal (152) and ventral (153) views. Figures 154–155. Female in dorsal (154) and ventral (155) views. 40 Euscorpius — 2016, No. 220 Euscorpius
2016, No. 220
40
Figures 156–157: Lychas srilankensis, male from locality 15CO in dorsal (156) and ventral (157) views. Figures 156–157: Lychas srilankensis, male from locality 15CO in dorsal (156) and ventral (157) views. para District, Lahugala-Kitulana National Park, 06°52'
46"N 081°43'21.8"E, 40 m a.s.l. (Locality 15CR, Fig. 596), 3.–4.V.2015, photos only, leg. Kovařík et al. para District, Lahugala-Kitulana National Park, 06°52'
46"N 081°43'21.8"E, 40 m a.s.l. (Locality 15CR, Fig. 596), 3.–4.V.2015, photos only, leg. Kovařík et al. COMMENTS ON DISTRIBUTION. I. maculatus has been re-
garded as cosmopolitan (see Fet & Lowe, 2000: 149)
and records of this species are so numerous that a com-
plete listing would be unreasonably long. A list of speci-
mens which the first author examined until 2003 is pub-
lished in Kovařík (2003: 3). Additonal records are given
in Kovařík & Ojanguren (2013: 182–184). Records
peaked in the earlier era of wooden sailing ships and
harbors full of wooden barracks, both ideal niches for
these scorpions. For that reason between 1758 and ca. 1940 this species was reported from many countries and
large harbors including Hamburg in Europe. With the
uilding materials and modes of
travel, reports of this species have greatly diminished,
transition to modern b DIAGNOSIS. Total length 30–75 mm. Females usually
reach ca. 45 mm. Manus of pedipalp very thin, in males
its width equals that of patella and femur. Pedipalps and
legs yellow, with spots. Manus of pedipalps yellow with
several spots, fingers dark. Mesosomal segments light-
colored. First (basal) middle lamella of female pecten
quadrangular. Posterior margin of sternite V straight
emales) to very slightly convex medially (males). Su-
7–20 in the examined Sri Lanka specimens). (f
baculear tooth spinoid. Pectinal teeth number 15–20
(1 Kovařík et al.: Scorpions of Sri Lanka Kovařík et al.: Scorpions of Sri Lanka 41 p
Figures 158–165:
I–III (158), sternopectinal region and sternite III (160), and distal segments of legs I–IV (162–165), retroventral view. Figures
159, 161. Female, chelicerae, carapace and tergites I–III (159) and s
Lychas srilankensis from locality 15CJ. Figures 158, 160, 162–165. Male, chelicerae, carapace and tergites
ternopectinal region and sternite III (161). Figures 158–165:
I–III (158), sternopectinal region and sternite III (160), and distal segments of legs I–IV (162–165), retroventral view. Figures
159, 161. Female, chelicerae, carapace and tergites I–III (159) and s
Lychas srilankensis from locality 15CJ. Figures 158, 160, 162–165. Male, chelicerae, carapace and tergites
ternopectinal region and sternite III (161). Euscorpius — 2016, No. 220
42
Figures 166–171: Lychas srilankensis from locality 15CJ. Figures 166–168. Male, metasoma and telson, lateral (166), ventral
s
(167), and dorsal (168) views. Figures 169–171. Female, meta
views. oma and telson, lateral (169), ventral (170), and dorsal (171) 42 Euscorpius — 2016, No. 220 Euscorpius — 2016, No. 220 Figures 166–171: Lychas srilankensis from locality 15CJ. Figures 166–168. Male, metasoma and telson, lateral (166), ventral
s
(167), and dorsal (168) views. Figures 169–171. Female, meta
views. oma and telson, lateral (169), ventral (170), and dorsal (171) region in the world where I. maculatus is found in really
wild conditions inland." This was pure speculation,
unsupported by any data. Firstly, their assertion implies
that the authors know all species of scorpions present in
every "wild condition inland" everywhere else on the
globe. Secondly, their logic is flawed because although
certain exotic species may fail to penetrate endemic
'wild' ecosystems of host countries, this is by no means
the rule. There are many counterexamples where in-
vasive species have become established in endemic eco-
systems and displaced native fauna or flora, causing nd in many places have ceased altogether. Apparently,
he only
a
relatively few of the old invasive populations were able
to persist without being resupplied by a constant influx
of new individuals via human transport. The distribution
of I. maculatus in southeast Asia is not well docu-
mented, but it is possibly indigenous to India and/ or Sri
Lanka because of the presence there of its most closely
related species: I. thurstoni and I. thwaitesi. Lourenço &
Huber (2002: 266) suggested that Sri Lanka was the
original home of this species before cosmotropical dis-
persal by human activity, because Sri Lanka "is t Kovařík et al.: Scorpions of Sri Lanka
43
Figures 172–182: Lychas srilankensis from locality 15CJ. Figures 174–180, 182. Male. Pedipalp chela, dorsal (174), external
(175), and ventral (176) views. Pedipalp patella, dorsal (177) and external (178) views. Pedipalp femur and trochanter dorsal
(179) view. The trichobothrial pattern is indicated in Figures 175–179. Pedipalp movable finger (180). Complete pedipalp (182). Figures 172–173, 181. Female. Pedipalp chela, dorsal (172) and external (173) views. Complete pedipalp (181). Kovařík et al.: Scorpions of Sri Lanka 43 Figures 172–182: Lychas srilankensis from locality 15CJ. Figures 174–180, 182. Male. Pedipalp chela, dorsal (174), external
(175), and ventral (176) views. Pedipalp patella, dorsal (177) and external (178) views. Pedipalp femur and trochanter dorsal
(179) view. The trichobothrial pattern is indicated in Figures 175–179. Pedipalp movable finger (180). Complete pedipalp (182). Figures 172–173, 181. Female. Pedipalp chela, dorsal (172) and external (173) views. Complete pedipalp (181). MATERIAL EXAMINED. Sri Lanka, Ratnapura District, 2
km S Hayes, 30.XI.1995, 1♀, FKCP, leg. S. Bečvář &
Košťál; North Central Province, Polonnaruwa District,
near Kaudulla National Park, 08°08'40.6"N 080°51'
04"E, 101 m a.s.l. (Locality 15CF, Fig. 581), 23.IV. 2015, 1♂ (Fig. 150, 549) 1♀ (Fig. 151), FKCP, leg. Kovařík et al.; Northern Province, Mannar District,
Madhu Road, 08°48'26.3"N 080°10'26"E, 90 m a.s.l. (Locality 15CH, Fig. 584), 24. –25.IV.2015, 1♂ (Fig-
ures 138–139, 142, 144, 146–149, 223, 239, 243, 405,
558–560) 1juv., FKCP, leg. Kovařík et al.; Northern
Province, Mannar District, Marichchukkaddi env, border
of Wilpattu National Park, 08°33'32.3"N 079°56'51"E, 7
m a.s.l. (Locality 15CI, Fig. 585), 25. –26.IV.2015, 1♀,
FKCP, leg. Kovařík et al.; North Central Province,
Anuradhapura District, Mihintale, 08°20'51.8"N 080°30'
27.7"E, 156 m a.s.l. (Locality 15CL, Fig. 589), 27. –
28.IV.2015, 1♂, FKCP, leg. Kovařík et al.; North Cen-
tral Province, Puttalam District, Eluwankulam, 08°17'
15"N 079°50'38.7"E, 38 m a.s.l. (Locality 15CO, Fig. 592), 28.IV.2015, 1♂1♀ (Figures 140–141, 143, 145,
197, 224, 240–241, 244, 253, 406) 1juv., FKCP, leg. major negative impacts. However, Veronika et. al. (2013: 75) cited this speculation as fact, claimimg that I. maculatus is an "Endemic species in Sri Lanka". A
proper test of this hypothesis may require comparative
DNA studies of widespread I. maculatus populations. Isometrus thwaitesi Pocock, 1897
(Figs. 13, 138–151, 197, 223–224, 239–241, 243–244,
253, 405–406, 549, 558–560) Isometrus thwaitesii Pocock, 1897: 114. Isometrus thwaitesii Pocock, 1897: 114. Isometrus (Isometrus) thwaitesi: Vachon, 1982: 88–90,
figs. 36–39; Fet & Lowe, 2000: 150 (complete
reference list until 1998); Kovařík, 2003: 4; Kovařík
& Ojanguren, 2013: 184, 347, figs. 1260–1263. = Isometrus (Isometrus) thwaitesi pallidus Lourenço &
Huber, 2002: 266, figs. 1–7 (syn. by Kovařík &
Ojanguren, 2013: 184). TYPE LOCALITY AND TYPE REPOSITORY. Ceylon, now Sri TYPE LOCALITY AND TYPE REPOSITORY. Ceylon, now Sri
Lanka; BMNH. Lanka; BMNH. Euscorpius — 2016, No. 220 44 Figures 183–186: Right hemispermatophore of Lychas sril nkensis from locality 15CN (presented as mirror image for
comparison to other species). External (183), dorsal (184), inte nal (185) and ventral (186) views. Extracted after 3rd ecdysis. Hemispermatophores examined from a second male after 4th ec ysis were similar. Scale bar: 500 µm. Abbreviations: bl, basal
lobe; f, flagellum; ml, median lobe; mlc, median lobe carina; pr, pars recta of flagellum; prf, pars reflecta of flagellum; t, trunk. Kovařík et al.; North Central Province, Sigiriya,
7°58'25.6"N 80°44'59.7"E, 1♀, UPSL, leg. S. Jay-
arathne
DIAGNOSIS. Total length 30–50 mm. Manus of pedipalp
very thin, width in male equal to that that of patella and
femur Pedipalps and legs yellow with spots Fingers
a
r
d Figures 183–186: Right hemispermatophore of Lychas sril nkensis from locality 15CN (presented as mirror image for
comparison to other species). External (183), dorsal (184), inte nal (185) and ventral (186) views. Extracted after 3rd ecdysis. Hemispermatophores examined from a second male after 4th ec ysis were similar. Scale bar: 500 µm. Abbreviations: bl, basal
lobe; f, flagellum; ml, median lobe; mlc, median lobe carina; pr, pars recta of flagellum; prf, pars reflecta of flagellum; t, trunk. a
r
d DIAGNOSIS. Total length 30–50 mm. Manus of pedipalp
very thin, width in male equal to that that of patella and
femur. Pedipalps and legs yellow, with spots. Fingers DIAGNOSIS. Total length 30–50 mm. Manus of pedipalp
very thin, width in male equal to that that of patella and
femur. Pedipalps and legs yellow, with spots. Fingers Kovařík et al.; North Central Province, Sigiriya,
7°58'25.6"N 80°44'59.7"E, 1♀, UPSL, leg. S. Jay-
arathne. Kovařík et al.: Scorpions of Sri Lanka
45 Kovařík et al.: Scorpions of Sri Lanka 45 Kovařík et al.: Scorpions of Sri Lanka
45 t
Figures 189. Male after 3rd ecdysis at l
Figures 187–190: Lychas srilankensis. Figures 187. Male af
ecdysis at locality 15CO. 15CJ. er 5th ecdysis at locality 15CJ. Figures 188. Female after 5th
ocality 15CN. Figures 190. Female after 5th ecdysis at locality t
Figures 189. Male after 3rd ecdysis at l
Figures 187–190: Lychas srilankensis. Figures 187. Male af
ecdysis at locality 15CO. 15CJ. er 5th ecdysis at locality 15CJ. Figures 188. Female after 5th
ocality 15CN. Figures 190. TYPE LOCALITY AND TYPE REPOSITORY. Ceylon, now Sri Female after 5th ecdysis at locality Euscorpius — 2016, No. 220 46 Figures 191–192: Lychas srilankensis, female from locality 1
after first e
5
cdysis (192). CN with newborns before first ecdysis (191), and with juveniles Figures 191–192: Lychas srilankensis, female from locality 1
after first e
5
cdysis (192). CN with newborns before first ecdysis (191), and with juveniles Kovařík et al.: Scorpions of Sri Lanka
47
Figures 193–200: Distal segments of leg IV, retroventral view of Sri Lankan Buthidae and Chaerilidae genera. Buthoscorpio
sarasinorum, male from locality 15CF (193), Charmus laneus, male from locality 15CO (194), Hottentotta tamulus, male from
locality 15CK (195), Isometrus maculatus, female from locality 15CI (196), Isometrus thwaitesi, female from locality 15CO
(197), Lychas srilankensis, male from locality 15CJ (198), Reddyanus basilicus, male from locality 15CS (199), and Chaerilus
ceylonensis, male from locality 15CD (200). Kovařík et al.: Scorpions of Sri Lanka Kovařík et al.: Scorpions of Sri Lanka 47 Figures 193–200: Distal segments of leg IV, retroventral view of Sri Lankan Buthidae and Chaerilidae genera. Buthoscorpio
sarasinorum, male from locality 15CF (193), Charmus laneus, male from locality 15CO (194), Hottentotta tamulus, male from
locality 15CK (195), Isometrus maculatus, female from locality 15CI (196), Isometrus thwaitesi, female from locality 15CO
(197), Lychas srilankensis, male from locality 15CJ (198), Reddyanus basilicus, male from locality 15CS (199), and Chaerilus
ceylonensis, male from locality 15CD (200). 122–123; Fet & Lowe, 2000: 158–169 (complete
reference list until 1998); Kovařík & Ojanguren,
2013: 194–211, 361–378, figs. 1410–1600 (refer-
ence and synonymy list until 2013). and manus of pedipalps of the same color, spotted. Mes-
osomal segments light-colored. First (basal) middle lam-
ella of both sexes pecten rounded. Posterior margin of
sternite V strongly convex medially. Subaculear tooth
spinoid. Pectinal teeth number 13–16. = Archisometrus Kraepelin, 1891: 75 (in part); type
species by subsequent designation (L. E. Koch,
1977: 123) Tityus marmoreus C. L. Koch, 1845
(syn. by Pocock, 1900: 35). HEMISPERMATOPHORE (Figs. 146–149). Trunk broad,
short, only slightly longer than capsule region. Capsule
region broad. Flagellum laminiform, short, basally
broadened and fused with median lobe. Median lobe
rather narrow, distally truncate, with strong, curved dor-
sal carina. Basal lobe well developed, a broad hook-like
process arising dorsally at base of median lobe. = Lychas (Distotrichus) Tikader & Bastawade, 1983:
41–51, figs. 99–129 (syn. by Vachon, 1986: 848). = Lychas (Alterotrichus) Tikader & Bastawade, 1983:
52–71, figs. 130–184) (syn. DISTRIBUTION. Sri Lanka. TYPE SPECIES. Lychas scutilus C. L. Koch, 1845. TYPE LOCALITY AND TYPE REPOSITORY. Ceylon, now Sri by Kovařík, 1995: 188). = Lychas (Endotrichus) Tikader & Bastawade, 1983:
71–107, figs. 185–285 (syn. by Kovařík, 1995:
188). = Lychas (Endotrichus) Tikader & Bastawade, 1983:
71–107, figs. 185–285 (syn. by Kovařík, 1995:
188). DISTRIBUTION. Sri Lanka. Lychas C. L. Koch, 1845
(Figs. 13, 152–192, 198, 251, 407–408, 550) DISTRIBUTION. Sri Lanka. Lychas C. L. Koch, 1845
(Figs. 13, 152–192, 198, 251, 407–408, 550) TYPE SPECIES. Lychas scutilus C. L. Koch, 1845. DIAGNOSIS. Total length 21.8–90 mm. Sternum type 1,
subpentagonal or subtriangular. Pedipalps orthoboth-
dorsomedian carina; chela with 3 Eb trichobothria on
riotaxic, type Aβ; patella trichobothrium d3 external to , 1845: 3 (in part), fig. 962, Tab. 9–285; Kovařík, 1997: 311–371, figs. 1–116,
Lychas C. L. Koch
CCCXCVIII; Tikader & Bastawade, 1983: 40–107,
figs. 9 , 1845: 3 (in part), fig. 962, Tab. 9–285; Kovařík, 1997: 311–371, figs. 1–116,
Lychas C. L. Koch
CCCXCVIII; Tikader & Bastawade, 1983: 40–107,
figs. 9 Euscorpius — 2016, No. 220
48
Figures 201–208: Distal segments of legs, retroventral view of Sri Lankan Reddyanus. Figures 201–204. R. ceylonensis sp. n.,
male holotype from locality 15CI, legs I–IV. Figures 205–208. Leg IV. R. besucheti, male from locality 15CD (205), R. jayarathnei sp. n., male paratype (206), R. loebli, male from locality 15CG (207), and R. ranawanai sp. n., male holotype (208). Euscorpius — 2016, No. 220 48 Figures 201–208: Distal segments of legs, retroventral view of Sri Lankan Reddyanus. Figures 201–204. R. ceylonensis sp. n.,
male holotype from locality 15CI, legs I–IV. Figures 205–208. Leg IV. R. besucheti, male from locality 15CD (205), R. jayarathnei sp. n., male paratype (206), R. loebli, male from locality 15CG (207), and R. ranawanai sp. n., male holotype (208). = Lychas ceylonensis Lourenço & Huber, 1999: 23–26,
figs. 1–7 (syn. by Kovařík & Ojanguren, 2013:
209). manus. Cheliceral fixed finger with a single ventral den-
ticle. Third and fourth legs with tibial spurs. Pectines
with conspicuous or inconspicuous fulcra, rarely without
fulcra. Pectinal teeth number 8–26. Movable fingers of
pedipalps with six rows of granules and external and
internal granules and apical row represented by 3 or 4
accessory granules. Total number of terminal granules 6
or 7. Carapace in lateral view with entire dorsal surface
horizontal, or nearly so. Telson with distinct subaculear
tooth. Fifth metasomal segment with carinae. manus. Cheliceral fixed finger with a single ventral den-
ticle. Third and fourth legs with tibial spurs. Pectines
with conspicuous or inconspicuous fulcra, rarely without
fulcra. Pectinal teeth number 8–26. Movable fingers of
pedipalps with six rows of granules and external and
internal granules and apical row represented by 3 or 4
accessory granules. Total number of terminal granules 6
or 7. Carapace in lateral view with entire dorsal surface
horizontal, or nearly so. Telson with distinct subaculear
tooth. Fifth metasomal segment with carinae. TYPE LOCALITY AND TYPE REPOSITORY. TYPE SPECIES. Lychas scutilus C. L. Koch, 1845. Sri Lanka, Nor-
thern Province, Mannar District, Occapu Kallu, Wil-
pattu; MHNG. MATERIAL EXAMINED. Sri Lanka, Northern Province,
Jaffna District, 09°42'51.6"N 080°04'44.8"E, 19 m a.s.l. (Locality 15CJ, Fig. 586), 26. –27.IV.2015, 1♂ after 5th
ecdysis (Figs. 152–153, 158, 160, 162–168, 174–180,
182, 198, 407) 1♀ (Figs. 154–155, 159, 161, 169–173,
181, 408) 1im.♂, FKCP, 1♂, UPSL, leg. Kovařík et al.;
North Central Province, Puttalam
District, Elu-
wankulam, 08°12'35.1"N 079°51'32"E, 52 m a.s.l. (Locality 15CN, Fig. 591), 28.IV.2015, 1♂ after 3rd
ecdysis (Figs. 183–186, 189, 550) 1♂ after 4th ecdysis
1♀ (Figs. 191–192), FKCP, leg. Kovařík et al.; North
Central Province, Puttalam District, Eluwankulam,
08°17'15"N 079°50'38.7"E, 38 m a.s.l. (Locality 15CO,
Fig. 592), 28.IV.2015, 2♂ (Figs. 156–157, 251) 1♀,
FKCP, 1♀1juv, UPSL, leg. Kovařík et al. DISTRIBUTION. Lychas is one of the most widely dis-
tributed genera of the family Buthidae, with species
present in Africa and Seychelles, and in the Oriental
region from India to Melanesia. Lychas srilankensis Lourenço, 1997
(Figs. 13, 152–192, 198, 251, 407–408, 550) Lychas srilankensis Lourenço, 1997: 831–836, figs. 1–9;
Lourenço & Huber, 1999: 26; Fet & Lowe, 2000:
168; Kovařík & Ojanguren, 2013: 209, 364, figs. 1462–1470. Lychas srilankensis Lourenço, 1997: 831–836, figs. 1–9;
Lourenço & Huber, 1999: 26; Fet & Lowe, 2000:
168; Kovařík & Ojanguren, 2013: 209, 364, figs. 1462–1470. Kovařík et al.: Scorpions of Sri Lanka
49 Kovařík et al.: Scorpions of Sri Lanka Kovařík et al.: Scorpions of Sri Lanka 49 Figures 209–224: Metasomal segments II–III, lateral view of Sri La
eddyanus basilicus, male (209) and female (210) from locality 1
male from loca
214. R. ceylonens
15CI
e (215) and f
217)
–220. R. ranawanai sp. n., male holotype (219) and female paratype
20). Figures 221–222. Isometrus maculatus, male from locality 15CP (221) and female from locality 15CI (222). Figures 223–
224. I. thwaitesi, male from locality 15CH (223) and female from
nkan Reddyanus and Isometrus species. Figures 209–210. R
fe
5CR. Figures 211–212. R. besucheti, male holotype (211) and
is sp. n., male holotype (213) and female paratype from locality
emale (216) paratypes. Figures 217–218. R. loebli, male
lity 15CG (212). Figures 213–
(214). Figures 215–216. R. jayarathnei sp. n., mal
and female (218) from locality 15CG. Figures 219
2
locality 15CO (224). Figures 209–224: Metasomal segments II–III, lateral view of Sri La
eddyanus basilicus, male (209) and female (210) from locality 1
male from loca
214. R. TYPE SPECIES. Lychas scutilus C. L. Koch, 1845. ceylonens
15CI
e (215) and f
(217)
–220. R. ranawanai sp. n., male holotype (219) and female paratype
20). Figures 221–222. Isometrus maculatus, male from locality 15CP (221) and female from locality 15CI (222). Figures 223–
224. I. thwaitesi, male from locality 15CH (223) and female from
nkan Reddyanus and Isometrus species. Figures 209–210. R
fe
5CR. Figures 211–212. R. besucheti, male holotype (211) and
is sp. n., male holotype (213) and female paratype from locality
emale (216) paratypes. Figures 217–218. R. loebli, male
lity 15CG (212). Figures 213–
(214). Figures 215–216. R. jayarathnei sp. n., mal
and female (218) from locality 15CG. Figures 219
(2
locality 15CO (224). Figures 209–224: Metasomal segments II–III, lateral view of Sri La
eddyanus basilicus, male (209) and female (210) from locality 1
male from loca
214. R. ceylonens
15CI
e (215) and f
(217)
–220. R. ranawanai sp. n., male holotype (219) and female paratype
20). Figures 221–222. Isometrus maculatus, male from locality 15CP (221) and female from locality 15CI (222). Figures 223–
224. I. thwaitesi, male from locality 15CH (223) and female from
nkan Reddyanus and Isometrus species. Figures 209–210. R
fe
5CR. Figures 211–212. R. besucheti, male holotype (211) and
is sp. n., male holotype (213) and female paratype from locality
emale (216) paratypes. Figures 217–218. R. loebli, male
lity 15CG (212). Figures 213–
(214). Figures 215–216. R. jayarathnei sp. n., mal
and female (218) from locality 15CG. Figures 219
(2
locality 15CO (224). Euscorpius — 2016, No. 220 Euscorpius — 2016, No. 220
50
Figures 225–240: Pedipalp, dorsal view of Sri Lankan Reddya
cus, male (225) and female (226) from locality 15CR. Figures 2
from locality 15CG (228). Figures 229–230. R. ceylonensis sp. n
(230). Figures 231–232. R. jayarathnei sp. n., male (231) and f
and female (234) from locality 15CG. Figures 235
nus a
etrus species. Figures 225–226. Reddyanus basili-
2
228. R. besucheti, male from locality 15CD (227) and female
–236. R. rana
igures 237–238. Isometrus maculatus, male from locality 15CP
rom locality 15CI (238). Figures 239–240. I. ity 5CO (240). nd Isom
7–
., male holotype (229) and female paratype from locality 15CI
emale (232) paratypes. Figures 233–234. R. loebli, male (233)
wanai sp. n., male holotype (235) and female paratype (236). (237) and female f
F
thwaitesi, male from locality 15CH (239) and female from local
1 Euscorpius — 2016, No. TYPE SPECIES. Lychas scutilus C. L. Koch, 1845. 220 50 Figures 225–240: Pedipalp, dorsal view of Sri Lankan Reddya
cus, male (225) and female (226) from locality 15CR. Figures 2
from locality 15CG (228). Figures 229–230. R. ceylonensis sp. n
(230). Figures 231–232. R. jayarathnei sp. n., male (231) and f
and female (234) from locality 15CG. Figures 235
nus a
etrus species. Figures 225–226. Reddyanus basili-
2
228. R. besucheti, male from locality 15CD (227) and female
–236. R. rana
igures 237–238. Isometrus maculatus, male from locality 15CP
rom locality 15CI (238). Figures 239–240. I. ity 5CO (240). nd Isom
7–
., male holotype (229) and female paratype from locality 15CI
emale (232) paratypes. Figures 233–234. R. loebli, male (233)
wanai sp. n., male holotype (235) and female paratype (236). (237) and female f
F
thwaitesi, male from locality 15CH (239) and female from local
1 tal compression. Meta-
ma app
exes, or
male with
of gran-
les o
with
DIAGNOSIS. Total length 38–65 mm. Sternum type 1,
subpentagonal, exhibiting horizon
so
roximately the same length in both s
slightly longer metasoma. Sixth row
u
n both movable and fixed fingers of pedipalps out external and internal granules. First through third
metasomal segments with 10 carinae, fourth with eight
carinae. Lateral inframedian carinae of second and third
metasomal segments may be incomplete. Fingers and
manus of pedipalps identically colored, light and spot- Kovařík et al.: Scorpions of Sri Lanka
5 Kovařík et al.: Scorpions of Sri Lanka
5
Figures 241–259: Pedipalp chela dorsal (241), movable finger (242–250), and fixed finger (251–259) of Sri Lankan Isometr
Lychas, and Reddyanus species. Figures 241–242, 252. Isometrus maculatus, female from locality 15CO (241) and male fro
ocality 15CP (242, 252). Figures 243–244, 253. I. thwaitesi, male from locality 15CH (243) and female from locality 15C
(244, 253). Figures 245, 254. Reddyanus basilicus, male from locality 15CS. Figures 246, 255. R. besucheti, male from local
ures 248, 257. R. jayarathnei sp. n., male paratype. Figur
49, 258. R. loebli, male from locality 15CG. Figures 250, 259. R. ranawanai sp. n., male holotype. Figure 251. Lych
srilankensis, male from locality 15CO. 15CD. Figures 247, 256. R. ceylonensis sp. n., male holotype. Fig
2 51 Figures 241–259: Pedipalp chela dorsal (241), movable finger (242–250), and fixed finger (251–259) of Sri Lankan Isometrus
Lychas, and Reddyanus species. Figures 241–242, 252. Isometrus maculatus, female from locality 15CO (241) and male from
locality 15CP (242, 252). Kovařík et al.: Scorpions of Sri Lanka COMMENTS. The taxonomic position of this species is
not clear. L. srilankensis is very similar to L. tricarinatus
(Simon, 1884) and there is a real possibility that further
study of L. tricarinatus (type locality India, Pondichéry)
could reveal that these two species are synonyms. Lourenço did not compare these two species and only
compared L. srilankensis with L. shoplandi (Oates,
1888) and L. feae (Thorell, 1889) (= L. shoplandi) from
Myanmar in the original description (Lourenço, 1997:
831–836, figs. 1–9). The description is superficial with
several errors (e.g. compare Fig. 179 with fig. 6 in
Lourenço 1997: 833, which depicts a spurious additional
trichobothrium between d4 and d5; and the shape and
densely hirsute setation of tarsomere II of leg IV in Fig. 165 with fig. 8 in Lourenço 1997: 833, which depicts
same setation for tarsomeres I and II). Kovařík et
Ojanguren (2013: 196) cited a difference in the length of
the metasoma in males, but our study of new Sri Lankan
specimens shows that there is little or no significant
difference. Both species L. srilankensis and L. tri-
carinatus have the metasoma approximately the same
length in both sexes, or the male has a slightly longer
metasoma. TYPE SPECIES. Isometrus acanthurus Pocock, 1899. TYPE SPECIES. Isometrus acanthurus Pocock, 1899. TYPE SPECIES. Isometrus acanthurus Pocock, 1899. etween et and est. Three to five pairs of lateral eyes. Tibial spurs absent on all legs. Pedipalp movable finger
with six rows of granules, several accessory granules
and external and internal accessory granules. Pedipalp
fixed finger with seven rows of granules and six external
and seven internal accessory granules. Cheliceral fixed
finger with a single ventral denticle. Third and fourth
legs with tibial spurs. Tibia and tarsomeres of legs I–III
with setae not arranged into bristle combs on dorsal
surfaces. Tarsomere II of leg IV with two sparse rows of
< 20 spiniform setae on ventral surface. Mesosoma
dorsally with one median carina. Telson with subaculear
tooth pointed or more often rounded. Males of most
species have longer metasomal segments and often also
a wider pedipalp chela manus than females. DIAGNOSIS. Medium sized buthids, adults 19–75 mm. Sternum type 1, triangular in shape. Pedipalps ortho-
bothriotaxic, type Aβ. Chelal trichobothrium db located
b SUBORDINATE TAXA. Currently, 27 species are formally
included under the genus (cf. Kovařík & Ojanguren
(2013) for information about species not treated here):
Reddyanus acanthurus (Pocock, 1899) comb. Kovařík et al.: Scorpions of Sri Lanka n., Reddy-
anus assamensis (Oates, 1888) comb. n., Reddyanus
basilicus (Karsch, 1879) comb. n., Reddyanus besucheti
(Vachon, 1982) comb. n., Reddyanus bilyi (Kovařík,
2003) comb. n., Reddyanus brachycentrus (Pocock,
1899) comb. n., Reddyanus ceylonensis Kovařík, Lowe,
Ranawana, Hoferek, Jayarathne, Plíšková et Šťáhlavský,
sp. n., Reddyanus corbeti (Tikader et Bastawade, 1983)
comb. n., Reddyanus deharvengi (Lourenço et Duhem,
2010) comb. n., Reddyanus feti (Kovařík, 2013) comb. n., Reddyanus heimi (Vachon, 1976) comb. n., Reddy-
anus jayarathnei Kovařík, sp. n., Reddyanus jendeki
(Kovařík, 2013) comb. n., Reddyanus khammamensis
(Kovařík, 2003) comb. n., Reddyanus krasenskyi
(Kovařík, 1998) comb. n., Reddyanus kurkai (Kovařík,
1997) comb. n., Reddyanus loebli (Vachon, 1982)
comb. n., Reddyanus melanodactylus (L. Koch, 1867)
comb. n., Reddyanus navaiae (Kovařík, 1998) comb. n.,
Reddyanus neradi (Kovařík, 2013) comb. n., Reddyanus
petrzelkai (Kovařík, 2003) comb. n., Reddyanus prob-
lematicus (Kovařík, 2003) comb. n., Reddyanus rana-
wanai Kovařík, sp. n., Reddyanus rigidulus (Pocock,
1897) comb. n., Reddyanus tibetanus (Lourenço et Zhu,
2008) comb. n., Reddyanus vittatus (Pocock, 1900)
comb. n. and Reddyanus zideki (Kovařík, 1994) comb. n.. In the original description, L. ceylonensis was
characterized as having the terminal accessory granules
on the movable finger of the pedipalp modified into
another (seventh) row of granules (see fig. 7 in Lourenço
& Huber, 1999: 24). The authors (p. 26) distinguished L. ceylonensis from L. srilankensis as follows: "... the
presence of 7 oblique rows of granules on the movable
finger of pedipalp, instead of 6 as in L. srilankensis. The
row of granules on the extremity of the finger is normal,
and not represented merely by 3 or 4 accessory granules
as it is in L. srilankensis". This is incorrect. The male
holotype of L. ceylonensis only has a right movable fin-
ger (the left is missing), and this finger only bears six
rows of granules and three external accessory granules
on its distal extremity (see Fig. 1465 in Kovařík &
Ojanguren, 2013: 364), which is the normal condition
for the genus Lychas (see genus diagnosis), including L. srilankensis. TYPE SPECIES. Lychas scutilus C. L. Koch, 1845. Figures 243–244, 253. I. thwaitesi, male from locality 15CH (243) and female from locality 15CO
(244, 253). Figures 245, 254. Reddyanus basilicus, male from locality 15CS. Figures 246, 255. R. besucheti, male from localit
ures 248, 257. R. jayarathnei sp. n., male paratype. Figure
49 258 R loebli male from locality 15CG Figures 250 259 R ranawanai sp n
male holotype Figure 251 Lycha
15CD. Figures 247, 256. R. ceylonensis sp. n., male holotype. Fig
2 Figures 241–259: Pedipalp chela dorsal (241), movable finger (242–250), and fixed finger (251–259) of Sri Lankan Isometrus,
Lychas, and Reddyanus species. Figures 241–242, 252. Isometrus maculatus, female from locality 15CO (241) and male from
locality 15CP (242, 252). Figures 243–244, 253. I. thwaitesi, male from locality 15CH (243) and female from locality 15CO
(244, 253). Figures 245, 254. Reddyanus basilicus, male from locality 15CS. Figures 246, 255. R. besucheti, male from locality
ures 248, 257. R. jayarathnei sp. n., male paratype. Figures
49, 258. R. loebli, male from locality 15CG. Figures 250, 259. R. ranawanai sp. n., male holotype. Figure 251. Lychas
srilankensis, male from locality 15CO. 15CD. Figures 247, 256. R. ceylonensis sp. n., male holotype. Fig
2 52 Euscorpius — 2016, No. 220
52
in dorsal (260) and ventral (261) views. Figures 260–263: Reddyanus basilicus from locality 15CS. Figures 260–261. Male
igures 262–263. Female in dorsal (262) and ventral (263) views. Scale bar: 10 mm. F
ted. Manus of pedipalps smooth without granules in
females and usually finely granulated in males. Pectinal
region broad. Flagellum short, laminiform, with broad
hyaline fin along internal margin of cylindrical core. Euscorpius — 2016, No. 220 Euscorpius — 2016, No. 220 in dorsal (260) and ventral (261) views. Figures 260–263: Reddyanus basilicus from locality 15CS. Figures 260–261. Male
igures 262–263. Female in dorsal (262) and ventral (263) views. Scale bar: 10 mm. F ted. Manus of pedipalps smooth without granules in
females and usually finely granulated in males. Pectinal
teeth number 21–25. cess arising dorsally at base of median lobe
carina. region broad. Flagellum short, laminiform, with broad
hyaline fin along internal margin of cylindrical core. Median lobe broad, with thin dorsal carina near internal
margin. Basal lobe strongly developed, a broad, blunt
hook-like pro ort, only slightly longer than capsule region. Capsule
HEMISPERMATOPHORE (Figs. 183–186). Trunk broad,
sh 53 DISTRIBUTION. Sri Lanka. Genus Reddyanus Vachon, 1972 stat. n. (Figs. 14, 199, 201–220, 225–236, 245–250, 254–402,
409–420, 551–554, 561–570, Tables 3–5) Isometrus (Reddyanus): Vachon, 1972: 176–177, figs. 14, 16, 18, 20; Vachon, 1982: 90–101, 108–110,
figs. 47–67, 88–91; Fet & Lowe, 2000: 150–154;
Kovařík, 2003: 5–14, figs. 1–8; Kovařík & Ojang-
uren, 2013: 184–193, 347–354, 357–360, figs. 1265–1367, 1384–1409. COMMENTS. Vachon (1972: 177) described the subgenus
Reddyanus and distinguished it from the nominotypic
subgenus Isometrus Ehrenberg, 1828 by the position of
chelal fixed finger trichobothria (trichobothrium db lo-
cated between dt and et in Isometrus, between et and est Isometrus (Raddyanus [sic]): Tikader & Bastawade,
1983: 254–311 (in part), figs. 727–770, 841–895. Isometrus (Raddyanus [sic]): Tikader & Bastawade,
1983: 254–311 (in part), figs. 727–770, 841–895. Euscorpius — 2016, No. 220 Euscorpius — 2016, No. 220 54 Euscorpius — 2016, No. 220
54
Figures 264–269: Reddyanus basilicus. Figures 264, 266. Male from locality 15CS, chelicerae, carapace and tergites I–III
(264), and sternopectinal region and sternite III (266). Figures 265, 267. Female from locality 15CS, chelicerae, carapace and
tergites I–III (265) and sternopectinal region and sternites III–IV (267). Figures 268–269. Male from locality 15CR, chelicera
dorsal (268) and ventral (269) aspects. p Figures 264–269: Reddyanus basilicus. Figures 264, 266. Male from locality 15CS, chelicerae, carapace and tergites I–III
(264), and sternopectinal region and sternite III (266). Figures 265, 267. Female from locality 15CS, chelicerae, carapace and
tergites I–III (265) and sternopectinal region and sternites III–IV (267). Figures 268–269. Male from locality 15CR, chelicera
dorsal (268) and ventral (269) aspects. characters for distinguishing these taxa: (i) ventral aspect
of tarsomere II of leg IV with two dense rows of > 30
longer, more filamentous setae in Isometrus (Figs. 196–
197), vs. two sparse rows of < 20 shorter, thicker, more
spiniform setae in Reddyanus (Figs. 199, 205–208); (ii)
hemispermatophore with a relatively short, broad trunk,
a short, broad, uncoiled flagellum, and a smaller, blunt
basal lobe in Isometrus (Figs. 146–149), vs. a narrow
trunk, a long, thin, coiled flagellum and a much
enlarged, pointed basal lobe in Reddyanus (Figs. 283–
286). The form of the hemispermatophore of Isometrus in Reddyanus). Vachon also cited the different positions
of the e2 trichobothria on the femur (see figs 15, 16 in
Vachon, 1972: 176). DISTRIBUTION. Sri Lanka. Subsequently, Vachon (1982) add-
ed another character for distinguishing the two sub-
genera: Isometrus has pedipalp fixed fingers with six
ws of granules and six external and internal accessory
ro
granules (Figs. 252–253 and figs. 64, 65 in Vachon,
1982: 100), and Reddyanus has pedipalp fixed fingers
with seven rows of granules and six external and seven
internal granules (Figs. 254–259 and figs. 66 and 67 in
Vachon, 1982: 100). Here we propose two additional Kovařík et al.: Scorpions of Sri Lanka 55 Kovařík et al.: Scorpions of Sri Lanka
55
Figures 270–275: Reddyanus basilicus from locality 15CS. Figures 270–272. Male, metasoma and telson, lateral (270),
ventral (271), and dorsal (272) views. Figures 273–275. Female, metasoma and telson, lateral (273), ventral (274), and dorsal
(275) views. Scale bar: 10 mm. Figures 270–275: Reddyanus basilicus from locality 15CS. Figures 270–272. Male, metasoma and telson, lateral (270),
ventral (271), and dorsal (272) views. Figures 273–275. Female, metasoma and telson, lateral (273), ventral (274), and dorsal
(275) views. Scale bar: 10 mm. Euscorpius — 2016, No. 220 56 Figures 276–282: Reddyanus basilicus from locality 15CS. external (277) views. Figures 278–282. Male. Pedipalp chela,
(280) and external (281) views. Pedipalp femur and trochante
Figures 279–282. d
r
Figures 276–277. Female. Pedipalp chela, dorsal (276) and
orsal (278) and external (279) views. Pedipalp patella, dorsal
dorsal view (282). The trichobothrial pattern is indicated in Figures 276–282: Reddyanus basilicus from locality 15CS. external (277) views. Figures 278–282. Male. Pedipalp chela,
(280) and external (281) views. Pedipalp femur and trochante
Figures 279–282. d
r
Figures 276–277. Female. Pedipalp chela, dorsal (276) and
orsal (278) and external (279) views. Pedipalp patella, dorsal
dorsal view (282). The trichobothrial pattern is indicated in TYPE MATERIAL EXAMINED. Ceylon (now Sri Lanka),
1♂ (holotype, Fig. 565, Table 3), leg. Nietner, ZMHB
No. 113. appears more similar to that of Lychas (Figs. 183–186),
than to Reddyanus. Differences in trichobothriotaxy,
pedipalp finger dentition, tarsal setation and hemisper-
matophore structure provide characters that are useful
for higher level classification of buthid scorpions
(Kovařík, 2009; Lamoral, 1979; Sissom, 1990; Stahnke,
1972; Stockwell, 198; Vachon, 1952). Taken together,
these differences support our taxonomic decision to ele-
vate Reddyanus to a genus distinct from Isometrus. OTHER MATERIAL EXAMINED. DISTRIBUTION. Sri Lanka. Sri Lanka, Uva Province,
Uva Hills, Badula District, Namunukula, 1970, 1♂A,
FKCP; Kandy, 1♂ after 4th ecdysis 2♂ after 5th ecdysis
2♀, FKCP; Eastern Province, Ampara District, Lahugala
Kitulana National Park, 06°52'46"N 081°43'21.8"E, 40
m a.s.l. (Locality 15CR, Fig. 596), 3.–4.V.2015, 1♂
(Figs. 209, 225, 268–269, 409, 552) 1♀ (Figs. 210, 226,
410) 1juv., FKCP, 1♂, UPSL, leg. Kovařík et al.; Eas-
tern Province, Ampara District, Ampara env., 07°20'
01.3"N 081°41'57.1"E, 56 m a.s.l. (Locality 15CS, Fig. 597), 4.V.2015, 1♂ (Figs. 199, 245, 254, 260–261, 264,
266, 270–272, 278–286, 290, 551) 2♀ (Figs. 262–263,
265, 267, 273–275, 276–277, 287–289), FKCP, leg. Kovařík et al. DISTRIBUTION. Oriental region from India, China (Tibet)
to Melanesia. Reddyanus basilicus (Karsch, 1879) comb. n. igs. 14, 19
25–226, 245, 254, 260–290,
, 565, Tables 3, 5)
(F
9, 209–210, 2
409–410, 551–552 I
I
fig
2000:
sometrus basilicus Karsch, 1879: 113. sometrus (Reddyanus) basilicus: Vachon, 1982: 91–92,
s. 40–46, 49?, 66, 88–89; Fet & Lowe,
151; Kovařík, 2003: 5 (in part); Kovařík & Ojan-
guren, 2013: 185, 350, 358, figs. 1296–1303, 1386–
1392 (reference list until 2013). Isometrus basilicus Karsch, 1879: 113. TYP
Lan
E LOCALITY AND TYPE REPOSITORY. Ceylon, now Sri
ka; ZMHB No. 113. Isometrus basilicus Karsch, 1879: 113. I
fig
2000:
sometrus (Reddyanus) basilicus: Vachon, 1982: 91–92,
s. 40–46, 49?, 66, 88–89; Fet & Lowe,
151; Kovařík, 2003: 5 (in part); Kovařík & Ojan-
guren, 2013: 185, 350, 358, figs. 1296–1303, 1386–
1392 (reference list until 2013). xes. Ratio of pedipalp chela length/ width 3.41–
n ma
DIAGNOSIS. Total length 27–46 mm. Male with longer
metasomal segments and telson than female. Segments
of pedipalps approximately the same length and width in
oth se
b
3.79 i
le. Pedipalp movable finger clearly longer
than chela manus in both sexes, but in largest male
specimens could be shorter than manus. Base coloration Kovařík et al.: Scorpions of Sri Lanka Kovařík et al.: Scorpions of Sri Lanka 57 Figures 283–286: Left hemispermatophore of Reddyanus basilicus from locality 15CR. External (283), dorsal (284), internal
(285) and ventral (286) views. Scale bar: 500 µm. Abbreviation : bl, basal lobe; f, flagellum; ml, median lobe; mlc, median lobe
carina; t, trunk. reddish. Pedipalps and legs with brown maculation,
cely larger than preceding tubercles. Subaculear tooth
s Figures 283–286: Left hemispermatophore of Reddyanus basilicus from locality 15CR. External (283), dorsal (284), internal
(285) and ventral (286) views. Scale bar: 500 µm. Abbreviation : bl, basal lobe; f, flagellum; ml, median lobe; mlc, median lobe
carina; t, trunk. s reddish. Pedipalps and legs with brown maculation,
identical on femur and patella. First metasomal segment
bears 10 carinae, second through fourth segments bear
eight carinae, fifth segment bears five carinae in female
and three to five in male. Terminal tubercle of each
dorsal carina on metasomal segments of both sexes scar- cely larger than preceding tubercles. Subaculear tooth
wide and rounded, dorsally with granules in three or four
rows; four or six symmetrical granules and one or two
granules on tip. Glabrous zone along posterior margin of
fifth sternite absent. Pectinal teeth number 13–18 in both
sexes. Euscorpius — 2016, No. 220 58 p cdysis (289), and male (290). Figures 287–290: Reddyanus basilicus from locality 15CS, female (287), female with newborns before first ecdysis (288),
female with juveniles after first e cdysis (289), and male (290). Figures 287–290: Reddyanus basilicus from locality 15CS, female (287), female with newborns before first ecdysis (288),
female with juveniles after first e cdysis (289), and male (290). Isometrus basilicus Karsch, 1879: 113. Figures 287–290: Reddyanus basilicus from locality 15CS, female (287), female with newborns before first ecdysis (288),
female with juveniles after first e 59 Kovařík et al.: Scorpions of Sri Lanka Figures 2
91–292. Ma
Figures 293–294. Female from locality 15CG in dorsal (293) and
91–294: Reddyanus besucheti. Figures 2
le from locality 15CD in dorsal (291) and ventral (292) views. ventral (294) views. Figures 2
91–292. Ma
Figures 293–294. Female from locality 15CG in dorsal (293) and
91–294: Reddyanus besucheti. Figures 2
le from locality 15CD in dorsal (291) and ventral (292) views. ventral (294) views. process arising dorsally, separated from base of median
lobe. HEM
ly n
regi
hyal
f cylindrical core,
istally coiled. Median lobe narrow, distally truncate,
w, angular, pointed hook-like
ISPERMATOPHORE (Figs. 283–286). Trunk moderate-
arrow, elongate, more than twice as long as capsule
on. Flagellum long, slender, laminiform, with broad
ine fin along internal margin o
d
with thin dorsal lamina near internal margin. Basal lobe
greatly enlarged, a narro HEM
ly n
regi
hyal
f cylindrical core,
istally coiled. Median lobe narrow, distally truncate,
w, angular, pointed hook-like
ISPERMATOPHORE (Figs. 283–286). Trunk moderate-
arrow, elongate, more than twice as long as capsule
on. Flagellum long, slender, laminiform, with broad
ine fin along internal margin o
d
with thin dorsal lamina near internal margin. Basal lobe
greatly enlarged, a narro VARIABILITY. There is high variability in morphometric
characters between small males 27–32 mm long after the
fourth ecdysis, and males 40–46 mm long after the fifth
ecdysis. TYPE LOCALITY AND TYPE REPOSITORY. Sri Lanka, Am-
bagaswewa; MHNG. OTHER MATERIAL EXAMINED. Sri Lanka, North Central
Province, Polonnaruwa District, ca 35 km from Dam-
bula, 07°57'15.1"N 080°54'45,4"E, 132 m a.s.l. (Locality
15CD, Fig. 578), 22.IV.2015, 2♂ (topotypes, Figs. 205,
227, 246, 255, 291–292, 295–297), FKCP, leg. Kovařík
trict, near Kaudulla National Park, 08°08'40.6"N 080°51'
et al.; North Central Province, Polonnaruwa Dis- Reddyanus besucheti (Vachon, 1982) comb. n.
(Figs. 14, 205, 211–212, 227–228, 246, 255, 291–302,
411–412, 562, 564, 566–567, Tables 3, 5) Isometrus (Reddyanus) besucheti Vachon, 1982: 93–97,
figs. 47, 51–57, 90–91; Fet & Lowe, 2000: 151;
Lourenço & Huber, 2002: 267; Kovařík, 2003: 5–6;
Kovařík & Ojanguren, 2013: 186 (in part). TYPE MATERIAL EXAMINED. Sri Lanka, Ambagaswewa,
Locality 44, 3.II.1970, leg. C. Besuchet et Y. Löbl, 1♂
(holotype, Figs. 211, 411, 566, Table 3), MHNG. DISTRIBUTION. Sri Lanka. DISTRIBUTION. Sri Lanka. 60 Euscorpius — 2016, No. 220 Figures 295–300: Reddyanus besucheti. Figures 295–297. Male from locality 15CD, metasoma and telson, lateral (295),
ventral (296), and dorsal (297) views. Figures 298–300. Female from locality 15CG, metasoma and telson, lateral (298), ventral
(299), and dorsal (300) views. Figures 295–300: Reddyanus besucheti. Figures 295–297. Male from locality 15CD, metasoma and telson, lateral (295),
ventral (296), and dorsal (297) views. Figures 298–300. Female from locality 15CG, metasoma and telson, lateral (298), ventral
(299), and dorsal (300) views. Kovařík et al.: Scorpions of Sri Lanka
61 Kovařík et al.: Scorpions of Sri Lanka 61 Kovařík et al.: Scorpions of Sri Lanka
61
Figures 301–302: Reddyanus besucheti, male at locality 15CF (301) and female at locality 15CG (302). Figures 301–302: Reddyanus besucheti, male at locality 15CF (301) and female at locality 15CG (302). Reddyanus besucheti (Vachon, 1982) comb. n. TYPE MATERIAL EXAMINED. Sri Lanka, Ambagaswewa,
Locality 44, 3.II.1970, leg. C. Besuchet et Y. Löbl, 1♂
(holotype, Figs. 211, 411, 566, Table 3), MHNG. TYPE MATERIAL EXAMINED. Sri Lanka, Ambagaswewa,
Locality 44, 3.II.1970, leg. C. Besuchet et Y. Löbl, 1♂
(holotype, Figs. 211, 411, 566, Table 3), MHNG. Reddyanus besucheti (Vachon, 1982) comb. n. TYPE MATERIAL EXAMINED. Sri Lanka, Ambagaswewa,
Locality 44, 3.II.1970, leg. C. Besuchet et Y. Löbl, 1♂
(holotype, Figs. 211, 411, 566, Table 3), MHNG. TYPE MATERIAL EXAMINED. Sri Lanka, Ambagaswewa,
Locality 44, 3.II.1970, leg. C. Besuchet et Y. Löbl, 1♂
(holotype, Figs. 211, 411, 566, Table 3), MHNG. y
(
)
(Figs. 14, 205, 211–212, 227–228, 246, 255, 291–302,
411–412, 562, 564, 566–567, Tables 3, 5) OTHER MATERIAL EXAMINED. Sri Lanka, North Central
Province, Polonnaruwa District, ca 35 km from Dam-
bula, 07°57'15.1"N 080°54'45,4"E, 132 m a.s.l. (Locality
15CD, Fig. 578), 22.IV.2015, 2♂ (topotypes, Figs. 205,
227, 246, 255, 291–292, 295–297), FKCP, leg. Kovařík
trict, near Kaudulla National Park, 08°08'40.6"N 080°51'
et al.; North Central Province, Polonnaruwa Dis- Isometrus (Reddyanus) besucheti Vachon, 1982: 93–97,
figs. 47, 51–57, 90–91; Fet & Lowe, 2000: 151;
Lourenço & Huber, 2002: 267; Kovařík, 2003: 5–6;
Kovařík & Ojanguren, 2013: 186 (in part). Euscorpius — 2016, No. 220 62 igures 303–306: Reddyanus ceylonensis sp. n. from locality 15CI. Figures 303–304. Male holotype in dorsal (303) and
entral (304) views. Figures 305–306. Female paratype in dorsal (
entral (306) views. F
v
305) and v igures 303–306: Reddyanus ceylonensis sp. n. from locality 15CI. Figures 303–304. Male holotype in dorsal (303) and
entral (304) views. Figures 305–306. Female paratype in dorsal (
entral (306) views. F
v
305) and v Kovařík et al.: Scorpions of Sri Lanka
63 Kovařík et al.: Scorpions of Sri Lanka 63 Kovařík et al.: Scorpions of Sri Lanka
63
Figures 307–312: Reddyanus ceylonensis sp. n. from locality 15CI. Figures 307, 309, 311–312. Male holotype, chelicerae,
carapace and tergites I–III (307), sternopectinal region and sternites III–IV (309), and chelicera in dorsal (311) and ventral (312)
views. Figures 308, 310. Female paratype, chelicerae, carapace and tergites I–III (308), and sternopectinal region and sternites
I–IV (310). II
04"E, 101 m a.s.l. (Locality 15CF, Fig. 581), 23.IV. a.s.l. (Locality 15CG, Fig. 582), 23. –24.IV.2015, 1♀ Figures 307–312: Reddyanus ceylonensis sp. n. from locality 15CI. Figures 307, 309, 311–312. 7), FKCP, leg. Kovařík et
l.; Central Province, Matale District, Habarana, Wanan-
ahana Resort, 07°59'25.8"N 080°43'24.6"E, 280 m
04"E, 101 m a.s.l. (Locality 15CF, Fig. 581), 23.IV.
2015, 1♂ (Fig. 562, 564, 56
a
iw 7), FKCP, leg. Kovařík et
l.; Central Province, Matale District, Habarana, Wanan-
ahana Resort, 07°59'25.8"N 080°43'24.6"E, 280 m
04"E, 101 m a.s.l. (Locality 15CF, Fig. 581), 23.IV.
2015, 1♂ (Fig. 562, 564, 56
a
iw (Figs. 212, 228, 293–294, 298–300, 302, 412), FKCP,
leg. Kovařík et al.; North Central Province, Anurad-
hapura District, Mihintale, 08°20'51.8"N 080°30'27.7"E,
a.s.l. (Locality 15CG, Fig. 582), 23. –24.IV.2015, 1♀ (Figs. 212, 228, 293–294, 298–300, 302, 412), FKCP,
leg. Kovařík et al.; North Central Province, Anurad-
hapura District, Mihintale, 08°20'51.8"N 080°30'27.7"E,
a.s.l. (Locality 15CG, Fig. 582), 23. –24.IV.2015, 1♀ Euscorpius — 2016, No. 220 156 m a.s.l. (Locality 15CL, Fig. 589), 27. –28.IV.2015,
1♀1juv.♂, FKCP, leg. Kovařík et al. DIAGNOSIS. Total length 27–39 mm. Male has slightly
longer metasomal segments and telson than female. Ped-
ipalp segments approximately the same length in both
sexes, pedipalp chela wider in male. Ratio of pedipalp
chela length/ width 2.97–3.17 in male (Fig. 213, Tab. 5). Pedipalp movable finger shorter than manus of chela in
male. Pedipalps and legs with brown maculation, iden-
tical on femur and patella. First metasomal segment with
10 carinae, second through fourth segments with eight
carinae, fifth segment with five carinae in female and
three to five in male. Terminal tubercle of each dorsal
carina on metasomal segments of both sexes scarcely
larger than preceding tubercles. Subaculear tooth wide
and rounded, dorsally with granules in three rows, four
symmetrical granules and one or two granules on tip. Ratio of metasomal segment II length/ width 1.85–1.97
in male. Glabrous zone along posterior margin of fifth
sternite absent. Pectinal teeth number 11–15 in both
sexes. DIAGNOSIS. Total length 27–39 mm. Male has slightly
longer metasomal segments and telson than female. Ped-
ipalp segments approximately the same length in both
sexes, pedipalp chela wider in male. Ratio of pedipalp
chela length/ width 2.97–3.17 in male (Fig. 213, Tab. 5). DIAGNOSIS. Total length 30–45 mm. Male with slightly
longer metasomal segments and telson than female. Seg-
ments of pedipalps approximately the same length in
both sexes, pedipalp chela wider in male. Pedipalps and
legs with brown maculation, identical on femur and
patella. First metasomal segment with 10 carinae, second
through fourth segments with eight carinae, fifth seg-
ment with five carinae in female and three to five in
male. Posterior terminal tubercle of each dorsal carina
on metasomal segments of both sexes scarcely larger
than preceding tubercles. Subaculear tooth wide and
rounded, dorsally with granules in three rows, four sym-
metrical granules and one or two granules on tip. Glabrous zone along posterior margin of fifth sternite
either absent or present. Pectinal teeth number 12–18 in
both sexes. DISTRIBUTION. Sri Lanka DISTRIBUTION. Sri Lanka DESCRIPTION. Total length 27–39 mm. The habitus is
shown in Figs. 313–306. For measurements and morpho-
metric ratios see Tables 3 and 5. For position and dis-
tribution of trichobothria of pedipalps see Figs. 320–326. The male has slightly longer metasomal segments and
telson (Figs. 331–318) and wider pedipalp chela than the
female (Figs. 319–320). DESCRIPTION. Total length 27–39 mm. The habitus is
shown in Figs. 313–306. For measurements and morpho-
metric ratios see Tables 3 and 5. For position and dis-
tribution of trichobothria of pedipalps see Figs. 320–326. The male has slightly longer metasomal segments and
telson (Figs. 331–318) and wider pedipalp chela than the
female (Figs. 319–320). Reddyanus ceylonensis Kovařík, Lowe, Ranawana,
Hoferek, Jayarathne, Plíšková et Šťáhlavský, sp. n. (Figs. 14, 201–204, 213–214, 229–230, 247, 256, 303–
333, 413–414, 553, 567, Tables 3, 5) http://www.zoobank.org/urn:lsid:zoobank.org:act:E
E798511-D2D8-4D5A-9094-BE5551EC1CCB Coloration (Figs. 303–306). Base color yellowish to
reddish, with brown to black spots. Chelicera strongly
reticulated, mainly anteriorly, with spotted fingers. Ventral surface of mesosoma and pedipalps yellowish
brown with a pair of black spots on seventh sternite. Carapace and pedipalps dorsally and laterally yellowish
to reddish, with brown to black spots, identical on
femur, patella and manus of pedipalps. Pedipalp fingers
reddish black. Legs with same color and pattern as ped-
ipalp femur and patella. Metasomal segments yellowish
to reddish with the spots. Older specimens have fourth
and fifth metasomal segments reddish brown to black,
both darker than the first to third segments. Telson
reddish brown with spots but in older specimens could
be black. TYPE LOCALITY AND TYPE REPOSITORY. Sri Lanka, Nor-
thern Province, Mannar District, Marichchukkaddi env,
border of Wilpattu National Park, 08°33'32.3"N 079°
56'51"E, 7 m a.s.l., Locality 15CI; UPSL. TYPE MATERIAL. Sri Lanka, Northern Province, Mannar
District, Madhu Road, 08°48'26.3"N 080°10'26"E, 90 m
a.s.l. (Locality 15CH, Fig. 584), 24. –25.IV.2015, 1♀
(paratype, Fig. 331), FKCP, leg. Kovařík et al.; Northern
Province, Mannar District, Marichchukkaddi env, border
of Wilpattu National Park, 08°33'32.3"N 079°56'51"E, 7
m a.s.l. (Locality 15CI, Fig. 585), 25. –26.IV.2015, 1♂
(holotype, Figs. 201–204, 213, 229, 247, 256, 303–304,
307, 309, 311–315, 320–326, 413, 567) 3♂ (paratypes,
igs. 327–330, 333, 553) 3♀ (paratypes, Figs. 214, 230,
type
et
a
w
ality
ara-
Prov
079
28.I
F
305–306, 308, 310, 316–319, 332, 414), 2juvs. (para-
s), FKCP, 1♂1♀ (paratypes), UPSL, leg. Reddyanus besucheti (Vachon, 1982) comb. n. Male holotype, chelicerae,
carapace and tergites I–III (307), sternopectinal region and sternites III–IV (309), and chelicera in dorsal (311) and ventral (312)
views Figures 308 310 Female paratype chelicerae carapace and tergites I III (308) and sternopectinal region and sternites Figures 307–312: Reddyanus ceylonensis sp. n. from locality 15CI. Figures 307, 309, 311–312. Male holotype, chelicerae,
carapace and tergites I–III (307), sternopectinal region and sternites III–IV (309), and chelicera in dorsal (311) and ventral (312)
views. Figures 308, 310. Female paratype, chelicerae, carapace and tergites I–III (308), and sternopectinal region and sternites
I–IV (310). II 64 DISTRIBUTION. Sri Lanka Kovařík
l.; North Central Province, Puttalam District, Elu-
ankulam, 08°12'35.1"N 079°51'32"E, 52 m a.s.l. (Lo-
15CN, Fig. 591), 28.IV.2015, 2♀1juv.♂ (p
c
types), FKCP, leg. Kovařík et al.; North Central
ince, Puttalam District, Eluwankulam, 08°17'15"N
°50'38.7"E, 38 m a.s.l. (Locality 15CO, Fig. 592),
V.2015, 4♀4juvs, UPSL, leg. Kovařík et al. TYPE MATERIAL. Sri Lanka, Northern Province, Mannar
District, Madhu Road, 08°48'26.3"N 080°10'26"E, 90 m
a.s.l. (Locality 15CH, Fig. 584), 24. –25.IV.2015, 1♀
(paratype, Fig. 331), FKCP, leg. Kovařík et al.; Northern
Province, Mannar District, Marichchukkaddi env, border
of Wilpattu National Park, 08°33'32.3"N 079°56'51"E, 7
m a.s.l. (Locality 15CI, Fig. 585), 25. –26.IV.2015, 1♂
(holotype, Figs. 201–204, 213, 229, 247, 256, 303–304,
307, 309, 311–315, 320–326, 413, 567) 3♂ (paratypes,
igs. 327–330, 333, 553) 3♀ (paratypes, Figs. 214, 230,
type
et
a
w
ality
ara-
Prov
079
28.I
F
305–306, 308, 310, 316–319, 332, 414), 2juvs. (para-
s), FKCP, 1♂1♀ (paratypes), UPSL, leg. Kovařík
l.; North Central Province, Puttalam District, Elu-
ankulam, 08°12'35.1"N 079°51'32"E, 52 m a.s.l. (Lo-
15CN, Fig. 591), 28.IV.2015, 2♀1juv.♂ (p
c
types), FKCP, leg. Kovařík et al.; North Central
ince, Puttalam District, Eluwankulam, 08°17'15"N
°50'38.7"E, 38 m a.s.l. (Locality 15CO, Fig. 592),
V.2015, 4♀4juvs, UPSL, leg. Kovařík et al. Mesosoma and carapace (Figs. 307–310). Carapace
without carinae but with large granules. Mesosoma with
e VII pentacarinate. venth sternite with four
num-
one granulated median carina. Tergit
Se
incomplete carinae, sparsely
granulate. The pectinal tooth count 11–15 in the females,
14–15 in the males. Pectines with three marginal lam-
llae, six or seven middle lamellae. Lamellae with
e
erous pale or reddish setae indicated or absent in the male. Ventral carina present on
telson. Intercarinal surfaces of metasoma granulated, in-
Metasoma and telson (Figs. 213–214, 313–318, 413–
414). First metasomal segment with 10 carinae, second
to the fourth segments with eight carinae, fifth segment
with five carinae well developed in the female, only or Sri Lanka. ETYMOLOGY. Named after country of occurrence. Cey-
lon is the older name f Kovařík et al.: Scorpions of Sri Lanka 65 p
Figures 313–318: Reddyanus ceylonensis sp. n. from locality 15CI. Figures 313–315. Male holotype, metasoma and telson,
lateral (313), ventral (314), and dorsal (315) views. Figures 316–318. Female paratype, metasoma and telson, lateral (316),
entral (317), and dorsal (318) views. v Figures 313–318: Reddyanus ceylonensis sp. n. from locality 15CI. Figures 313–315. DISTRIBUTION. Sri Lanka Telson
elongate, with subaculear tooth wide and rounded, dor-
sally with granules in three rows, four symmetrical
granules and one or two granules on the tip. Pedipalps (Figs. 229–230, 319–326). Femur and patella
only very sparsely hirsute, with complete carinae, gran-
ulated. Dorsal carinae indicated on the chela manus in
female, absent in male. Sixth row of granules on mov-
able finger with one external granule. Seventh row of
granules on fixed finger without additional granules. Legs (Figs. 201–204). Femur and patella with complete
carinae, granulated. Legs hirsute, without bristle combs. Hemispermatophore (Figs. 327–330). Trunk moderate-
ly narrow, elongate, more than twice as long as capsule
region. Flagellum long, slender, laminiform, with broad
hyaline fin along internal margin of cylindrical core,
distally coiled. Median lobe narrow, distally truncate,
with thin dorsal lamina near internal margin. Basal lobe
greatly enlarged, a narrow, angular, pointed hook-like
process arising dorsally, separated from base of median
lobe. AFFINITIES. The described features distinguish R. cey-
lonensis sp. n. from all other species of the genus, and
are recounted in the key. The morphologically closest
species is R. besucheti, from which R. ceylonensis sp. n. differs mainly by the longer and narrower metasoma in
males, and other measurements compared in Tab. 5. R. besucheti and R. ceylonensis sp. n. are the only two Sri
Lankan Reddyanus species with the movable finger
shorter than the manus of the pedipalp chela in the male,
and with sexual dimorphism in the width of the chela. Pedipalps (Figs. 229–230, 319–326). Femur and patella
only very sparsely hirsute, with complete carinae, gran-
ulated. Dorsal carinae indicated on the chela manus in
female, absent in male. Sixth row of granules on mov-
able finger with one external granule. Seventh row of
granules on fixed finger without additional granules. Reddyanus jayarathnei Kovařík, sp. n. (Figs. 14, 206, 215–216, 231–232, 248, 257, 334–357,
415–416, 569, Tables 4–5)
http://www.zoobank.org/urn:lsid:zoobank.org:act:F
E1515A9-8302-46D4-8298-11BFB4555882 Reddyanus jayarathnei Kovařík, sp. n. (Figs. 14, 206, 215–216, 231–232, 248, 257, 334–357,
415–416, 569, Tables 4–5) http://www.zoobank.org/urn:lsid:zoobank.org:act:F
E1515A9-8302-46D4-8298-11BFB4555882 http://www.zoobank.org/urn:lsid:zoobank.org:act:F
E1515A9-8302-46D4-8298-11BFB4555882 E; UPSL. TYPE LOCALITY AND TYPE REPOSITORY. Sri Lanka, Galle
district, Kanneliya Rain Forest, 06°15'04" N 80°20'18" TYPE MATERIAL. Sri Lanka, Galle District, Kanneliya
Rain Forest, 06°15'04" N 80°20'18" E (Fig. 357), 1♂
(holotype, Fig. 356) 1♀ (paratype, Fig. 355), UPSL, 1♂
(paratype, Figs. 206, 215, 231, 248, 257, 334–335, 338, lts after the third to fifth ecdysis. DISTRIBUTION. Sri Lanka Male holotype, metasoma and telson,
lateral (313), ventral (314), and dorsal (315) views. Figures 316–318. Female paratype, metasoma and telson, lateral (316),
entral (317), and dorsal (318) views. v Euscorpius — 2016, No. 220
66
Figures 319–326: Reddyanus ceylonensis sp. n. from locality 15CI. Figures 319. Female partatype, pedipalp chela, dorsal
view. Figures 320–326. Male holotype. Pedipalp chela, dorsal (320), external (321) and ventral (322) views. Pedipalp patella,
dorsal (323) and external (324) views. Pedipalp femur and trochanter internal (325) and dorsal (326) views. The trichobothrial
pattern is indicated in Figures 320–326. Euscorpius — 2016, No. 220
66 Euscorpius — 2016, No. 220 66 Figures 319–326: Reddyanus ceylonensis sp. n. from locality 15CI. Figures 319. Female partatype, pedipalp chela, dorsal
view. Figures 320–326. Male holotype. Pedipalp chela, dorsal (320), external (321) and ventral (322) views. Pedipalp patella,
dorsal (323) and external (324) views. Pedipalp femur and trochanter internal (325) and dorsal (326) views. The trichobothrial
pattern is indicated in Figures 320–326. nositic characters such as fully developed shape of chela
or telson. cluding dorsal surface mainly in the female. In both
sexes, posterior terminal tubercle of each dorsal carina
on metasomal segments is not enlarged but only very
slightly larger on second and third segments. Telson
elongate, with subaculear tooth wide and rounded, dor-
sally with granules in three rows, four symmetrical
granules and one or two granules on the tip. Pedipalps (Figs. 229–230, 319–326). Femur and patella
only very sparsely hirsute, with complete carinae, gran-
ulated. Dorsal carinae indicated on the chela manus in
female, absent in male. Sixth row of granules on mov-
able finger with one external granule. Seventh row of
granules on fixed finger without additional granules. Legs (Figs. 201–204). Femur and patella with complete
carinae, granulated. Legs hirsute, without bristle combs. Hemispermatophore (Figs. 327–330). Trunk moderate-
ly narrow, elongate, more than twice as long as capsule
region. Flagellum long, slender, laminiform, with broad
hyaline fin along internal margin of cylindrical core,
distally coiled. Median lobe narrow, distally truncate,
with thin dorsal lamina near internal margin. Basal lobe
greatly enlarged, a narrow, angular, pointed hook-like
process arising dorsally, separated from base of median
lobe. cluding dorsal surface mainly in the female. In both
sexes, posterior terminal tubercle of each dorsal carina
on metasomal segments is not enlarged but only very
slightly larger on second and third segments. DISTRIBUTION. Sri Lanka Small
ales after the third ecdysis may not exhibit some diag-
VARIABILITY. Males of Sri Lankan Reddyanus species
could be adu
m Kovařík et al.: Scorpions of Sri Lanka Kovařík et al.: Scorpions of Sri Lanka 67 o
af
Figures 327–330: Left hemispermatophore of Reddyanus ceyl
dorsal (328), internal (329) and ventral (330) views. Extracted
second male after 5th ecdysis was similar. Scale bar: 250 µm. A
median lobe carina; t, trunk. nensis sp. n. male paratype from locality 15CI. External (327),
ter 4th ecdysis. A second hemispermatophore examined from a
bbreviations: bl, basal lobe; f, flagellum; ml, median lobe; mlc, o
af
Figures 327–330: Left hemispermatophore of Reddyanus ceyl
dorsal (328), internal (329) and ventral (330) views. Extracted
second male after 5th ecdysis was similar. Scale bar: 250 µm. A
median lobe carina; t, trunk. nensis sp. n. male paratype from locality 15CI. External (327),
ter 4th ecdysis. A second hemispermatophore examined from a
bbreviations: bl, basal lobe; f, flagellum; ml, median lobe; mlc, Euscorpius — 2016, No. 220 68 Euscorpius — 2016, No. 220
68
Figures 331–333: Reddyanus ceylonensis sp. n., female from locality 15CH (331), female from locality 15CI with newborns
before first ecdysis (332), and male from locality 15CI (333). Figures 331–333: Reddyanus ceylonensis sp. n., female from locality 15CH (331), female from locality 15CI with newborns
before first ecdysis (332), and male from locality 15CI (333). 340, 343–351, 415, 569) 1♀ (paratype, Figs. 216, 232,
336–337, 339, 341–342, 352–354, 416), FKCP, V.–
XI.2015, leg. S. Jayarathne. 340, 343–351, 415, 569) 1♀ (paratype, Figs. 216, 232,
336–337, 339, 341–342, 352–354, 416), FKCP, V.–
XI.2015, leg. S. Jayarathne. imately the same length and width in both sexes. Pedipalp movable finger longer than manus of chela in
both sexes. Pedipalps and legs with brown maculation,
identical on femur and patella. First metasomal segment
bears 10 carinae, second through fourth segments bear
eight carinae, fifth segment bears five carinae in female
and three to five in male. Posterior terminal tubercle of
each dorsal carina on metasomal segments of both sexes
scarcely larger than preceding tubercles. Subaculear
tooth wide and rounded, dorsally with granules in four ETYMOLOGY. Named after V. A. Sanjeewa Jayarathne
who collected the types. ETYMOLOGY. Named after V. A. Sanjeewa Jayarathne
who collected the types. DIAGNOSIS. Total length 37.1 mm (female) – 45.5 mm
(male). Male with slightly longer metasomal segments
and telson than female. Pedipalp segments approx- Kovařík et al.: Scorpions of Sri Lanka Kovařík et al.: Scorpions of Sri Lanka Kovařík et al.: Scorpions of Sri Lanka 69 Reddyanus jayarathnei sp. n., paratypes. Figures 334–335. Kovařík et al.: Scorpions of Sri Lanka Male in dorsal (334) and ventral (335) views. Figures 334–337:
Figures 336–337. Female in dorsal (336) and ventral (337) views. Scale bar: 10 mm. Reddyanus jayarathnei sp. n., paratypes. Figures 334–335. Male in dorsal (334) and ventral (335) views. Figures 334–337:
Figures 336–337. Female in dorsal (336) and ventral (337) views. Scale bar: 10 mm. Euscorpius — 2016, No. 220 70 s 338–348: Reddyanus jayarathnei sp. n., paratypes. Fig
. Male, chelicerae, carapace and tergites
III (338), sternopectinal region (340). Pedipalp chela, dorsal (
a
Figure
ures 338, 340, 343–348
I–
343), external (344) and ventral (345) views. Pedipalp patella,
nter dorsal (348) view. The trichobothrial pattern is indicated in
pace and tergites I–III (339), sternopectinal region and sternite
dorsal (346) and external (347) views. Pedipalp femur and trocha
Figures 344–348. Figures 339, 341–342. Female, chelicerae, car
III (341), and pedipalp chela dorsal view (342). s 338–348: Reddyanus jayarathnei sp. n., paratypes. Fig
. Male, chelicerae, carapace and tergites
III (338), sternopectinal region (340). Pedipalp chela, dorsal (
a
Figure
ures 338, 340, 343–348
I–
343), external (344) and ventral (345) views. Pedipalp patella,
nter dorsal (348) view. The trichobothrial pattern is indicated in
pace and tergites I–III (339), sternopectinal region and sternite
dorsal (346) and external (347) views. Pedipalp femur and trocha
Figures 344–348. Figures 339, 341–342. Female, chelicerae, car
III (341), and pedipalp chela dorsal view (342). s 338–348: Reddyanus jayarathnei sp. n., paratypes. Fig
. Male, chelicerae, carapace and tergites
III (338), sternopectinal region (340). Pedipalp chela, dorsal (
a
Figure
ures 338, 340, 343–348
I–
343), external (344) and ventral (345) views. Pedipalp patella,
nter dorsal (348) view. The trichobothrial pattern is indicated in
pace and tergites I–III (339), sternopectinal region and sternite
dorsal (346) and external (347) views. Pedipalp femur and trocha
Figures 344–348. Figures 339, 341–342. Female, chelicerae, car
III (341), and pedipalp chela dorsal view (342). Kovařík et al.: Scorpions of Sri Lanka
71
Figures 349–354: Reddyanus jayarathnei sp. n., paratypes. Figures 349–351. Male, metasoma and telson, lateral (349),
ventral (350), and dorsal (351) views. Figures 352–354. Female, metasoma and telson, lateral (352), ventral (353), and dorsal
(354) views. Scale bar: 10 mm. ws; six symmetrical granules in three rows and one or
, 14 in male. DESCRIPTION. Total length 37.1 mm (female paratype) –
see Figs. 344–348. The male has slightly longer meta-
ro
two granules on tip. DESCRIPTION. Total length 37.1 mm (female paratype) –
see Figs. 344–348. The male has slightly longer meta-
45.5 mm (male holotype). The habitus is shown in Figs.
334–337. For measurements and ratios see Tables 4–5.
For position and distribution of trichobothria of pedipalps DESCRIPTION. Total length 37.1 mm (female paratype) –
see Figs. 344–348. The male has slightly longer meta-
45.5 mm (male holotype). The habitus is shown in Figs.
334–337. For measurements and ratios see Tables 4–5.
For position and distribution of trichobothria of pedipalps Kovařík et al.: Scorpions of Sri Lanka Ratio of metasomal segment II
length/ width 1.81 in male. Glabrous zone on posterior
part of fifth sternite present medially in male. Pectinal
eeth number 12 in female
45.5 mm (male holotype). The habitus is shown in Figs. 334–337. For measurements and ratios see Tables 4–5. For position and distribution of trichobothria of pedipalps
t Kovařík et al.: Scorpions of Sri Lanka 71 Figures 349–354: Reddyanus jayarathnei sp. n., paratypes. Figures 349–351. Male, metasoma and telson, lateral (349),
ventral (350), and dorsal (351) views. Figures 352–354. Female, metasoma and telson, lateral (352), ventral (353), and dorsal
(354) views. Scale bar: 10 mm. ws; six symmetrical granules in three rows and one or
, 14 in male. ro
two granules on tip. Ratio of metasomal segment II
length/ width 1.81 in male. Glabrous zone on posterior
part of fifth sternite present medially in male. Pectinal
eeth number 12 in female
t Euscorpius — 2016, No. 220
72
at
Galle district, Kanneliya Rain Forest, 06°15'04" N 80°20'18" E. Figures 355–357: Reddyanus jayarathnei sp. n., female par ype (355) and male holotype (356) at the type locality (357), Euscorpius — 2016, No. 220 Euscorpius — 2016, No. 220 72 at
Galle district Kanneliya Rain Forest 06°15'04" N 80°20'18" E
Figures 355–357: Reddyanus jayarathnei sp. n., female par ype (355) and male holotype (356) at the type locality (357), at
Galle district, Kanneliya Rain Forest, 06°15'04" N 80°20'18" E. Figures 355–357: Reddyanus jayarathnei sp. n., female par ype (355) and male holotype (356) at the type locality (357), 73 Kovařík et al.: Scorpions of Sri Lanka Kovařík et al.: Scorpions of Sri Lanka
73
Figures 358–361: Reddyanus loebli from locality 15CG. Figures 358–359. Male in dorsal (358) and ventral (359) views. Figures 360–361. Female in dorsal (360) and ventral (361) views. somal segments and telson than the female (Figs. 349–
354). chela fingers reddish black. Legs with same color and
pattern as pedipalp femur and patella. Metasomal seg- Figures 358–361: Reddyanus loebli from locality 15CG. Figures 358–359. Male in dorsal (358) and ventral (359) views. Figures 360–361. Female in dorsal (360) and ventral (361) views. somal segments and telson than the female (Figs. 349–
354). somal segments and telson than the female (Figs. 349–
354). oloration (Figs. 334–337). Entire mesosoma and cara-
edipalps dorsally and laterally reddish, with brown to
lack spots, identical on femur and patella. Kovařík et al.: Scorpions of Sri Lanka Pedipalp
C
pace dark, almost black with yellow to reddish spots. helicera strongly reticulated, with spotted fingers. C
P
b chela fingers reddish black. Legs with same color and
pattern as pedipalp femur and patella. Metasomal seg-
ments reddish brown with the spots. Telson reddish
brown with spots. carapace (Figs. 338–341). Carapace
without carinae but with large granules. Mesosoma with
ne granulated median carina. Tergite VII pentacarinate. Mesosoma and
o Euscorpius — 2016, No. 220 Euscorpius — 2016, No. 220 74 Euscorpius
2016, No. 220
74 Figures 362–365: Reddyanus loebli from locality 15CG. Figures 362, 364. Male, chelicerae, carapace and tergites I–III (362),
ernopectinal region and sternite III (364). Figures 363, 365. Female, chelicerae, carapace and tergites I–III (363), and
ernopectinal region (365). st
st Figures 362–365: Reddyanus loebli from locality 15CG. Figures 362, 364. Male, chelicerae, carapace and tergites I–III (362),
ernopectinal region and sternite III (364). Figures 363, 365. Female, chelicerae, carapace and tergites I–III (363), and
ti
l
i
(365)
st
t Figures 362–365: Reddyanus loebli from locality 15CG. Figures 362, 364. Male, chelicerae, carapace and tergites I–III (362),
ernopectinal region and sternite III (364). Figures 363, 365. Female, chelicerae, carapace and tergites I–III (363), and
ernopectinal region (365). st
st Kovařík et al.: Scorpions of Sri Lanka Kovařík et al.: Scorpions of Sri Lanka 75 Kovařík et al.: Scorpions of Sri Lanka
75
Figures 366–371: Reddyanus loebli from
367), and dorsal (368) views. Figures 369
locality 15CG. Figu
–371. Female, metas
iews. res 366–368. Male, metasoma and telson, lateral (366), ventral
ma and telson, lateral (369), ventral (370), and dorsal (371)
(
v
o Figures 366–371: Reddyanus loebli from
367), and dorsal (368) views. Figures 369
locality 15CG. Figu
–371. Female, metas
iews. res 366–368. Male, metasoma and telson, lateral (366), ventral
ma and telson, lateral (369), ventral (370), and dorsal (371)
(
v
o Euscorpius — 2016, No. 220
76
Figures 372–375: Distal trunk, capsule region and flagellum of left hemispermatophore of Reddyanus loebli from locality
15CH. External (372), dorsal (373), internal (374) and ventral (375) views. Extracted after 5th ecdysis. Note: this hemi-
spermatopore was folded along a dorsal line and flattened; dorsal and ventral views show it as unfolded and flat, hence the broad
appearance of the trunk. Compare with Figs. 283–286 and 327–330 which show typical Reddyanus hemispermatophores with
trunk in natural curved conformation. Scale bar: 500 µm. Kovařík et al.: Scorpions of Sri Lanka Abbreviations: bl, basal lobe; f, flagellum; ml, median lobe; mlc,
median lobe carina; t, trunk. 76 Euscorpius — 2016, No. 220 Figures 372–375: Distal trunk, capsule region and flagellum of left hemispermatophore of Reddyanus loebli from locality
15CH. External (372), dorsal (373), internal (374) and ventral (375) views. Extracted after 5th ecdysis. Note: this hemi-
spermatopore was folded along a dorsal line and flattened; dorsal and ventral views show it as unfolded and flat, hence the broad
appearance of the trunk. Compare with Figs. 283–286 and 327–330 which show typical Reddyanus hemispermatophores with
trunk in natural curved conformation. Scale bar: 500 µm. Abbreviations: bl, basal lobe; f, flagellum; ml, median lobe; mlc,
median lobe carina; t, trunk. rows; six symmetrical granules in three rows and one or
two granules on the tip. Fifth sternite with glabrous zone on posterior medial part
in the male. Seventh sternite with four incomplete car-
inae, sparsely granulate. Pectinal tooth count is 12 in the
female, 14 in the male. Pectines with three marginal
lamellae and seven middle lamellae. The lamellae bear
numerous pale setae. two granules on the tip. Pedipalps (Figs. 231–232, 342–348). Femur and patella
only very sparsely hirsute, with complete carinae, gran-
ulated. Dorsal carinae indicated on chela manus in both
sexes. Sixth row of granules on movable finger with one
external granule. The seventh row of granules on the
fixed finger without additional granules. Legs (Fig. 206). Femur and patella bear complete cari-
nae and are granulated. Legs hirsute without bristle
combs. Pedipalps (Figs. 231–232, 342–348). Femur and patella
only very sparsely hirsute, with complete carinae, gran-
ulated. Dorsal carinae indicated on chela manus in both
sexes. Sixth row of granules on movable finger with one
external granule. The seventh row of granules on the
fixed finger without additional granules. Metasoma and telson (Figs. 215–216, 349–354, 415–
416). The first metasomal segment bears 10 carinae and
the second to the fourth segments bear eight carinae, the
fifth segment bears five carinae well developed in the
female and only indicated or absent in the male. Lateral
inframedian carinae may be indicated on second meta-
somal segment. Ventral carina present on telson. Inter-
carinal surfaces of metasoma granulated including dorsal
surface mainly in the female. In both sexes, posterior
terminal tubercle of each dorsal carina on metasomal
gments not enlarged. Isometrus (Reddyanus) acanthurus loebli Vachon, 1982:
98–99, figs 48, 58–63, 92–93; Fet & Lowe, 2000:
151. Isometrus (Reddyanus) loebli: Lourenço & Huber, 2002:
267; Kovařík, 2003: 10; Kovařík & Ojanguren, Kovařík et al.: Scorpions of Sri Lanka Telson elongate, with subaculear
tooth wide and rounded, dorsally with granules in four
se Legs (Fig. 206). Femur and patella bear complete cari-
nae and are granulated. Legs hirsute without bristle
combs. AFFINITIES. The described features distinguish R. jayar-
athnei sp. n. from all other species of the genus. R. jayarathnei sp. n. is well characterized by the glabrous
zone on the posterior medial part of the fifth sternite in
the male, which is not as extensive in R. ranawanai sp. n. (Fig. 570 versus Fig. 569) and is absent in other Sri
Lankan Reddyanus species (Figs. 567–568). Kovařík et al.: Scorpions of Sri Lanka Kovařík et al.: Scorpions of Sri Lanka 77 Figures 376–377: Reddyanus loebli from locality 15CH, male (
376) and female with newborns (377). Figures 376–377: Reddyanus loebli from locality 15CH, male (
376) and female with newborns (377). 78 Euscorpius — 2016, No. 220 Figures 378–381: Reddyanus ranawanai sp. n. Figures 378–379. Male holotype in dorsal (378) and ventral (379) views. Figures 380–381. Female paratype in dorsal (380) and ventral (381) views. Figures 378–381: Reddyanus ranawanai sp. n. Figures 378–379. Male holotype in dorsal (378) and ventral (379) views. Figures 380–381. Female paratype in dorsal (380) and ventral (381) views. Figures 378–381: Reddyanus ranawanai sp. n. Figures 378–379. Male holotype in dorsal (378) and
Figures 380–381. Female paratype in dorsal (380) and ventral (381) views. Reddyanus loebli (Vachon, 1982) comb. n. (Figs. 14, 207, 217–218, 233–234, 249, 258, 358–377,
417–418, 554, 561, 563, Table 5) = Isometrus (Reddyanus) garyi Lourenço & Huber,
2002: 270–273, figs. 13–18; Kovařík & Ojanguren,
2013: 187–188, 347, 350, figs. 1266, 1304–1307
(reference list until 2013). Syn. n. = Isometrus (Reddyanus) garyi Lourenço & Huber,
2002: 270–273, figs. 13–18; Kovařík & Ojanguren,
2013: 187–188, 347, 350, figs. 1266, 1304–1307
(reference list until 2013). Syn. n. Isometrus (Reddyanus) acanthurus loebli Vachon, 1982:
98–99, figs 48, 58–63, 92–93; Fet & Lowe, 2000:
151. TYPE LOCALITY AND TYPE REPOSITORY. Sri Lanka,
Kandy; MNHN RS 1164. TYPE LOCALITY AND TYPE REPOSITORY. Sri Lanka,
Kandy; MNHN RS 1164. Isometrus (Reddyanus) loebli: Lourenço & Huber, 2002:
267; Kovařík, 2003: 10; Kovařík & Ojanguren, Kovařík et al.: Scorpions of Sri Lanka 79 Figures 382–386: Reddyanus ranawanai sp. n. Figures 382, 384–385. Male holotype, chelicerae, carapace and tergites I–III
(382), sternites IV–VII (384), and sternopectinal region (385). Figures 383, 386. Kovařík et al.: Scorpions of Sri Lanka Female paratype, chelicerae, carapace and
tergites I–III (383), and sternopectinal region with sternite III (386). Figures 382–386: Reddyanus ranawanai sp. n. Figures 382, 384–385. Male holotype, chelicerae, carapace and tergites I–III
(382), sternites IV–VII (384), and sternopectinal region (385). Figures 383, 386. Female paratype, chelicerae, carapace and
tergites I–III (383), and sternopectinal region with sternite III (386). TYPE MATERIAL EXAMINED. Sri Lanka, Mannar District,
beside an irrigation canal, Madhu Road, 50 ft., 23.III. 1970, leg. Davis et Rowe, 1♀juv., holotype of Isometrus
(Reddyanus) garyi (figs. 1266, 1304–1307 in Kovařík &
Ojanguren, 2013: 187–188, 347, 350), MHNG. 080°10'26"E, 90 m a.s.l. (Locality 15CH, Fig. 584), 24. –25.IV.2015, 2♂ (Figs. 372–375, 376, 554) 1♀ (Fig. 377), FKCP, leg. Kovařík et al.; Northern Province,
Mannar District, Marichchukkaddi env, border of Wil-
pattu National Park, 08°33'32.3"N 079°56'51"E, 7 m
a.s.l. (Locality 15CI, Fig. 585), 25. –26.IV.2015, 1♂
1♀1juv., FKCP, leg. Kovařík et al.; North Central Pro-
vince, Anuradhapura District, Mihintale, 08°20'51.8"N
080°30'27.7"E, 156 m a.s.l. (Locality 15CL, Fig. 589),
27. –28.IV.2015, 1♂1♀1juv.♀, FKCP, 2♀, UPSL, leg. Kovařík et al.; Central Province, Matale District, Pal-
Kovařík et al.; North Central Province, Puttalam Dis-
legama, 07°32'49.5"N 080°47'50"E, 434 m a.s.l. (Lo-
cality 15CM, Fig. 590), 28.IV.2015, 1♀, FKCP, leg. 080°10'26"E, 90 m a.s.l. (Locality 15CH, Fig. 584), 24. –25.IV.2015, 2♂ (Figs. 372–375, 376, 554) 1♀ (Fig. 377), FKCP, leg. Kovařík et al.; Northern Province,
Mannar District, Marichchukkaddi env, border of Wil-
pattu National Park, 08°33'32.3"N 079°56'51"E, 7 m
a.s.l. (Locality 15CI, Fig. 585), 25. –26.IV.2015, 1♂
1♀1juv., FKCP, leg. Kovařík et al.; North Central Pro-
vince, Anuradhapura District, Mihintale, 08°20'51.8"N
080°30'27.7"E, 156 m a.s.l. (Locality 15CL, Fig. 589),
27. –28.IV.2015, 1♂1♀1juv.♀, FKCP, 2♀, UPSL, leg. Kovařík et al.; Central Province, Matale District, Pal-
Kovařík et al.; North Central Province, Puttalam Dis-
legama, 07°32'49.5"N 080°47'50"E, 434 m a.s.l. (Lo-
cality 15CM, Fig. 590), 28.IV.2015, 1♀, FKCP, leg. OTHER MATERIAL EXAMINED. Sri Lanka, Central Pro-
vince, Matale District, Habarana, Wananiwahana Resort,
07°59'25.8"N 080°43'24.6"E, 280 m a.s.l. (Locality
15CG, Figs. 582–583), 23. –24.IV.2015, 1♂ (Figs. 207,
17, 233, 249, 258, 358–359, 362, 364, 366–368, 417,
t, Madhu Road, 08°48'26.3"N
2
561, 563) 1♀ (Figs. 218, 234, 360–361, 363, 365, 369–
371, 418) 1juv, FKCP, leg. Kovařík et al.; Northern
Province, Mannar Distric Euscorpius — 2016, No. 220 80 Figures 387–392: Reddyanus ranawanai sp. n. Figures 387–389. Male holotype, metasoma and telson, lateral (387), ventral
(388), and dorsal (389) views. trict, Eluwankulam, 08°12'35.1"N 079°51'32"E, 52 m
a.s.l. (Locality 15CN, Fig. 591), 28.IV.2015, 1juv.,
FKCP, leg. Kovařík et al.; Uva Province, Monaragala
istrict, Monaragala, 06°52'30.7"N 081°21'17"E, 288 m
D
a.s.l. (Locality 15CQ, Fig. 595), 2.–3.V.2015, 1♂1♀,
FKCP, 1♂4♀, UPSL, leg. Kovařík et al.; Eastern Pro- vince, Ampara District, Lahugala Kitulana National
Park, 06°52'46"N 081°43'21.8"E, 40 m a.s.l. (Locality
15CR, Fig. 596), 3.–4.V.2015, 1♂1♀1juv., FKCP,
1♂4♀2juvs, UPSL, leg. Kovařík et al.; Eastern Pro-
vince, Ampara District, Ampara env., 07°20'01.3"N
081°41'57.1"E, 56 m a.s.l. (Locality 15CS, Fig. 597), Kovařík et al.: Scorpions of Sri Lanka Figures 390–392. Female paratype, metasoma and telson, lateral (390), ventral (391), and dorsal
(392) views. Scale bar: 10 mm. trict, Eluwankulam, 08°12'35.1"N 079°51'32"E, 52 m
vince, Ampara District, Lahugala Kitulana National Figures 387–392: Reddyanus ranawanai sp. n. Figures 387–389. Male holotype, metasoma and telson, lateral (387), ventral
(388), and dorsal (389) views. Figures 390–392. Female paratype, metasoma and telson, lateral (390), ventral (391), and dorsal
(392) views. Scale bar: 10 mm. vince, Ampara District, Lahugala Kitulana National
Park, 06°52'46"N 081°43'21.8"E, 40 m a.s.l. (Locality
15CR, Fig. 596), 3.–4.V.2015, 1♂1♀1juv., FKCP,
1♂4♀2juvs, UPSL, leg. Kovařík et al.; Eastern Pro-
vince, Ampara District, Ampara env., 07°20'01.3"N
081°41'57.1"E, 56 m a.s.l. (Locality 15CS, Fig. 597), Kovařík et al.: Scorpions of Sri Lanka
81
Figures 393–400: Reddyanus ranawanai sp. n. Figure 393. Female paratype, pedipalp chela dorsal. Figures 394–400. Male
holotype. Pedipalp chela, dorsal (394), external (395) and ventral (396) views. Pedipalp patella, dorsal (397) and external (398)
views. Pedipalp femur internal (399) and trochanter and femur dorsal (400) views. The trichobothrial pattern is indicated in
Figures 395–400. Kovařík et al.: Scorpions of Sri Lanka
81
Figures 393–400: Reddyanus ranawanai sp. n. Figure 393. Female paratype, pedipalp chela dorsal. Figures 394–400. Male
holotype. Pedipalp chela, dorsal (394), external (395) and ventral (396) views. Pedipalp patella, dorsal (397) and external (398)
views. Pedipalp femur internal (399) and trochanter and femur dorsal (400) views. The trichobothrial pattern is indicated in
Figures 395–400. Kovařík et al.: Scorpions of Sri Lanka 81 Figures 393–400: Reddyanus ranawanai sp. n. Figure 393. Female paratype, pedipalp chela dorsal. Figures 394–400. Male
holotype. Pedipalp chela, dorsal (394), external (395) and ventral (396) views. Pedipalp patella, dorsal (397) and external (398)
views. Pedipalp femur internal (399) and trochanter and femur dorsal (400) views. The trichobothrial pattern is indicated in
Figures 395–400. .V.2015, 1♂1♀, FKCP, 2♀1juv., UPSL, leg. Kovařík
4
et al.; Central Province, Kandy District, Tree Centre,
Wildlife Trust Sri Lanka “Rantambe”, 07°12'22.1"N
080°57'20.7"E, 171 m a.s.l. (Locality 15CT, Fig. 598),
5.V.2015, 2♀, FKCP, leg. Kovařík et al. with broad hyaline fin along internal margin of cy-
sally, separated from base of median lobe. Translucent
structures with a light violet tint. lindrical core, distally coiled. Median lobe broad,
distally truncate, with thin dorsal lamina near internal
margin terminating in distal cusp. Basal lobe enlarged, a
narrow, angular, pointed hook-like process arising dor- IAGNOSIS. Total length 26–45 mm. Euscorpius — 2016, No. 220 84 1♂3♀ (paratypes, Figs. 220, 236, 380–381, 383, 386,
390–393, 401, 420, 570), FKCP, leg. V. Fura. 1♂3♀ (paratypes, Figs. 220, 236, 380–381, 383, 386,
390–393, 401, 420, 570), FKCP, leg. V. Fura. COMMENTS. Isometrus (Reddyanus) garyi Lourenço et
Huber, 2002 was based on two juveniles (the holotype is
28 mm long, the second juvenile is smaller), which the
authors compared only to I.(R.) besucheti. They ignored
R. loebli, the only species of Sri Lankan Reddyanus with
a pointed (spinoid) subaculear tooth (Figs. 417–418) in
juveniles. Although R. loebli is characterized by a mark-
edly enlarged terminal tubercle of the dorsal carina on
the second metasomal segment of the male (Fig. 217),
this character is missing in females (Fig. 218) and juv-
eniles. Thus, we consider Isometrus (Reddyanus) garyi
to be a junior synonym of Reddyanus loebli. ETYMOLOGY. Named after Prof. Kithsiri B. Ranawana
who organized our Sri Lankan expedition. DIAGNOSIS. Total length 35–43 mm. Male with very
slightly longer metasomal segments than female. Ped-
ipalp segments and telson approximately the same length
and width in both sexes. Pedipalp movable finger longer
than manus of chela in both sexes. Pedipalps and legs
with brown maculation, identical on femur and patella. First metasomal segment with 10 carinae, second
through fourth segments with eight carinae, fifth seg-
ment with five carinae in female and three to five in
male. Posterior terminal tubercle of each dorsal carina
on metasomal segments of both sexes scarcely larger
than preceding tubercles. Subaculear tooth wide and
rounded, dorsally with granules in four rows; six sym-
metrical granules in three rows and one or two granules
on tip. Ratio metasomal segment II length/ width 1.78–
1.80 in male. Glabrous zone stretching over almost
whole posterior margin of fifth sternite. Pectinal teeth
number 12–14 in both sexes. DISTRIBUTION. Sri Lanka. Figures 403–429: Telson, lateral view of Sri Lankan
Buthidae and Chaerilidae species. Figures 403–404. Isometrus
maculatus, male from locality 15CP (403) and female from
locality 15CI (404). Figures 405–406. I. thwaitesi, male from
locality 15CH (405) and female from locality 15CO (406). Figures 407–408. Lychas srilankensis, male (407) and female
(408) from locality 15CJ. Figures 409–410. Reddyanus
basilicus, male (409) and female (410) from locality 15CR. Figures 411–412. R. besucheti, male holotype (411) and
female from locality 15CG (412). Figures 413–414. R. ceylonensis sp. n., male holotype (413) and female paratype
(414) from locality 15CI. Figures 415–416. R. Euscorpius — 2016, No. 220 jayarathnei sp. n., male (415) and female (416) paratypes. Figures 417–418. R. loebli, male (417) and female (418) from locality 15CG. Figures 419–420. R. ranawanai sp. n., male holotype (419)
and female paratype (420). Figures 421–422: Buthoscorpio
sarasinorum, male from locality 15CF (421) and female from
locality 15CG (422). Figures 423–424. Charmus laneus, male
(423) and female (424) from locality 15CO. Figures 425–426. Charmus saradieli sp. n. male paratype (425) (MHNG) and
female holotype (426). Figurs 427. Hottentotta tamulus, male
from locality 15CK (427). Figures 428–429. Chaerilus
ceylonensis, male (428) and female (429) from locality 15CD. DESCRIPTION. Total length 35–43 mm. The habitus is
shown in Figs. 378–381. For measurements and ratios
see Tables 4–5. For positions and distribution of tri-
chobothria see Figs. 395–400. The male has very slightly
longer metasomal segments than the female. Length and
width of the telson is approximately the same in both
sexes (Figs. 419–420). Coloration (Figs. 378–381). Base color reddish, with
brown to black spots. Chelicera strongly reticulated
mainly anteriorly, with spotted fingers. Ventral surface
of mesosoma and pedipalps yellowish brown with a pair
of black spots mainly on the sixth and seventh sternites. The carapace and pedipalps dorsally and laterally yel-
lowish to reddish, with brown to black spots, identical
dipalp femur and patella. Metasomal segments and
telson are yellowish to reddish with spots. on pedipalp femur, patella and manus. Pedipalp fingers
reddish black. Legs with the same color and pattern as
the pe 419–420, 570, Tables 4–5)
Reddyanus ranawanai Kovařík, sp. n. (Figs. 14, 208, 219–220, 235–236, 250, 259, 378–401, http://www.zoobank.org/urn:lsid:zoobank.org:act:D
6B4D355-F67F-4D3C-94A5-6782DD9E1986 Mesosoma and carapace (Figs. 382–386). Carapace
without carinae but with large granules. Tergites I–VI
with one granulated median carina, tergite VII penta-
carinate. Fifth sternite with glabrous zone stretching
over almost the whole posterior margin. Seventh sternite
with four incomplete carinae, sparsely granulate. Pec-
tinal tooth count 12–14 in females, 13–14 in males. Pectines with three marginal lamellae and six to seven
middle lamellae. The lamellae bear numerous pale setae. Metasoma and telson (Figs. 219–220, 387–392, 419–
420). The first metasomal segment bears 10 carinae and
the second to the fourth segments bear eight carinae, the
fifth segment bears five carinae developed in both sexes. Isometrus (Reddyanus) basilicus: Kovařík, 2003: 5 (in
part); Kovařík & Ojanguren, 2013: 186 (in part),
347, 350, 359, figs. 1267, 1308–1311, 1393–1395. Kovařík et al.: Scorpions of Sri Lanka Male with longer
omal segment of male markedly
D
metasomal segments and telson. Pedipalp segments
approximately the same length and width in both sexes. Pedipalps and legs yellow with several small brown
spots. Base color yellow with black spots. Pedipalp fe-
mur and patella spotted, patella mostly black, femur
mostly yellow. Posterior terminal tubercle of dorsal car-
ina on second metas
enlarged; on third metasomal segment of male partly
enlarged; terminal tubercle of dorsal carina on other
metasomal segments of male scarcely larger than pre-
ceding granules. Subaculear tooth more or less pointed,
termination not rounded, dorsally with two pairs of
granules and one or two terminal granules. Pectinal teeth
number 14–17 in both sexes. characters (c.f. d Figs. 417–418, showing the pointed subacu-
ooth in both sexes) and in behavior (all other Sri
COMPARISONS. The hemispermatophore of R. loebli
comb. n. differs from those of R. basilicus comb. n. and
R. ceylonensis sp. n. by having a median lobe term-
inating distally in a pointed cusp (vs. a rounded margin),
and a smaller basal lobe with apex blunt (vs. sharp) in
lateral view. R. loebli also differs from all other Sri
Lankan Reddyanus species in external
Figs. 217, 561, showing the enlarged terminal tubercle
of dorsal carina on the second metasomal segment of the
male; an
lear t
Lankan Reddyanus species forage at ground level among
leaf litter, whereas R. loebli usually sits under the scales
of bark of standing tree trunks, c.f. Fig. 583). Also, first
instar juveniles R. loebli are different colored than those
of other Sri Lankan Reddyanus species (c.f. Fig. 377
versus Figs. 288 and 332). HEMISPERMATOPHORE (Figs. 372–375). Trunk moderate-
ly narrow, at least twice as long as capsule region. Cap-
sule region narrow. Flagellum long, slender, laminiform, Euscorpius — 2016, No. 220 82 Euscorpius — 2016, No. 220
82
Figures 401–402: Reddyanus ranawanai sp. n., female paratype (401) and male holotype (402). Figures 401–402: Reddyanus ranawanai sp. n., female paratype (401) and male holotype (402). Kovařík et al.: Scorpions of Sri Lanka 83 TYPE LOCALITY AND TYPE REPOSITORY. Sri Lanka, Kan-
dy; FKCP. besucheti and R. ceylonensis sp. n. Abbreviations:
length (L), width (W, in carapace it corresponds to posterior width), depth (D). R. ranawanai sp. n. R. ranawanai sp. n. R. jayarathnei sp. n. Dimensions (mm)
♂ holotype
♀ paratype
♂ holotype
♀ paratype
Carapace
L / W
4.55 / 4.40
4.20 / 4.35
5.10 / 5.35
4.97 / 5.15
Mesosoma
L
13.00
11.7
11.4
8.80
Tergite VII
L / W
3.00 / 3.90
2.60 / 4.10
3.05 / 4.15
2.20 / 4.25
Metasoma & telson
L
25.35
20.6
29.0
23.33
Segment I
L / W / D 2.90 / 2.20 / 2.05
2.40 / 2.20 / 2.05
3.35 / 2.60 / 2.50
2.58 / 2.45 / 2.35
Segment II
L / W / D 3.65 / 2.05 / 2.05
2.95 / 2.15 / 1.95
4.35 / 2.40 / 2.50
3.15 / 2.45 / 2.35
Segment III
L / W / D 3.95 / 2.00 / 2.10
3.25 / 2.00 / 1.90
4.70 / 2.45 / 2.50
3.60 / 2.42 / 2.35
Segment IV
L / W / D 4.35 / 2.00 / 2.10
3.55 / 1.95 / 1.85
5.10 / 2.40 / 2.45
4.05 / 2.30 / 2.25
Segment V
L / W / D 5.75 / 2.05 / 2.10
4.50 / 1.95 / 1.80
6.40 / 2.40 / 2.35
5.30 / 2.22 / 2.20
Telson
L / W / D 4.75 / 1.85 / 1.95
3.95 / 1.70 / 1.65
5.10 / 2.15 / 2.20
4.65 / 1.85 / 1.95
Pedipalp
L
17.35
15.50
20.02
18.12
Femur
L / W
4.25 / 1.40
3.40 / 1.35
4.97 / 1.52
4.32 / 1.50
Patela
L / W
5.05 / 1.85
4.60 / 1.80
5.60 / 1.97
5.15 / 2.03
Chela
L
8.05
7.50
9.45
8.65
Manus
L / W / D 3.20 / 2.25 / 2.00
3.05 / 1.90 / 1.56
4.20 / 2.70 / 2.25
3.25 / 2.27 / 1.95
Movable finger
L
4.85
4.45
5.25
5.40
Total
L
42.9
36.5
45.5
37.1
Table 4: Comparative measurements of adults of Reddyanus ranawanai sp. n. and R. jayarathnei sp. n. Abbreviations: length
(L), width (W, in carapace it corresponds to posterior width), depth (D). R. jayarathnei sp. n. Table 4: Comparative measurements of adults of Reddyanus ranawanai sp. n. and R. jayarathnei sp. n. Abbreviations: length
(L), width (W, in carapace it corresponds to posterior width), depth (D). TYPE LOCALITY AND TYPE REPOSITORY. Sri Lanka, Kan-
dy; FKCP. TYPE LOCALITY AND TYPE REPOSITORY. Sri Lanka, Kan-
dy; FKCP. TYPE MATERIAL. Sri Lanka, Kandy, IV.2001, 1♂ after
fifth ecdysis (holotype, Figs. 208, 219, 235, 250, 259,
378–379, 382, 384–385, 387–389, 394–400, 402, 419), Kovařík et al.: Scorpions of Sri Lanka
85
R. basilicus
R. besucheti
R.ceylonensis sp. n. Dimensions (mm)
♂ holotype
♂ holotype
♂ holotype
♀ paratype
Carapace
L / W
4.10 / 3.70
4.50 / 3.90
3.65 / 3.55
3.30 / 3.30
Mesosoma
L
9.00
10.1
12.65
10.5
Tergite VII
L / W
2.30 / 3.30
2.80 / 3.45
2.75 / 3.10
2.30 / 3.20
Metasoma & telson
L
26.75
24.78
22.7
16.35
Segment I
L / W / D
3.25 / 1.80 / 1.80
2.65 / 2.20 / 1.95
2.75 / 1.80 / 1.60
1.95 / 1.80 / 1.65
Segment II
L / W / D
4.00 / 1.75 / 1.70
3.45 / 2.20 / 2.10
3.35 / 1.70 / 1.63
2.30 / 1.65 / 1.70
Segment III
L / W / D
4.35 / 1.70 / 1.75
4.55 / 2.20 / 2.05
3.80 / 1.80 / 1.75
2.55 / 1.70/ 1.70
Segment IV
L / W / D
4.80 / 1.75 / 1.75
4.23 / 2.25 / 1.88
4.10 / 1.75 / 1.73
2.90 / 1.68 / 2.10
Segment V
L / W / D
5.85 / 1.85 / 1.75
5.30 / 2.30 / 2.07
4.80 / 1.90 / 1.70
3.60 / 1.70 / 1.55
Telson
L / W / D
4.50 / 1.80 / 1.57
4.60 / 1.90 / 1.65
3.90 / 1.70 / 1.35
3.05 / 1.35 / 1.30
Pedipalp
L
15.35
15.35
12.8
11.25
Femur
L / W
3.85 / 1.25
3.75 / 1.23
3.40 / 1.05
2.90 / 1.05
Patela
L / W
4.45 / 1.55
4.35 / 1.65
3.80 / 1.45
2.80 / 1.40
Chela
L
7.05
7.25
6.50
5.55
Manus
L / W / D
3.30 / 1.90 / 1.60
3.70 / 2.30 / 2.15
3.30 / 2.05 / 1.73
2.40 / 1.65 / 1.40
Movable finger
L
3.75
3.55
3.20
3.15
Total
L
39.85
39.38
39.0
30.2
Table 3: Comparative measurements of adults of Reddyanus basilicus, R. besucheti and R. ceylonensis sp. n. Abbreviations:
length (L), width (W, in carapace it corresponds to posterior width), depth (D). Kovařík et al.: Scorpions of Sri Lanka 85 R.ceylonensis sp. n. Table 3: Comparative measurements of adults of Reddyanus basilicus, R. TYPE LOCALITY AND TYPE REPOSITORY. Sri Lanka, Kan-
dy; FKCP. Ratios of adult males
R. basilicus
(n = 6)
R. besucheti
(n = 4)
R. ceylonensis
sp. n. (n = 2)
R. jayarathnei
sp. n. (n = 1)
R. loebli
(n = 9)
R. ranawanai
sp. n. (n = 2)
Metasomal segment I (L/W)
1.50–1.80
1.20–1.35
1.40–1.52
1.29
1.58–2.03
1.30–1.31
Metasomal segment II (L/W)
2.04–2.32
1.56–1.79
1.85–1.97
1.81
2.08–2.60
1.78–1.80
Metasomal segment IV (L/W)
2.47–2.75
1.88–2.09
2.24–2.34
2.12
2.69–3.35
2.16–2.18
3.04–3.34
2.56–2.72
2.81–2.82
2.72
3.11–3.74
2.73–2.80
elson (L/D)
2.57–3.05
2.42–2.36
2.29–2.40
2.32
2.76–3.35
2.43
Metasomal segment IV (L/D)
2.57–2.79
2.25–2.28
2.29–2.36
2.08
2.45–3.46
2.07–2.15
Metasomal segment V (L/W)
2.85–3.16
2.30–2.31
2.45–2.53
2.66
2.90–3.82
2.79–2.80
Metasomal segment V (L/D)
T
Pedipalp chela (L/W)
3.41–3.79
2.94–3.15
2.97–3.17
3.50
3.35–3.66
3.57–3.61
Pedipalp chela (L)/Mov. Fing.(L)
1.98–1.88
2.04–2.44
2.03–2.10
1.80
1.70–2.03
1.66–1.69
Total (L)
28.3–45.85
34–46
36–39.1
45.5
28–45
40–42.9
Table 5: Comparison among Sri Lankan Reddyanus species specimens), based upon selected morphometric ratios of adult
( Table 5: Comparison among Sri Lankan Reddyanus species specimens), based upon selected morphometric ratios of adult
(
males. Abbreviations: length (L), width (W), depth (D). Euscorpius — 2016, No. 220 Euscorpius — 2016, No. 220 Euscorpius — 2016, No. 220 86 Euscorpius — 2016, No. 220
86
Figures 430–435: Chaerilus ceylonensis from locality 15CD. F
Figures 432–435. Female, dorsal (432) and ventral (433) views,
Ventral carina present on telson. Intercarinal surfaces of
metasoma granulated, including dorsal surface. In both
sexes, posterior terminal tube
igures 430–431. Male in dorsal (430) and ventral (431) views. nd chelicera ventral (434) and dorsal (435) views. granules in four rows; six symmetrical granules in three
rows and one or two granules on the tip. Pedipalps (Figs. 235–236, 393–400). Femur and patella
only very sparsely hirsute, with complete carinae, gran-
l t d Ch l
i b th
l t d
ith d
a
rcle of each dorsal carina
n metasomal segments not enlarged. Telson elongate,
ith
b
l
t
th
id
d
d d d
ll
ith
o Figures 430–435: Chaerilus ceylonensis from locality 15CD. F
Figures 432–435. Female, dorsal (432) and ventral (433) views,
igures 430–431. Male in dorsal (430) and ventral (431) views. nd chelicera ventral (434) and dorsal (435) views. a Ventral carina present on telson. Intercarinal surfaces of
metasoma granulated, including dorsal surface. In both
sexes, posterior terminal tubercle of each dorsal carina
n metasomal segments not enlarged. TYPE LOCALITY AND TYPE REPOSITORY. Sri Lanka, Kan-
dy; FKCP. Telson elongate,
with subaculear tooth wide and rounded, dorsally with
o granules in four rows; six symmetrical granules in three
rows and one or two granules on the tip. Pedipalps (Figs. 235–236, 393–400). Femur and patella
only very sparsely hirsute, with complete carinae, gran-
ulated. Chela manus in both sexes granulated, with dor- Kovařík et al.: Scorpions of Sri Lanka 87 gures 436–439: Chaerilus ceylonensis from locality 15CD. Figures 436, 438. Male, chelicerae, carapace and tergites
6), and sternopectinal region with sternite III (438). Figures 437, 439. Female, chelicerae, carapace and tergites I–II (43
sternopectinal region (439). Figures 436–439: Chaerilus ceylonensis from locality 15CD. Figures 436, 438. Male, chelicerae, carapace and tergites I–II
(436), and sternopectinal region with sternite III (438). Figures 437, 439. Female, chelicerae, carapace and tergites I–II (437),
and sternopectinal region (439). Euscorpius — 2016, No. 220 88 Euscorpius — 2016, No. 220
88
Figures 440–445: Chaerilus ceylonensis from locality 15CD. Figures 440–442. Male, metasoma and telson, lateral (440),
ventral (441), and dorsal (442) views. Figures 443–445. Female, metasoma and telson, lateral (443), ventral (444), and dorsal
(445) views. Figures 440–445: Chaerilus ceylonensis from locality 15CD. Figures 440–442. Male, metasoma and telson, lateral (440),
ventral (441), and dorsal (442) views. Figures 443–445. Female, metasoma and telson, lateral (443), ventral (444), and dorsal
(445) views. guren, 2013: 131–145, figs. ZN.1–7, 617–776 (ref-
erence list until 2013); Kovařík et al., 2015: 1–21,
figs. 1–91. = Chelomachus Thorell, 1889: 583 (syn. by Kraepelin,
1899: 157). = Uromachus Pocock, 1890: 250 (syn. by Kraepelin,
1899: 157). guren, 2013: 131–145, figs. ZN.1–7, 617–776 (ref-
erence list until 2013); Kovařík et al., 2015: 1–21,
figs. 1–91. sal carinae indicated. Sixth row of granules on movable
finger with one external granule. Seventh row of gran-
ules on the fixed finger without additional granules. Legs (Fig. 208). Femur and patella with complete car-
inae, granulated. Legs hirsute, without bristle combs. = Chelomachus Thorell, 1889: 583 (syn. by Kraepelin,
1899: 157). = Uromachus Pocock, 1890: 250 (syn. by Kraepelin,
1899: 157). AFFINITIES. The described features distinguish R. rana-
wanai sp. n. from all other species of the genus. All
other Sri Lankan Reddyanus species exhibit sexual di-
morphism in the length of the telson, which is absent in
R. ranawanai sp. n. (c.f. Figs. 419–420 versus Figs. 409–418). TYPE SPECIES. Chaerilus variegatus Simon, 1877. TYPE SPECIES. Chaerilus variegatus Simon, 1877. DIAGNOSIS. Total length 15–75.4 mm. Orthobothriotaxy
type B; pedipalp femoral d3–d4 trichobothria config-
uration points toward dorsoexternal carina; pedipalp
patella with 3 ventral trichobothria and pedipalp femur
with 9 trichobothria, 4 of them dorsal. Cheliceral fixed
finger with median and basal denticles flush on surface,
not conjoined on common trunk; ventral edge of che-
liceral movable finger crenulated; dorsal edge of
th a single subdistal den-
cheliceral movable finger wi Family Chaerilidae Pocock, 1893
Chaerilus Simon, 1877
(Figs. 13, 200, 428–457) Chaerilus Simon, 1877: 238; Fet, 2000: 323–328 (com-
plete reference list until 1998); Kovařík & Ojan- Kovařík et al.: Scorpions of Sri Lanka 89 Figures 446–453: Chaerilus ceylonensis from locality 15CD. Figures 446–449. Male. Pedipalp chela, dorsal (446), external
(447) and ventral (448) views. Movable finger (449). Figures 450–453. Female. Pedipalp chela, dorsal (450), external (451) and
ventral (452) views. Movable finger (453). ticles; ventral surface of cheliceral fixed finger with
denticle. Sternum, type 1, exhibits subtle wide horizontal
compression; maxillary lobes I spatulate. Hemisper-
t
h
f
if
P di
l
t ll
ith "6
i
"
configuration. Median denticle row of pedipalp chelal
finger arranged in oblique groups; pedipalp chela
exhibits "8-carinae" configuration. Fifth metasomal seg-
t
ith
i
l
t l
i
L
ith
t tibi l Figures 446–453: Chaerilus ceylonensis from locality 15CD. Figures 446–449. Male. Pedipalp chela, dorsal (446), external
(447) and ventral (448) views. Movable finger (449). Figures 450–453. Female. Pedipalp chela, dorsal (450), external (451) and
ventral (452) views. Movable finger (453). ticles; ventral surface of cheliceral fixed finger with
denticle. Sternum, type 1, exhibits subtle wide horizontal
compression; maxillary lobes I spatulate. Hemisper-
atophore fusiform. Pedipalp patella with "6-carinae"
m configuration. Median denticle row of pedipalp chelal
finger arranged in oblique groups; pedipalp chela
exhibits "8-carinae" configuration. Fifth metasomal seg-
ment with single ventral carina. Legs without tibial Euscorpius — 2016, No. 220 90 Figures 454–457: Chaerilus ceylonensis from locality 15CD, male (454), female (455), female with juveniles after first ecdysis
(456), and female with newborns (457). Figures 454–457: Chaerilus ceylonensis from locality 15CD, male (454), female (455), female with juveniles after first ecdysis
(456), and female with newborns (457). Kovařík et al.: Scorpions of Sri Lanka 91 Kovařík et al.: Scorpions of Sri Lanka Figures 458–461: Heterometrus gravimanus from locality 15CE. Figures 458–459. Male in dorsal (458) and ventral (459)
views. Figures 460–461. Female, dorsal (460) and ventral (461) views. Scale bar: 10 mm. Figures 458–461: Heterometrus gravimanus from locality 15CE. Figures 458–459. Male in dorsal (458) and ventral (459)
views. Figures 460–461. Female, dorsal (460) and ventral (461) views. Scale bar: 10 mm. Euscorpius — 2016, No. 220 92 Figures 462–469: Heterometrus gravimanus. Figures 462, 464. Male from locality 15CE, chelicerae, carapace and tergites I–
g
68). Figure 469. l
II (462), and sternopectinal region with sternites III–IV (464). Fi ures 463, 465. Family Chaerilidae Pocock, 1893
Chaerilus Simon, 1877
(Figs. 13, 200, 428–457) Female from locality 15CJ, chelicerae, carapace
and tergites I–II (463), and sternopectinal region with sternites III–IV (465). 466–468. Male from locality 15CJ, tarsomeres of
third (466) and fourth (467) legs retroventral views, and telson with metasomal segment V in lateral view (4
Female from locality 15CJ, telson with metasomal segment V in
ateral view. Figures 462–469: Heterometrus gravimanus. Figures 462, 464. Male from locality 15CE, chelicerae, carapace and tergites I–
g
68). Figure 469. l
II (462), and sternopectinal region with sternites III–IV (464). Fi ures 463, 465. Female from locality 15CJ, chelicerae, carapace
and tergites I–II (463), and sternopectinal region with sternites III–IV (465). 466–468. Male from locality 15CJ, tarsomeres of
third (466) and fourth (467) legs retroventral views, and telson with metasomal segment V in lateral view (4
Female from locality 15CJ, telson with metasomal segment V in ateral view. 93 Kovařík et al.: Scorpions of Sri Lanka Figures 470–475: Heterometrus gravimanus. Figures 470–472. Male 105 mm long from locality 15CE, pedipalp chela in
orsal (470), external (471) and ventral (472) views. Figure 473. Female from locality 15CE, pedipalp chela in dorsal view. d
Figures 474. Male 106 mm long from locality 15CJ, pedipalp chela in dorsal view. Figures 475. Male 77 mm long from locality
15CE, pedipalp chela in dorsal view. Figures 470–475: Heterometrus gravimanus. Figures 470–472. Male 105 mm long from locality 15CE, pedipalp chela in
orsal (470), external (471) and ventral (472) views. Figure 473. Female from locality 15CE, pedipalp chela in dorsal view. d
Figures 474. Male 106 mm long from locality 15CJ, pedipalp chela in dorsal view. Figures 475. Male 77 mm long from locality
15CE, pedipalp chela in dorsal view. Euscorpius — 2016, No. 220 94 Figures 476–479: Right hemispermatophore of Heterometrus
ev
gravimanus from locality 15CE. External (476), dorsal (477),
iations: bl, basal lobe; dl, distal lamina; dtdl, dorsal trough of
ugh; pl, proximal lobe; t, trunk. internal (478) and ventral (479) views. Scale bar: 2 mm. Abbr
distal lamina; h, hook; ml, median lobe; mtt, median transverse tro Figures 476–479: Right hemispermatophore of Heterometrus
ev
gravimanus from locality 15CE. External (476), dorsal (477),
iations: bl, basal lobe; dl, distal lamina; dtdl, dorsal trough of
ugh; pl, proximal lobe; t, trunk. internal (478) and ventral (479) views. Scale bar: 2 mm. Heterometrus gravimanus (Pocock, 1894)
(Figs. 15, 458–481, 572) Heterometrus gravimanus (Pocock, 1894)
(Figs. 15, 458–481, 572) Scorpio gravimanus Pocock, 1894: 75–76. Scorpio gravimanus Pocock, 1894: 75–76. Heterometrus (Srilankametrus) gravimanus: Tikader &
Bastawade, 1983: 550–555, figs. 1469–1482; Fet,
2000: 446;
Heterometrus gravimanus: Kovařík 2004: 15 17 fig Heterometrus (Srilankametrus) gravimanus: Tikader &
Bastawade, 1983: 550–555, figs. 1469–1482; Fet,
2000: 446; Heterometrus gravimanus: Kovařík, 2004: 15, 17, fig. 15; Kovařík, 2009: 38, 79, figs. 40–42. TYPE LOCALITY AND TYPE REPOSITORY. Ceylon, now Sri
Lanka; BMNH. Euscorpius — 2016, No. 220 Family Scorpionidae Latreille, 1802
Heterometrus Ehrenberg, 1828
(Figs. 15, 458–546, 571–574) spurs, but with prolateral and retrolateral pedal spurs. Tarsi of legs bear two rows of ventral setae and median
row of spinules. Telson without subaculear tubercle. DISTRIBUTION. Oriental region from India to Melanesia. Buthus (Heterometrus) Ehrenberg in Hemprich &
Ehrenberg, 1828: pl. 1, figs 1–2 (part, only fig. 2);
Hemprich & Ehrenberg, 1829: 351. DISTRIBUTION. Oriental region from India to Melanesia. Chaerilus ceylonensis Pocock, 1894
(Figs. 13, 200, 428–457) Chaerilus ceylonensis Pocock, 1894
(Figs. 13, 200, 428–457) Heterometrus: Kovařík, 2004: 1–60, figs. 1–32 (com-
plete reference and synonymy list until 2004);
Kovařík, 2009: 34–49, 73–113, figs. 1–283. Chaerilus ceylonensis Pocock, 1894: 83–84; Vachon,
1982: 102, figs. 94–95; Tikader & Bastawade, 1983:
326–332, figs. 925–939; Fet, 2000: 325 (complete
reference list until 1998); Kovařík, 2000: 44–45, fig. 14; Lourenço & Huber, 2002: 273; Kovařík & Ojan-
guren, 2013: 135, 284, figs. 732–733. TYPE SPECIES. Buthus (Heterometrus) spinifer Ehren-
berg, 1828, by subsequent designation (Karsch, 1879:
20). DIAGNOSIS: Total length 60–176 mm. Orthobothriotaxy
type C. Pedipalp femur with three trichobothria, only
one of which resides on internal surface. Pedipalp patella
with 19 trichobothria, 3 ventral, and 13 external. Chela
of pedipalp with 26 trichobothria. Retrolateral pedal
spurs absent. Lateroapical margins of tarsi produced into
rounded lobes. Metasomal segments I–IV with paired
ventral submedian carinae. Stridulatory organ located on
opposing surfaces of pedipalp coxa and first leg. TYPE LOCALITY AND TYPE REPOSITORY. Sri Lanka (Cey-
lon), Trincomalee; BMNH. Family Chaerilidae Pocock, 1893
Chaerilus Simon, 1877
(Figs. 13, 200, 428–457) Abbr
distal lamina; h, hook; ml, median lobe; mtt, median transverse tro Kovařík et al.: Scorpions of Sri Lanka
95 Kovařík et al.: Scorpions of Sri Lanka Kovařík et al.: Scorpions of Sri Lanka 95 Kovařík et al.: Scorpions of Sri Lanka
95
Figures 480–481: Heterometrus gravimanus, male (480) and female (481) at locality 15CE. Figures 480–481: Heterometrus gravimanus, male (480) and female (481) at locality 15CE. Euscorpius — 2016, No. 220
Family Scorpionidae Latreille, 1802
Heterometrus Ehrenberg, 1828
(Figs. 15, 458–546, 571–574) Euscorpius — 2016, No. 220 96 TYPE LOCALITY AND TYPE REPOSITORY. Sri Lanka (Cey-
lon), Trincomalee; BMNH. TYPE MATERIAL EXAMINED. Sri Lanka, Trincomalee, 1♂
(holotype, figs. 732–733 in Kovařík & Ojanguren, 2013:
284), BMNH No. 1893.10.20.4. OTHER MATERIAL EXAMINED. Sri Lanka, North Central
Province, Polonnaruwa District, ca 35 km from Dam-
bula, 07°57'15.1"N 080°54'45,4"E, 132 m a.s.l. (Locality
15CD, Fig. 578), 22.IV.2015, 2♂ (Figs. 200, 428, 430–
431, 436, 438, 440–442, 446–449, 454) 1♀ (Figs. 429,
432–435, 437, 439, 443–445, 450–453, 455), FKCP,
1♂1♀1juv., UPSL, leg. Kovařík et al.; North Central
Province, Polonnaruwa District, near Kaudulla National
Park, 08°08'40.6"N 080°51'04"E, 101 m a.s.l. (Locality
15CF, Fig. 581), 23.IV.2015, 1♂1♀1juv., FKCP,
1♂1juv., UPSL, leg. Kovařík et al.; North Central Pro-
vince, Puttalam District, Eluwankulam, 08°17'15"N
079°50'38.7"E, 38 m a.s.l. (Locality 15CO, Fig. 592),
28.IV.2015, 2♂, FKCP, 3juvs., UPSL, leg. Kovařík et
al. OTHER MATERIAL EXAMINED. Sri Lanka, North Central
Province, Polonnaruwa District, ca 35 km from Dam-
bula, 07°57'15.1"N 080°54'45,4"E, 132 m a.s.l. (Locality
15CD, Fig. 578), 22.IV.2015, 2♂ (Figs. 200, 428, 430–
431, 436, 438, 440–442, 446–449, 454) 1♀ (Figs. 429,
432–435, 437, 439, 443–445, 450–453, 455), FKCP,
1♂1♀1juv., UPSL, leg. Kovařík et al.; North Central
Province, Polonnaruwa District, near Kaudulla National
Park, 08°08'40.6"N 080°51'04"E, 101 m a.s.l. (Locality
15CF, Fig. 581), 23.IV.2015, 1♂1♀1juv., FKCP,
1♂1juv., UPSL, leg. Kovařík et al.; North Central Pro-
vince, Puttalam District, Eluwankulam, 08°17'15"N
079°50'38.7"E, 38 m a.s.l. (Locality 15CO, Fig. 592),
28.IV.2015, 2♂, FKCP, 3juvs., UPSL, leg. Kovařík et
al. DISTRIBUTION. Oriental region from India to Indonesia. Heterometrus gravimanus (Pocock, 1894)
(Figs. 15, 458–481, 572) TYPE LOCALITY AND TYPE REPOSITORY. Ceylon, now Sri
Lanka; BMNH. DIAGNOSIS. Total length 27–45 mm. Two pairs of lateral
eyes and one pair of median eyes. Movable fingers of
pedipalps with 11–12 rows of granules. Fingers straight
and short. Trichobothrium d2 of pedipalp patella located
on dorsal surface, trichobothrium id on internal surface. Pedipalp chela with 9 carinae. Pectinal teeth number 4–
6. Entire carapace evenly covered by granules. Ratio of
median eye distance from anterior and posterior margins
of carapace = 1 : 1.55–1.60. Mesosoma without carinae. Sternites smooth. First and second metasomal segments
with 10 carinae, third and fourth segments with 8 car-
inae, fifth segment with 7 carinae. All carinae consist of
large, denticulate granules. SRI LANKAN MATERIAL EXAMINED. Sri Lanka, Southern
Province, Galle, 1♂, FKCP, IV.1994, leg. P. Senft;
North Central Province, Polonnaruwa District, Giritale,
08°01'26.0"N 080°54'37.2"E, 233 m a.s.l. (Locality
15CE, Fig. 579), 22. –23.IV.2015, 2♂ (Figs. 458–459,
462, 464, 470–472, 475–479, 480, 572) 1♀ (Figs. 460–
461, 463, 465, 473), FKCP, 1♀7juvs., UPSL, leg. Kovařík et al.; Northern Province, Jaffna District, 09°
42'51.6"N 080°04'44.8"E, 19 m a.s.l. (Locality 15CJ,
Figs. 586–587), 26. –27.IV.2015, 2♂ (Figs. 466–468,
474), 1♀ (Fig. 469), FKCP, leg. Kovařík et al. 13–16 in males and 11–13 in females. Male with slightly
DIAGNOSIS. Total length 75–110 mm long. Color of
adults uniformly reddish brown. Pectinal teeth number 13–16 in males and 11–13 in females. Male with slightly
DIAGNOSIS. Total length 75–110 mm long. Color of
adults uniformly reddish brown. Pectinal teeth number DISTRIBUTION. Sri Lanka. DISTRIBUTION. Sri Lanka. Kovařík et al.: Scorpions of Sri Lanka Kovařík et al.: Scorpions of Sri Lanka 97 igures 482–485: Heterometrus indus from locality 15CA. Figures 482–483. Male in dorsal (482) and ventral (483) views. F
Figures 484–485. Female, dorsal (484) and ventral (485) views. Scale bar: 10 mm. igures 482–485: Heterometrus indus from locality 15CA. Figures 482–483. Male in dorsal (482) and ventral (483) views. F
Figures 484–485. Female, dorsal (484) and ventral (485) views. Scale bar: 10 mm. Euscorpius — 2016, No. 220 98 Figures 486–489: Heterometrus indus from locality 15CA. Figures 486, 488. Male, chelicerae, carapace and tergites I–II
86), and sternopectinal region (488). Figures 487, 489. Female, chelicerae, carapace and tergites I–II (487), and sternopectina
gion (489). (4
re Figures 486–489: Heterometrus indus from locality 15CA. Figures 486, 488. Male, chelicerae, carapace and tergites I–II
86), and sternopectinal region (488). Figures 487, 489. TYPE LOCALITY AND TYPE REPOSITORY. Ceylon, now Sri
Lanka; BMNH. Female, chelicerae, carapace and tergites I–II (487), and sternopectina
i
(
)
(4 Figures 486–489: Heterometrus indus from locality 15CA. Figures 486, 488. Male, chelicerae, carapace and tergites I–III
86), and sternopectinal region (488). Figures 487, 489. Female, chelicerae, carapace and tergites I–II (487), and sternopectinal
gion (489). (4
re Kovařík et al.: Scorpions of Sri Lanka 99 Figures 490–493: Heterometrus indus from locality 15CA. Figure 490. Male, metasoma and telson, lateral view. Figures
491–493. Female, metasoma and telson, lateral (491), ventral (492), and dorsal (493) views. Scale bar: 10 mm. Figures 490–493: Heterometrus indus from locality 15CA. Figure 490. Male, metasoma and telson, lateral view. Figures
491–493. Female, metasoma and telson, lateral (491), ventral (492), and dorsal (493) views. Scale bar: 10 mm. Euscorpius — 2016, No. 220
100
Figures 494–499: Heterometrus indus from locality 15CA. Figures 494, 496–499. Male, telson with metasomal segment V in
lateral view (494), chelicera dorsal (496) and ventral (497) views, and tarsomeres of third (498) and fourth (499) legs retroventral
views. Figure 495. Female, telson with metasomal segment V in lateral view. Euscorpius — 2016, No. 220 100 Figures 494–499: Heterometrus indus from locality 15CA. Figures 494, 496–499. Male, telson with metasomal segment V in
lateral view (494), chelicera dorsal (496) and ventral (497) views, and tarsomeres of third (498) and fourth (499) legs retroventral
views. Figure 495. Female, telson with metasomal segment V in lateral view. capsule and distal lamina, parallel-sided, with mod-
erately sclerotized diagonal axial rib. longer pedipalp femur and patella. Chela lobiform, nar-
rower in male than female; chela length/ width ratio
1.79–2.05 in males, 1.65–1.75 in females. Entire manus
covered by rounded granules, with five carinae mainly in
anterolateral part. Pedipalp patella smooth, without pro-
nounced internal tubercle. Carapace smooth, glossy. Spi-
nation formula of tarsomeres II of legs: 4/4-5 : 4/5 : 4/5-6
: 4/5-6. Telson elongate, vesicle shorter than aculeus. The hemispermatophore is quite similar to those of
several other species of Heterometrus previously il-
lustrated as line drawings: H. longimanus (Herbst,
1800), H. cyaneus (C.L. Koch, 1836) and H. spinifer
(Ehrenberg, 1828) (Couzijn, 1981; Stockwell, 1989). The relative proportions of the distal lamina and trunk
are comparable and the same capsular structures or lobes
are present. Some differences can be seen: (i) in H. cyaneus and H. spinifer, the distal lamina is straight, not
internally deflected; (ii) the hook is smaller in H. TYPE LOCALITY AND TYPE REPOSITORY. Ceylon, now Sri
Lanka; BMNH. longimanus and H. cyaneus, and has a shorter tip in H. spinifer; (iii) the distal lamina is not terminally dilated in
H. longimanus and H. cyaneus. However, the extent of
intraspecific morphological variation in Heterometrus
hemispermatophores is unknown, so it is unclear if these
differences have diagnostic value. HEMISPERMATOPHORE (Figs. 476–479). Lamelliform. Distal lamina long, slender, basal capsular portion one
fifth of total lamina length, wider, dorsal trough nearly
flat with slightly elevated transverse strip, bordered
internally by strong carina; portion of lamina just distal
to hook constricted, curved, deflected internally; distal
terminus of lamina dilated, truncated and spatulate. Hook prominent, with narrowed tip and wide triangular
base, lacking dorsal trough. Median lobe elongate, ex-
tending from base of hook to truncal flexure, with fine
longitudinal carina and trough along internal side. Inner
nd median transverse trough large, prominent. lobe a
Basal and proximal lobes smaller but well developed,
blunt, rounded. All lobes of capsule region smooth,
ithout barbs or spicules. Trunk broader than both
w VARIABILITY. The figures 470–475 show variability in
the shape of the chela in adult males. The greatest dif-
ference is between the 77 mm long male (Fig. 475) and
the 105 mm long male (Fig. 470) from the same locality
15CE. Kovařík et al.: Scorpions of Sri Lanka 101 Kovařík et al.: Scorpions of Sri Lanka
101
Figures 500–501: Heterometrus indus, female (500) and male (501) at locality 15CA. Figures 500–501: Heterometrus indus, female (500) and male (501) at locality 15CA. 102 Euscorpius — 2016, No. 220 etrus indus, female from Peradeni
03). Figures 502–503: Heterom
ya with newborns (502) and with juveniles after first ecdysis
(5 etrus indus, female from Peradeni
03). Figures 502–503: Heterom
ya with newborns (502) and with juveniles after first ecdysis
(5 etrus indus, female from Peradeni
3: Heterom
ya with newborns (502) and with juveniles after first ecdysi Kovařík et al.: Scorpions of Sri Lanka
103
Figures 504–511: Pedipalp chela. Figures 504–507: Heterometrus indus from locality 15CA, pedipalp chela dorsal (504) view
of female, and pedipalp chela dorsal (505), external (506) and ventral (507) views of male. Figures 508–511: Heterometrus
serratus from locality 15CP, pedipalp chela dorsal (508) view of female, and pedipalp chela dorsal (509), external (510) and
ventral (511) views of male. DISTRIBUTION. India and Sri Lanka. = Scorpio ceylonicus Herbst, 1800: 83–84, fig. 1, Tab. V
(syn. by Kraepelin, 1899: 113). DISTRIBUTION. India and Sri Lanka. = Scorpio ceylonicus Herbst, 1800: 83–84, fig. 1, Tab. V
(syn. by Kraepelin, 1899: 113). = Heterometrus (Heterometrus) spinifer solitarius Cou-
zijn, 1981: 93–94, fig. 23. Syn. n. DISTRIBUTION. India and Sri Lanka. = Scorpio ceylonicus Herbst, 1800: 83–84, fig. 1, Tab. V
(syn. by Kraepelin, 1899: 113). Heterometrus indus (De Geer, 1778)
(Figs. 15, 482–507, 573) = Heterometrus (Heterometrus) spinifer solitarius Cou-
zijn, 1981: 93–94, fig. 23. Syn. n. TYPE LOCALITY AND TYPE REPOSITORY. "India" (in-
correct type locality; see Kovařík, 2004: 20); NHRS. Scorpio indus De Geer, 1778: 341–343. TYPE LOCALITY AND TYPE REPOSITORY. Ceylon, now Sri
Lanka; BMNH. Kovařík et al.: Scorpions of Sri Lanka
103 103 Kovařík et al.: Scorpions of Sri Lanka Kovařík et al.: Scorpions of Sri Lanka Figures 504–511: Pedipalp chela. Figures 504–507: Heterometrus indus from locality 15CA, pedipalp chela dorsal (504) view
of female, and pedipalp chela dorsal (505), external (506) and ventral (507) views of male. Figures 508–511: Heterometrus
serratus from locality 15CP, pedipalp chela dorsal (508) view of female, and pedipalp chela dorsal (509), external (510) and
ventral (511) views of male. Scorpio indus De Geer, 1778: 341–343. Scorpio indus De Geer, 1778: 341–343. TYPE LOCALITY AND TYPE REPOSITORY. "India" (in-
correct type locality; see Kovařík, 2004: 20); NHRS. Scorpio indus De Geer, 1778: 341–343. Heterometrus indus indus: Couzijn, 1981: 121–123, fig. 35 (in part). Heterometrus indus indus: Couzijn, 1981: 121–123, fig. 35 (in part). Heterometrus indus indus: Couzijn, 1981: 121–123, fig. 35 (in part). MATERIAL EXAMINED. Sri Lanka, Kandy, 30.III.1902,
1♂, FKCP; Peradeniya, 3♂2♀ (Figs. 502–503) 6juvs.,
IV.1994, FKCP; leg. P. Senft; Central Province, Kandy
14'54.7"N 080°36'54"E, 760 m a.s.l. (Locality 15CA,
District, Hantana (Peradeniya), University land, 07° MATERIAL EXAMINED. Sri Lanka, Kandy, 30.III.1902,
1♂, FKCP; Peradeniya, 3♂2♀ (Figs. 502–503) 6juvs.,
IV.1994, FKCP; leg. P. Senft; Central Province, Kandy
14'54.7"N 080°36'54"E, 760 m a.s.l. (Locality 15CA,
District, Hantana (Peradeniya), University land, 07° Heterometrus indus: Fet, 2000: 446–448 (complete ref-
erence and synonymy list until 1998); Kovařík,
in part) (complete reference and sy-
figs. 50–56, 249–252. 2004: 17–20 (
nonymy list until 2004); Kovařík, 2009: 38, 80, 105, Euscorpius — 2016, No. 220 104 Figures 512–515: Heterometrus serratus from locality 15CP. Figures 512–513. Male in dorsal (512) and ventral (513) views. Figures 514–515. Female, dorsal (514) and ventral (515) views. Scale bar: 10 mm. Fig. 575), 19. –21.IV.2015, 1♂ (Figs. 482–483, 486,
488, 490, 494, 496–499, 505–507) 1♀ (Figs. 484–485,
487, 489, 491–493, 495, 504, 573), FKCP, 4♀1juv.,
20'41.9"N 080°39'30.6"E, 534 m a.s.l. (Locality 15CC,
Fig. 577), 21.IV.2015, 1♀, FKCP, leg. Kovařík et al. Figures 512–515: Heterometrus serratus from locality 15CP. Figures 512–513. Male in dorsal (512) and ventral (513) views. Figures 514–515. Female, dorsal (514) and ventral (515) views. Scale bar: 10 mm. 20'41.9"N 080°39'30.6"E, 534 m a.s.l. (Locality 15CC,
Fig. 577), 21.IV.2015, 1♀, FKCP, leg. Kovařík et al. 20'41.9"N 080°39'30.6"E, 534 m a.s.l. (Locality 15CC,
Fig. 577), 21.IV.2015, 1♀, FKCP, leg. Kovařík et al. Fig. 575), 19. –21.IV.2015, 1♂ (Figs. 482–483, 486,
488, 490, 494, 496–499, 505–507) 1♀ (Figs. 484–485,
487, 489, 491–493, 495, 504, 573), FKCP, 4♀1juv.,
PSL, leg. Kovařík et al.; Central Province, Kandy Dis-
U
trict, 20 km S Kandy, Meegammana, Wattegama, 07° DIAGNOSIS. Total length 90–130 mm long. Color of
adults uniformly reddish black to greenish black. Pec- Kovařík et al.: Scorpions of Sri Lanka 105 Figures 516–519: Heterometrus serratus from locality 15CP. Figures 516, 518. Male, chelicerae, carapace and tergites I–I
16), and sternopectinal region (518). Figures 517, 519. Female, chelicerae, carapace and tergites I–II (517), and sternopectina
gion (519). Scorpio indus De Geer, 1778: 341–343. (5
re Figures 516–519: Heterometrus serratus from locality 15CP. Figures 516, 518. Male, chelicerae, carapace and tergites I–II
16), and sternopectinal region (518). Figures 517, 519. Female, chelicerae, carapace and tergites I–II (517), and sternopectinal
gion (519). (5
re 106 Euscorpius — 2016, No. 220 Figures 520–525: Heterometrus serratus from locality 15CP
ventral (521), and dorsal (522) views. Figures 523–525. Femal
(525) views. Scale bar: 10 mm. . e
Figures 520–522. Male, metasoma and telson, lateral (520),
, metasoma and telson, lateral (523), ventral (524), and dorsal Figures 520–525: Heterometrus serratus from locality 15CP
ventral (521), and dorsal (522) views. Figures 523–525. Femal
(525) views. Scale bar: 10 mm. . e
Figures 520–522. Male, metasoma and telson, lateral (520),
, metasoma and telson, lateral (523), ventral (524), and dorsal tinal teeth number 10–15 in both sexes. Sexual dimor-
phism in proportions of pedipalps not noticeable. Ped-
ipalp chela hirsute, lobiform, without carinae on dorso-
external surface, but may bear rows of granules. Chela
length/ width ratio 1.8–2.0 in adults. Entire manus
covered by rounded granules that may merge and appear
as rows. Pedipalp patella without pronounced internal
tubercle. Carapace smooth, glossy, occasionally with
granules at margins. Second metasomal segment approx-
imately as long as wide. Fifth segment of metasoma
approximately as long as pedipalp femur, fourth segment /4 : 2-4/3-4 : 4/4-5 :
/5. of metasoma shorter than pedipalp femur. Dorsal and
dorsolateral carinae of metasomal segments smooth. Vesicle of telson usually longer than aculeus. Spination
formula of tarsomeres II of legs: 3
4 mmon and H. swammerdami is not very
common. The original description of H. spinifer soli-
COMMENTS. Heterometrus (Heterometrus) spinifer soli-
tarius Couzijn, 1981 was described from a female (109
mm long) from Sri Lanka, Peradeniya, where H. indus is
very co Kovařík et al.: Scorpions of Sri Lanka
107
Figures 526–529: Heterometrus serratus, female from locality 15CP, tarsomeres of legs I–IV, ventral view. Kovařík et al.: Scorpions of Sri Lanka 107 Figures 526–529: Heterometrus serratus, female from locality 15CP, tarsomeres of legs I–IV, ventral view. TYPE MATERIAL EXAMINED. Sri Lanka, Ceylon, 1♂ (ho-
lotype, fig. 16 in Kovařík, 2004: 19), leg. Dr. Ondaatje,
BMNH No. 1888.55. TYPE MATERIAL EXAMINED. Sri Lanka, Ceylon, 1♂ (ho-
lotype, fig. 16 in Kovařík, 2004: 19), leg. Dr. Ondaatje,
BMNH No. 1888.55. tarius lacks clear diagnostic characters and any con-
nection to H. spinifer (Ehrenberg, 1828). The type
locality of H. Scorpio indus De Geer, 1778: 341–343. spinifer (Ehrenberg, 1828) 'India' must be
regarded as erroneous. The species is known from Ma-
laysia and Thailand, and probably also from Cambodia
and Vietnam (Kovařík, 2009: 35, 44). TYPE MATERIAL EXAMINED. Sri Lanka, Ceylon, 1♂ (ho-
lotype, fig. 16 in Kovařík, 2004: 19), leg. Dr. Ondaatje,
BMNH No. 1888.55. OTHER MATERIAL EXAMINED. Sri Lanka, Southern
Province, Matara District, Kekanadura village, 05°
58'28.2"N 080°36'20.5"E, 40 m a.s.l. (Locality 15CP,
Fig. 594), 30.IV.2015, 1♂ (Figs. 509–513, 516, 518,
520–522, 571) 1♀ (Figs. 508, 514–515, 517, 519, 523–
529, 574), FKCP, 1♀im., UPSL, leg. Kovařík et al.; Uva
Province, Monaragala District, Monaragala, 06°52'
30.7"N 081°21'17"E, 288 m a.s.l. (Locality 15CQ, Fig. 595), 2.–3.V.2015, 2♀, FKCP, leg. Kovařík et al. Heterometrus serratus (Pocock, 1900)
(Figs. 15, 508–534, 571, 574) Palamnaeus serratus Pocock, 1900: 86, 97. Heterometrus serratus: Takashima, 1945: 94. Heterometrus (Srilankametrus) serratus: Tikader &
Bastawade, 1983: 555–561, figs. 1483–1496; Fet,
2000: 448. Palamnaeus serratus Pocock, 1900: 86, 97. DISTRIBUTION. Sri Lanka. DISTRIBUTION. Sri Lanka. Heterometrus serratus (Pocock, 1900)
(Figs. 15, 508–534, 571, 574) YPE LOCALITY AND TYPE REPOSITORY. Ceylon, now Sri
T
Lanka; BMNH. Palamnaeus serratus Pocock, 1900: 86, 97. Palamnaeus serratus Pocock, 1900: 86, 97. Heterometrus serratus: Takashima, 1945: 94. Heterometrus serratus: Takashima, 1945: 94. DIAGNOSIS. Total length 100–130 mm long. Color of
adults uniformly reddish black to greenish black. Pec-
tinal teeth number 12–15 in both sexes. Male with
slightly narrower chela than female; chela length/ width
ratio 1.79–1.94 in males, 1.69 in female. Chela hirsute,
lobiform, without carinae on dorsoexternal surface, but
may bear rows of granules. Chela length/ width ratio
1.8–2.0 in adults. Entire manus covered by rounded
granules that may merge and appear as rows. Pedipalp
patella without pronounced internal tubercle. Carapace
ally, with granules at margins. Se-
smooth, glossy medi Heterometrus (Srilankametrus) serratus: Tikader &
Bastawade, 1983: 555–561, figs. 1483–1496; Fet,
2000: 448. Heterometrus indus indus (in part): Couzijn, 1981: 121–
123, fig. 35 (in part). Heterometrus indus (in part): Kovařík, 2004: 17–20, fig. 16; Kovařík, 2009: 38. YPE LOCALITY AND TYPE REPOSITORY. Ceylon, now Sri
T
Lanka; BMNH. YPE LOCALITY AND TYPE REPOSITORY. Ceylon, now Sri
T
Lanka; BMNH. 108 Euscorpius — 2016, No. 220 at locality 15CP. Figures 530–534: Heterometrus serratus. Figures 530–533. Juvenile from locality 15CQ in phases of ecdysis. Figures 534. Female at locality 15CP
Figures 530–534: Heterometrus serratus. Figures 530–533. Juvenile from locality 15CQ in phases of ecdysis. Figures 534. Female at locality 15CP. Figures 530–534: Heterometrus serratus. Figures 530–533. Juvenile from locality 15CQ in phases of ecdysis. Figures 534. Female Kovařík et al.: Scorpions of Sri Lanka Kovařík et al.: Scorpions of Sri Lanka 109 Kovařík et al.: Scorpions of Sri Lanka
109
Figures 535–536: Heterometrus swammerdami from locality 15CB, female in dorsal (535) and ventral (536) views. Scale bar:
10 mm. Figures 535–536: Heterometrus swammerdami from locality 15CB, female in dorsal (535) and ventral (536) views. Scale bar:
10 mm Figures 535–536: Heterometrus swammerdami from locality 15CB, female in dorsal (535) and ventral (536) views. Scale bar:
10 mm. Species
2n
Relative Size of
Chromosomes
Locality
Buthoscorpio sarasinorum
14
2x 11.2 + 7.4 – 5.7
15CF
Charmus laneus
9
19.2 + 12.9 – 6.7
15CO
6.4 – 13.0 + 2x 6.3
15CF
Isometrus thwaitesi
8
1
Lychas srilankensis
16
2x 9.2 + 7.0 – 4.2
15CN
8.0 – 4.6
15CS
Reddyanus basilicus
16
Reddyanus basilicus
15
11.5 + 8.0 – 4.6
15CR
Reddyanus ceylonensis sp. n. le 6: The diploid numbers
set (in %) of chromosomes of Sri Lankan buthid scorpions.
Tab
and the relative size of the diploid Palamnaeus serratus Pocock, 1900: 86, 97. 16
Reddyanus loebli
1
le 6: The diploid numbers
8.9 – 4.2
15CI
7.7 – 5.0 + 3.5
15CH
set (in %) of chromosomes of Sri Lankan buthid scorpions
7
Tab
and the relative size of the diploid diploid numbers
set (in %) of chromosomes of Sri Lankan buthid scorpions. and the relative size of the diploid le 6: The diploid numbers
set (in %) of chromosomes of Sri Lankan buthid scorpions. Tab
and the relative size of the diploid le 6: The diploid numbers
set (in %) of chromosomes of Sri Lankan
Tab
and the relative size of the diploid Euscorpius — 2016, No. 220 110 Figures 537–538: Heterometrus swammerdami from locality 15CB, female, chelicerae, carapace and tergites I–III (537), and
sternopectinal region with sternites III–IV (538). Figures 537–538: Heterometrus swammerdami from locality 15CB, female, chelicerae, carapace and tergites I–III (537), and
sternopectinal region with sternites III–IV (538). the Sri Lankan expedition of 2015 convinced us that H. serratus is in fact a valid species. Pocock (1900: 86)
differentiated H. serratus and H. indus as follows: 1)
dorsal and dorsolateral carinae of metasomal segments
granulated in H. serratus (Fig. 574), smooth in H. indus
(Fig. 573). We found other minor differences: 2) sexual
H. indus, male with slightly narrower chela than female
in H. serratus (chela length to width ratio 1.79–1.94 in
H. dus (Figs. 486–487); carapace smooth and glossy
dimorphism in proportions of pedipalps not noticeable in
males, 1.69 in the females); 3) carapace smooth and
glossy, only occasionally with granules at margins in
in
medially, always with more granules at margins in H. serratus (Figs. 516–517); 4) spination formula of tar- the Sri Lankan expedition of 2015 convinced us that H. serratus is in fact a valid species. Pocock (1900: 86)
differentiated H. serratus and H. indus as follows: 1)
dorsal and dorsolateral carinae of metasomal segments
granulated in H. serratus (Fig. 574), smooth in H. indus
(Fig. 573). We found other minor differences: 2) sexual
H. indus, male with slightly narrower chela than female
in H. serratus (chela length to width ratio 1.79–1.94 in
H. dus (Figs. 486–487); carapace smooth and glossy
dimorphism in proportions of pedipalps not noticeable in
males, 1.69 in the females); 3) carapace smooth and
glossy, only occasionally with granules at margins in
in
medially, always with more granules at margins in H. serratus (Figs. Palamnaeus serratus Pocock, 1900: 86, 97. 516–517); 4) spination formula of tar- cond metasomal segment approximately as long as wide. Fifth segment of metasoma about as long pedipalp fe-
mur, fourth segment of metasoma shorter than pedipalp
femur. Dorsal and dorsolateral carinae of metasomal
segments granulated. Vesicle of telson usually longer
tion formula of tarsomeres II of legs:
than aculeus. Spina
3-4/4-6 : 4/4-5 : 4/5-6 : 4/5-7. COMMENTS. Palamnaeus serratus Pocock, 1900 was sy-
nonymized with H. indus by Couzijn (1981: 121). The
first author (Kovařík, 2004: 17 and Kovařík, 2009: 38)
accepted the synonymy, whereas Tikader & Bastawade
(1983: 555) considered H. serratus a valid species. Examination of additional specimens collected during Kovařík et al.: Scorpions of Sri Lanka
111
Figures 539–543: Heterometrus swammerdami from locality 15CB, female, pedipalp chela dorsal (539), external (540) and
ventral (541) views, tarsomeres of third (542) and fourth (543) legs retroventral views. 111 Kovařík et al.: Scorpions of Sri Lanka Figures 539–543: Heterometrus swammerdami from locality 15CB, female, pedipalp chela dorsal (539), external (540) and
ventral (541) views, tarsomeres of third (542) and fourth (543) legs retroventral views. list until 2004); Kovařík, 2009: 44, 93, 113, figs. 139–145, 275–283; Veronika & al., 2013: 72–73,
figs. 1, 3–8, tab. 1. someres II of legs: 3/4 : 2-4/3-4 : 4/4-5 : 4/5 in H. indus;
3-4/4-6 : 4/4-5 : 4/5-6 : 4/5-7 in H. serratus. The distribution of H. serratus was previously unknown,
as the type locality was imprecise and the holotype male
was the only known specimen. We collected additional
specimens at localities cited here as 15CP (Fig. 594) and
15CQ (Fig. 595). Our data suggest that H. serratus is
distributed in the southern part, and H. indus in the
central part of Sri Lanka (Fig. 15). g
Heterometrus (Gigantometrus) swammerdami: Tikader
& Bastawade, 1983: 562–567, figs. 1497–1510; = Heterometrus (Gigantometrus) swammerdami titan-
icus Couzijn, 1981: 165–167, figs. 4f, 55, 56 (syn. by Kovařík, 2004: 42). TYPE LOCALITY AND TYPE REPOSITORY. East Indies;
MNHN. Heterometrus swammerdami Simon, 1872
(Figs. 15, 535–546) SRI LANKAN MATERIAL EXAMINED. Sri Lanka, North
Western Province, Puttalam, IV.1994, 1♂1♀1juv.,
080°36'54"E, 760 m a.s.l. (Locality 15CA, Fig. 575), 19. FKCP, leg. P. Senft; Central Province, Kandy District,
Hantana (Peradeniya), University land, 07°14'54.7"N SRI LANKAN MATERIAL EXAMINED. Sri Lanka, North
Western Province, Puttalam, IV.1994, 1♂1♀1juv.,
080°36'54"E, 760 m a.s.l. (Locality 15CA, Fig. 575), 19. FKCP, leg. P. Senft; Central Province, Kandy District,
Hantana (Peradeniya), University land, 07°14'54.7"N 4: 42–44, fig. 28 (complete reference
Heterometrus swammerdami Simon, 1872: 56–59; Fet,
2000: 443–444 (complete reference list until 1998);
Kovařík, 200 Euscorpius — 2016, No. 220
112
Figures 544–546: Heterometrus swammerdami, two differently
at locality 15CE. colored females (544, 546) at locality 15CJ and juvenile (545 Euscorpius — 2016, No. 220 Euscorpius — 2016, No. 220 112 Figures 544–546: Heterometrus swammerdami, two differently
at locality 15CE
colored females (544, 546) at locality 15CJ and juvenile (545) Figures 544–546: Heterometrus swammerdami, two differently
at locality 15CE. colored females (544, 546) at locality 15CJ and juvenile (545) Kovařík et al.: Scorpions of Sri Lanka Kovařík et al.: Scorpions of Sri Lanka 113 Figures 547–554: The mitotic metaphases of buthid males from Sri Lanka. Buthoscorpio sarasinorum from locality 15CF
(2n=14) (547), Charmus laneus from locality 15CO (2n=9) (548), Isometrus thwaitesi from locality 15CF (2n=8) (549), Lychas
srilankensis from locality 15CN (2n=16) (550), Reddyanus basilicus from locality 15CS (2n=16) (551), Reddyanus basilicus
from locality 15CR (2n=15) (552), Reddyanus ceylonensis from locality 15CI (2n=16) (553), Reddyanus loebli from locality
15CH (2n=17) (554). Arrowheads indicate extra large odd chromosomes. Scale bar = 5 µm for 547–554. Figures 547–554: The mitotic metaphases of buthid males from Sri Lanka. Buthoscorpio sarasinorum from locality 15CF
(2n=14) (547), Charmus laneus from locality 15CO (2n=9) (548), Isometrus thwaitesi from locality 15CF (2n=8) (549), Lychas
srilankensis from locality 15CN (2n=16) (550), Reddyanus basilicus from locality 15CS (2n=16) (551), Reddyanus basilicus
from locality 15CR (2n=15) (552), Reddyanus ceylonensis from locality 15CI (2n=16) (553), Reddyanus loebli from locality
15CH (2n=17) (554). Arrowheads indicate extra large odd chromosomes. Scale bar = 5 µm for 547–554. –21.IV.2015, 1♀, UPSL, leg. Kovařík et al.; Central
15CB, Fig. 576), 21.IV.2015, 1♀ (Figs. 535–543),
l. (Locality 15CJ,
ig. 587), 26. –27.IV.2015, 1♀, FKCP, leg. Kovařík et
nce, Anuradhapura District,
ihintale, 08°20'51.8"N 080°30'27.7"E, 156 m a.s.l. (Locality
tos on-
ly, leg. Heterometrus swammerdami Simon, 1872
(Figs. 15, 535–546) Kova
e, Puttalam
District, Eluwankulam, 08° 2'35.1"N 079°51'32"E, 52
015, photos
ince, Put-
IV.2
Province, Matale District, Maragamuwa/Kumaragala,
7°41'58.5"N 080°42'26.7"E, 662 m a.s.l. (Locality
0
FKCP, leg. Kovařík et al.; North Central Province,
Polonnaruwa District, Giritale, 08°01'26.0"N 080°54'
37.2"E, 233 m a.s.l. (Locality 15CE, Figs. 579–580), 22. –23.IV.2015, 1juv. (Fig. 545), FKCP, 3juvs., UPSL, leg. Kovařík et al.; Northern Province, Jaffna District,
09°42'51.6"N 080°04'44.8"E, 19 m a.s. F
al.; North Central Provi
M
15CL, Fig. 589), 27. –28.IV.2015, pho
řík et al.; North Central Provinc
1
m a.s.l. (Locality 15CN, Fig. 591), 28.IV.2
only, leg. Kovařík et al.; North Central Prov
t
talam Distric , Eluwankulam, 08°17'15"N 079°50'
38.7"E, 38 m a.s.l. (Locality 15CO, Fig. 592), 28. 015, 1juv., UPSL, leg. Kovařík et al. with disc smooth, margins and posterior portion gran-
erculate; occas-
. Fifth segment
of metasoma longer than pedipalp femur, fourth segment
ulate, anterior portion granulate and tub
ionally entire surface sparsely granulate
of metasoma approximately as long as pedipalp femur. Telson bulbous, vesicle as long as or longer than acu-
leus. NOTE. For photos of the male see figs. 139–142 and 275
in Kovařík, 2009: 93 and 113. NOTE. For photos of the male see figs. 139–142 and 275
in Kovařík, 2009: 93 and 113. DISTRIBUTION. India and Sri Lanka. DISTRIBUTION. India and Sri Lanka. Hemispermatophores of the Buthids Sri
nka, th
ave been relatively well described, including for the
We obtained new data on hemispermatophore mor-
hologies of a variety of Old World buthids from
p
La
at have not previously been described. Our
findings may be significant in the context of the larger
problem of defining monophyletic genera in the family
and understanding relationships of major lineages of
buthids. A preliminary buthid phylogeny was derived by
Fet et. al. (2005) from the cladistic analysis of certain
trichobothrial characters. They proposed a major family
subdivision based on whether patella trichobothrium d3
is positioned internal to the dorsomedian carina, which
defined the 'Buthus' group, or external to it, which is the
character state for the remaining five 'non-Buthus'
roups. Hemispermatophores of 'Buthus' group genera
g
h DIA
28–176 mm long. Base color
be r
ectinal teeth number 16–20. exual dimorphism in proportions of pedipalps not
ticeable. Chela strongly lobiform, l
that do not form true carinae. Pedipalp
atella without pronounced internal tubercle. Carapace
GNOSIS. Total length 1
uniformly reddish brown to reddish black. Juveniles may
ed with yellow telson. P
S
no
ength/ width ratio
1.6–1.8 in both sexes. Entire manus covered by large,
ounded granulae
r
p Euscorpius — 2016, No. 220 114 igures 555–570: Key characters for distinguishing males of Sri Lankan Isometrus and Reddyanus. Figures 555–556, 56
66. Pedipalp chela dorsal view, I. maculatus from locality 15CP (555), I. thwaitesi from locality 15CH (556), R. basilicu
olotype (565), and R. besucheti, holotype (566). Figures 557–558. Sternopectinal area, I. maculatus from locality 15CP (557
nd I. thwaitesi from locality 15CH (558). Figures 559–560, 567–570. Sternite V, I. maculatus from locality 15CP (559),
waitesi from locality 15CH (560), R. besucheti, from locality 15CF (567), R. ceylonensis sp. n., holotype (568), R. jayarathn
h
p. n., paratype (569), and R. ranawanai sp. n., paratype (570). Figures 561–564. Metasomal segment II lateral (561–562) a
edipalp patella dorsal (563–564), R. loebli from locality 15CG (561, 563) and R. besucheti, from locality 15CF (562, 564). Figures 555–570: Key characters for distinguishing males of Sri Lankan Isometrus and Reddyanus. Figures 555–556, 565–
566. Pedipalp chela dorsal view, I. maculatus from locality 15CP (555), I. thwaitesi from locality 15CH (556), R. basilicus,
holotype (565), and R. besucheti, holotype (566). Figures 557–558. Sternopectinal area, I. maculatus from locality 15CP (557),
and I. thwaitesi from locality 15CH (558). Figures 559–560, 567–570. Sternite V, I. Hemispermatophores of the Buthids maculatus from locality 15CP (559), I. waitesi from locality 15CH (560), R. besucheti, from locality 15CF (567), R. ceylonensis sp. n., holotype (568), R. jayarathnei
th
sp. n., paratype (569), and R. ranawanai sp. n., paratype (570). Figures 561–564. Metasomal segment II lateral (561–562) and
pedipalp patella dorsal (563–564), R. loebli from locality 15CG (561, 563) and R. besucheti, from locality 15CF (562, 564). Kovařík et al.: Scorpions of Sri Lanka
115
Figures 571–574: Key characters for distinguishing Sri Lankan Heterometrus. Figures 571–572. Pedipalp chela dorsal view of
male, Heterometrus serratus from locality 15CP (571) and Heterometrus gravimanus from locality 15CE (572). Figures 573–
574. Metasomal segments IV-II lateral view of female, Heterometrus indus from locality 15CA (573) and Heterometrus serratus
from locality 15CP (574). Kovařík et al.: Scorpions of Sri Lanka Kovařík et al.: Scorpions of Sri Lanka 115 Figures 571–574: Key characters for distinguishing Sri Lankan Heterometrus. Figures 571–572. Pedipalp chela dorsal view of
male, Heterometrus serratus from locality 15CP (571) and Heterometrus gravimanus from locality 15CE (572). Figures 573–
574. Metasomal segments IV-II lateral view of female, Heterometrus indus from locality 15CA (573) and Heterometrus serratus
from locality 15CP (574). Locket, 1990; Lourenço et. al., 2006; Maury, 1969,
1970, 1974; Ojanguren-Affilastro, 2005; Prendini et. al.,
2009; Probst, 1972; Stockwell, 1989; Teruel & Armas,
2012). Here we document this lobe configuration also in
the non-Buthus group genera, Buthoscorpio, Charmus,
Lychas and Reddyanus stat. n. genera Androctonus, Apistobuthus, Buthacus, Buthiscus,
Buthus, Cicileus, Compsobuthus, Femtobuthus, Gint,
Hottentotta, Leiurus, Lissothus, Mesobuthus, Micro-
buthus, Neobuthus, Odontobuthus, Orthochirus, Pico-
buthus and Vachoniolus (Kovařík & Lowe, 2012;
Kovařík, Lowe et. al., 2013; Levy & Amitai, 1980;
Lowe, 2009, 2010a, 2010b, 2010c; Lowe et. al., 2014;
Navidpour & Lowe, 2009; Vachon, 1952a, 1952b, 1958;
Vachon & Stockmann, 1968). Their capsule region has a
stereotypic 4-lobed configuration, in which the sperm
hemi-duct is composed of 3 lobes (i.e. external lobe,
carinated median lobe, and internal lobe), and a single
basal lobe arises dorsally near the base of the median
agellum is well separated from these
ll Buthus group members, suggesting that it is a synap-
morphy for the group. lobe carina. The fl
lobes. Hemispermatophores of the Buthids So far, this '3+1' configuration has been found in
a
o y
y
Following Stockwell (1989), we have proposed that
the carinated lobe of buthids is homologous to and
derived from the carinated lobe of the chaerilid hemi-
spermatophore (Kovařík, Teruel & Lowe, 2016). In the
'Buthus' group, this lobe sits between two other lobes
with more external and more internal positions, and
hence was termed the 'median lobe' (Vachon, 1952). Taking the carina as a conserved landmark, we hypo-
inated lobe in non-Buthus group genera that is fused to
the base of the flagellum. We further hypothesize that
he fu
morphic because it approximates
the ch
n in which the carinated lobe is
o
e distal lamina. Some excep-
tions to the fused configuration are seen in the genera
An
, Grosp
Uroplectes (Lam-
or
; Ko
Lowe et. al., 2015,
0
stro,
on, 1950, 1969),
flagellum appea
separated from the
a
dian lobe. How
in contrast to the
u
rinate
al lobe is not inter-
o
een the flagellum and the median lobe. This
r
present
rsor to the Buthus
n. thesize that the median lobe is homologous to the car-
t
j
2
in
c
B
p
a
gr
sed state is plesio
aerilid conditio
ined continuously to th
anteris, Babycurus
hus and
al, 1979; Lowe, 2000
vařík,
16; Ojanguren-Affila
2005;Vach
which the
rinated me
rs well
ever,
thus group, a non-ca
d extern
lated betw
rangement could re
oup configuratio
a precu Hemispermatophores of n
s group genera
including most of the Sri Lankan uthids (except Hot-
tentotta tamulus
s. In th
majority of genera, the base of the flagellum is broadly
fused to a large carinated lobe, and there may be one or
more additional
ternal side. A
basal lobe is us
s size and shap
varies considerab
has been des
cribed in the gen
buthus, Butheo
loides, Centruro
ilychas, Isomet
roides, Isometrus
tyus an
Zabius (Botero-T
aza, 2011; Franck
Stockwell, 198
roller, 1968; Koch
owe, 2016; Lamoral, 1979; Lenarducci et. al., 2005;
on-Buthu
,
b
), are more heterogeneou
e
distinct lobes on the in
ually present, and it
e
ly. Key to Scorpions of Sri Lanka 1. Pedipalp patella without ventral trichobothria. .......... Buthidae C. L. Koch, 1837 ..……………………......... 3
– Pedipalp patella with three ventral trichobothria. ...... 2 1. Pedipalp patella without ventral trichobothria. .......... Buthidae C. L. Koch, 1837 ..……………………......... 3
– Pedipalp patella with three ventral trichobothria. ...... 2 Euscorpius — 2016, No. 220 116 ensis (Fig. 550) (both with one extra larger pair of
chromosomes) and Isometrus thwaitesi (Fig. 549) (with
one extra shorter pair of chromosomes). Moreover,
heterozygous chromosomal rearrangements may also
explain odd diploid numbers of chromosomes in kar-
yotypes of Isometrus thwaitesi (Fig. 549), Reddyanus
loebli (Fig. 554) and one male of Reddyanus basilicus
(Fig. 552). In Reddyanus basilicus we found 2n=16 in a
male from locality 15CS. In this case the chromosomes
gradually decrease in size (Fig. 551, Table 6). However,
the male of R. basilicus from locality 15CR has 2n=15,
including one extra large chromosome (Fig. 552, Table
6). The intraspecific variability has also been docu-
mented in another 8 species from different genera in the
family Buthidae (see Schneider et al., 2016). Due to the
intraspecific variability and high similarity of basic
cytogenetic characteristics (such as number and size of
chromosomes) it seems difficult to apply standard cyto-
genetic techniques to the taxonomy of buthid scorpions. In the future, application of more refined molecular
cytogenetic techniques should lead to a better under-
standing of the organization of genome, which may be
the key to detecting specific differences between closely
related species. metrus, Lychas and Reddyanus stat. n.) exhibit a basic
1+1 lobe configuration (fused median lobe + basal lobe),
without additional internal lobes developed. This con-
figuration is shared with a number of other non-Buthus
group genera that have been reported, i.e. some species
of Ananteris (Botero-Trujillo & Florez-Daza, 2011;
Ojanguren-Affilastro, 2005) and Tityus (Kovařík, Ter-
uel, et. al., 2015; Ojanguren-Affilastro, 2005); and
Australobuthus, Hemilychas, Isometroides and Isomet-
rus (Gysin & Le Coroller, 1968; Locket, 1990; Probst,
1972). This may be a plesiomorphic state from which
additional internal or external lobes have arisen inde-
pendently in different lineages. Even under a basic 1+1
configuration, we observe hemispermatophore differ-
ences that are diagnostic at the generic level, e.g. enabling us to differentiate Reddyanus from Isometrus
by the size of the basal lobe, and the length and shape of
the flagellum. Two novel, unique hemispermatophore
morphologies that we found are: (i) a highly elongated
capsule in Buthoscorpio, with a long narrow median
lobe fused to the base of the flagellum, and a round,
blunt basal lobe; and (ii) a short capsule in Charmus,
with a truncated median lobe, and a bulging, bilobate
basal lobe. Euscorpius — 2016, No. 220 These findings reveal an unexplored diversity
in buthid hemispermatophores, that could provide new
characters for analyzing phylogenetic relationships in
this large and ancient family. Hemispermatophores of the Buthids This configuration
era Ananteris, Australo
-
-
ides, Chaneke, Hem
-
, Parabuthus, Rhopalurus, Ti
d
rujillo & Florez-D
7; Gysin & Le Co
e
,
&
1977; Kovařík, Teruel, et al., 2015; Kovařík, Teruel &
L p
genera so far examined (Buthoscorpio, Charmus, Iso-
All Sri Lankan representatives of non-Buthus grou Cytogenetic Data on Sri Lankan Buthid
Scorpions Buthidae C. L. Koch, 1837 ..……………………......... 3
– Pedipalp patella with three ventral trichobothria. ...... 2
2. Adults 27–45 mm long. Pedipalp femur with 9 tri-
chobothria, of which 4 are dorsal. ……....... Chaerilidae
Pocock, 1893, …... Chaerilus ceylonensis Pocock, 1894
– Adults 75–176 mm long. Pedipalp femur with 3 or 4
trichobothria, of which only one is dorsal ..................... Scorpionidae Latreille, 1802 ....…………………...... 15
3. Legs III and IV with well developed long tibial spurs
(Figs. 193–195, 198) .....……………………................ 4
– Legs III and IV without tibial spurs (Figs. 196–197,
199) .......…………………............................................. 8
4. Telson with a distinct subaculear tooth (Figs. 407–
408). ..........…........ Lychas srilankensis Lourenço, 1997
– Telson without subaculear tooth (Fig. 421–427). ....... 5 Altogether we analyzed seven buthid species (Figs. 547–554, Table 6) using standard cytogenetic methods
(e.g. Kovařík et al., 2009; Šťáhlavský et al., 2014). The
karyotype characteristics of all analyzed species cor-
respond to the cytogenetic attributes typical for the fam-
ily Buthidae. Chromosomes do not have visible centro-
y known
the family Buthidae within scorpions (e.g. Mattos et
diploid numbers of chro-
somes l
mere regions which is a typical feature for holocentric
chromosomes. This type of organization is onl
in
al., 2013). This family is also characterized by low num-
bers of chromosomes. Half of the cytogenetically inves-
tigated species show diploid numbers from 14 to 24
(Schneider et al., 2016). In view of this fact, Charmus
laneus (2n=9) (Fig. 548) and Isometrus thwaitesi (2n=8)
(Fig. 549) represent exceptions with very low number of
chromosomes. Only three known species from the genus
ityus C. L. Koch, 1836 have
T
mo
ower than 10 (see Schneider et al., 2016). This phenomenon has been documented in T. bahiensis
(Perty, 1834) and was explained as an effect of intensive
fusion of holocentric chromosomes (Schneider et al.,
2009). This type of chromosomal rearrangement may
also explain the differences in chromosome size within
uthoscorpio sarasinorum (Fig. 547), Lychas srilank-
B 2. Adults 27–45 mm long. Pedipalp femur with 9 tri-
chobothria, of which 4 are dorsal. ……....... Chaerilidae
Pocock, 1893, …... Chaerilus ceylonensis Pocock, 1894
– Adults 75–176 mm long. Pedipalp femur with 3 or 4
trichobothria, of which only one is dorsal ..................... Scorpionidae Latreille, 1802 ....…………………...... 15 4. Telson with a distinct subaculear tooth (Figs. 407–
408). ..........…........ Lychas srilankensis Lourenço, 1997
– Telson without subaculear tooth (Fig. 421–427). ....... 5. Metasomal segments IV–V punctate without deve-
loped carinae (Figs. 24–29). Dentate margin of pedipalp
chela movable finger with distinct granules divided into
8–11 linear rows, apical rows of 3–6 granules, and 3
terminal granules (Figs. 39–44). ...……......................... 6 Kovařík et al.: Scorpions of Sri Lanka Glabrous zone present in middle part of fifth
sternite only (Fig. 569). ....……..... R. jayarathnei sp. n. – Mesosoma lighter, yellowish brown (Figs. 382–383). Glabrous zone stretches almost over whole posterior
margin of fifth sternite (Fig. 570).... R. ranawanai sp. n. 14. Ratio metasomal segment II length/width 1.56–1.79
in male (Fig. 211, Table 5). ............................…........... ………...………. R. besucheti (Vachon, 1982) comb. n. – Ratio metasomal segment II length/width 1.85–1.97 in
male (Fig. 213, Table 5). ……….... R. ceylonensis sp. n. 8. Chelal trichobothrium db located between dt and et. Fixed fingers of pedipalps with six rows of granules and
six external and internal granules (Figs. 252–253). Tarsomeres II of leg IV with two rows of dense setae
(Figs. 196–197). ........ Isometrus Ehrenberg, 1828 ….. 9
– Chelal trichobothrium db located between et and est
(Fig. 321). Fixed fingers of pedipalps with seven rows of
granules and six external and seven internal granules
(Figs. 254–259). Tarsomeres II of leg IV with two rows
of no more than 20 spiniform setae (Figs. 199, 201–
208). ............ Reddyanus Vachon, 1972 stat. n. ….... 10 15. Adults 128–176 mm long. Pectinal teeth number 16–
20. Fifth segment of metasoma longer than pedipalp
femur, fourth segment of metasoma about as long as
pedipalp femur ..…........ H. swammerdami Simon, 1872
– Adults 75–130 mm long. Pectinal teeth number 10–16. Fifth segment of metasoma about as long as pedipalp
femur, fourth segment of metasoma shorter than ped-
ipalp femur. .....…………………………………….... 16 9. First (basal) middle lamella of pectine in both sexes
rounded (Fig. 558). Fingers and manus of pedipalp chela
the same color, spotted (Fig. 556). Posterior margin of
sternite V strongly convex medially (Fig. 560) …….. ….....………................……… I. thwaitesi Pocock, 1897 First (basal) middle lamella of pectine in both sexes
margin
–
quadrangular (Fig. 557). Manus of pedipalp yellow with
several spots, fingers dark (Fig. 555). Posterior
of sternite V almost straight to very slightly convex
medially (Fig. 559). …..... I. maculatus (De Geer, 1778) ur mostly yellow. aculear tooth spinoid (Figs. 417–418). ..………...... bli (V
h
1982)
b
10. Terminal tubercle of dorsal carina on second and
third metasomal segment of male markedly enlarged
(Fig. 561). Pedipalp femur and patella spotted, patella
mostly black (Fig. 563), fem
Sub
R l Cytogenetic Data on Sri Lankan Buthid
Scorpions 5 – Metasomal segments IV–V not punctate with well
developed carinae (Figs. 120–121). Dentate margin of
edipalp chela movable finger with distinct granules
p 117 Kovařík et al.: Scorpions of Sri Lanka divided into 13–15 linear rows and 5–6 terminal gran-
ules (Fig. 46). ..... Hottentotta tamulus (Fabricius, 1798)
6. Adults 25–52 mm long. Pedipalps, metasoma and tel-
son glabrous (Figs. 24–29). ..………….......................... ……...…….. Buthoscorpio sarasinorum (Karsch, 1892)
– Adults 12–25 mm long. Pedipalps, metasoma and tel-
son densely hirsute (Figs. 71–73). .……………............ 7
7. Pedipalp patella yellowish with several black spots
(Figs. 118–119); metasomal segment V length/ width
ratio is 1.28–1.43 in female (Fig. 83); pedipalp chela
length/ fixed finger length ratio is 1.69–1.79 in female
(Figs. 42–43). .……….... Charmus laneus Karsch, 1879
– Pedipalp patella black with several little yellow spots
(Figs. 111, 116); metasomal segment length/ width ratio
is 1.80 in female (Fig. 84); pedipalp chela length/ fixed
finger length ratio is 1.45 in female (Fig. 44). .............. ……………………………..... Charmus saradieli sp. n. 8. Chelal trichobothrium db located between dt and et. Fixed fingers of pedipalps with six rows of granules and
six external and internal granules (Figs. 252–253). Tarsomeres II of leg IV with two rows of dense setae
(Figs. 196–197). ........ Isometrus Ehrenberg, 1828 ….. 9
– Chelal trichobothrium db located between et and est
(Fig. 321). Fixed fingers of pedipalps with seven rows of
granules and six external and seven internal granules
(Figs. 254–259). Tarsomeres II of leg IV with two rows
of no more than 20 spiniform setae (Figs. 199, 201–
208). ............ Reddyanus Vachon, 1972 stat. n. ….... 10
9. First (basal) middle lamella of pectine in both sexes
rounded (Fig. 558). Fingers and manus of pedipalp chela
the same color, spotted (Fig. 556). Posterior margin of
First (basal) middle lamella of pectine in both sexes
margin
ur mostly yellow. aculear tooth spinoid (Figs. 417–418). ..………...... bli (Vachon, 1982) comb. n. Ter
omal
segme
palps
i h b
d
ll
(Fi
11. Glabrous zone on posterior part of fifth sternite
present medially in male (Figs. 569–570). ………….. 12
– Glabrous zone along posterior margin of fifth sternite
absent or indicated (R. besucheti) (Figs. 567–568). .... 13
12. Whole mesosoma dark, almost black (Figs. 338–
339). Glabrous zone present in middle part of fifth
sternite only (Fig. 569). ....……..... R. jayarathnei sp. n. – Mesosoma lighter, yellowish brown (Figs. 382–383). Glabrous zone stretches almost over whole posterior
margin of fifth sternite (Fig. 570).... R. ranawanai sp. n. 13. Manus of pedipalp chela wide in male. Ratio
pedipalp chela length/width 2.94–3.28 (Fig. 566, Table
5). Kovařík et al.: Scorpions of Sri Lanka Chela wider in male than in female. .......…........... 14
– Manus of pedipalp chela narrow in male. Ratio
pedipalp chela length/width 3.41–3.79 (Fig. 565, Table
5). Chela the same width in both sexes. .................... ………......……… R. basilicus (Karsch, 1879) comb. n. 14. Ratio metasomal segment II length/width 1.56–1.79
in male (Fig. 211, Table 5). ............................…........... ………...………. R. besucheti (Vachon, 1982) comb. n. – Ratio metasomal segment II length/width 1.85–1.97 in
male (Fig. 213, Table 5). ……….... R. ceylonensis sp. n. 15. Adults 128–176 mm long. Pectinal teeth number 16–
20. Fifth segment of metasoma longer than pedipalp
femur, fourth segment of metasoma about as long as
pedipalp femur ..…........ H. swammerdami Simon, 1872
– Adults 75–130 mm long. Pectinal teeth number 10–16. Fifth segment of metasoma about as long as pedipalp
femur, fourth segment of metasoma shorter than ped-
ipalp femur. .....…………………………………….... 16
......... H. gravimanus (Pocock, 1894)
– Pedipalp chela without carinae on dorsoexternal sur-
face
17. Dorsal and dorsolateral carinae of metasomal seg-
– D
Ack
ndra, Suraj Goonewardene, Chamalka Halyala,
jith Hemachandra, Sanjaya Karunarathne, P. Rajku-
sternite V strongly convex medially (Fig. 560) …….. ….....………................……… I. thwaitesi Pocock, 1897
16. Pedipalp chela with carinae on dorsoexternal surface
(Fig. 572). .………
–
quadrangular (Fig. 557). Manus of pedipalp yellow with
several spots, fingers dark (Fig. 555). Posterior
of sternite V almost straight to very slightly convex
medially (Fig. 559). …..... I. maculatus (De Geer, 1778)
10. Terminal tubercle of dorsal carina on second and
third metasomal segment of male markedly enlarged
(Fig. 561). Pedipalp femur and patella spotted, patella
mostly black (Fig. 563), fem
Sub
…………………….. R. loe
–
minal tubercle of dorsal carina on metas
nts of male
. 562). Pedi
are not enlarged (Fig
id
i
l
f
(Fig. 571). .......…………..................................... 17
ments smooth (Fig. 573). ….. H. indus (De Geer, 1778)
orsal and dorsolateral carinae of metasomal segments
granulated (Fig. 574). ..….... H. serratus (Pocock, 1900)
nowledgments
Thanks are due to Dilan Chathuranga, Paramuditha
Devasure
A
mar, A. N. Thudugala, Sewwandi Wijesooriya and a lot
f l
l
l
f
S i
k
h
i i
d
d 11. Glabrous zone on posterior part of fifth sternite
present medially in male (Figs. 569–570). ………….. 12
– Glabrous zone along posterior margin of fifth sternite
absent or indicated (R. besucheti) (Figs. 567–568). .... 13 12. Whole mesosoma dark, almost black (Figs. 338–
339). Acknowledgments ndra, Suraj Goonewardene, Chamalka Halyala,
jith Hemachandra, Sanjaya Karunarathne, P. Rajku-
Thanks are due to Dilan Chathuranga, Paramuditha
Devasure
A
mar, A. N. Thudugala, Sewwandi Wijesooriya and a lot
of local people from Sri Lanka who participated and
helped in the expedition to Sri Lanka; Victor Fet and
Michael Soleglad for their help in processing the manu- Ter
omal
segme
palps
with brown spots
nd patella (Fig. 416,
–
minal tubercle of dorsal carina on metas
nts of male
. 562). Pedi
are not enlarged (Fig
, identical on femur a
564). Subaculear tooth wide and rounded (Figs. 409–
419–420). ….............……................................... 11 11 Euscorpius — 2016, No. 220 118 Figures 575–576: Figure 575. Locality 15CA, Sri Lanka, Central Province, Kandy District, Hantana (Peradeniya), Universit
land, 07°14'54.7"N 080°36'54"E, 760 m a.s.l. Specimens of Heterometrus indus were collected along a road during a day unde
stones and under bark on the photo. A male was collected also in a kitchen inside a house. During night collecting on 19. 21.IV.2015 (UV detection), we recorded a lot of specimens around their burrows. On the locality we recorded night temperatur
25.6 ºC– 21.4 ºC (minimum temperature) and night humidity varied between 72% (evening) and 87% (morning). There also live
eterometrus swammerdami and Buthoscorpio sarasinorum (type locality). Figure 576. Locality 15CB, Sri Lanka, Centra
s thwaitesi. H
Province, Matale District, Maragamuwa/Kumaragala, 07°41'58.5"N 080°42'26.7"E, 662 m a.s.l. During a day (21.IV.2015
temperature 26.1 ºC– 33.2 ºC, humidity 63%–88%) we recorded Heterometrus swammerdami inside termite constructions. Ther
also lives Isometru Figures 575–576: Figure 575. Locality 15CA, Sri Lanka, Central Province, Kandy District, Hantana (Peradeniya), University
land, 07°14'54.7"N 080°36'54"E, 760 m a.s.l. Specimens of Heterometrus indus were collected along a road during a day under
stones and under bark on the photo. A male was collected also in a kitchen inside a house. During night collecting on 19.–
21.IV.2015 (UV detection), we recorded a lot of specimens around their burrows. On the locality we recorded night temperature
25.6 ºC– 21.4 ºC (minimum temperature) and night humidity varied between 72% (evening) and 87% (morning). There also lives
eterometrus swammerdami and Buthoscorpio sarasinorum (type locality). Figure 576. Locality 15CB, Sri Lanka, Central
s thwaitesi. H
Province, Matale District, Maragamuwa/Kumaragala, 07°41'58.5"N 080°42'26.7"E, 662 m a.s.l. During a day (21.IV.2015,
temperature 26.1 ºC– 33.2 ºC, humidity 63%–88%) we recorded Heterometrus swammerdami inside termite constructions. There
also lives Isometru Figures 575–576: Figure 575. Acknowledgments Locality 15CA, Sri Lanka, Central Province, Kandy District, Hantana (Peradeniya), University
land, 07°14'54.7"N 080°36'54"E, 760 m a.s.l. Specimens of Heterometrus indus were collected along a road during a day under
stones and under bark on the photo. A male was collected also in a kitchen inside a house. During night collecting on 19.–
21.IV.2015 (UV detection), we recorded a lot of specimens around their burrows. On the locality we recorded night temperature
25.6 ºC– 21.4 ºC (minimum temperature) and night humidity varied between 72% (evening) and 87% (morning). There also lives
eterometrus swammerdami and Buthoscorpio sarasinorum (type locality). Figure 576. Locality 15CB, Sri Lanka, Central
s thwaitesi. H
Province, Matale District, Maragamuwa/Kumaragala, 07°41'58.5"N 080°42'26.7"E, 662 m a.s.l. During a day (21.IV.2015,
temperature 26.1 ºC– 33.2 ºC, humidity 63%–88%) we recorded Heterometrus swammerdami inside termite constructions. There
also lives Isometru Kovařík et al.: Scorpions of Sri Lanka Kovařík et al.: Scorpions of Sri Lanka 119 y
Figures 577–578: Figure 577. Locality 15CC, Sri Lanka, Cen
Wattegama, 07°20'41.9"N 080°39'30.6"E, 534 m a.s.l.. We co
21.IV.2015 evening. There also lives Isometrus sp. Figure 578. L
District, ca 35 km from Dambula, 07°57'15.1"N 080°54'45,4"E, 1
Reddyanus besucheti (close to the type locality) and Chaerilus ce
tral Province, Kandy District, 20 km S Kandy, Meegammana,
llected a female of Heterometrus indus close to a house on
ocality 15CD, Sri Lanka, North Central Province, Polonnaruwa
32 m a.s.l. During the day 22.IV.2015 under stones we recorded
lonensis. y
Figures 577–578: Figure 577. Locality 15CC, Sri Lanka, Cen
Wattegama, 07°20'41.9"N 080°39'30.6"E, 534 m a.s.l.. We co
21.IV.2015 evening. There also lives Isometrus sp. Figure 578. L
District, ca 35 km from Dambula, 07°57'15.1"N 080°54'45,4"E, 1
Reddyanus besucheti (close to the type locality) and Chaerilus ce
tral Province, Kandy District, 20 km S Kandy, Meegammana,
llected a female of Heterometrus indus close to a house on
ocality 15CD, Sri Lanka, North Central Province, Polonnaruwa
32 m a.s.l. During the day 22.IV.2015 under stones we recorded
lonensis. Euscorpius — 2016, No. 220 120 Euscorpius — 2016, No. 220
120 Euscorpius — 2016, No. 220
120
Figures 579–581: Figures 579–580. Locality 15CE, Sri Lanka, North Central Province, Polonnaruwa District, Giritale
08°01'26.0"N 080°54'37.2"E, 233 m a.s.l. During night collecting on 22.–23.IV.2015 in the hotel garden (UV detection) w
corded Heterometrus swammerdami (inside the termite constructions, Fig. 580) and Heterometrus gravimanus inside burrow
re
n open terrain. Figures 579–581: Figures 579–580. Locality 15CE, Sri Lanka, North Central Province, Polonnaruwa District, Giritale,
08°01'26.0"N 080°54'37.2"E, 233 m a.s.l. During night collecting on 22.–23.IV.2015 in the hotel garden (UV detection) we
corded Heterometrus swammerdami (inside the termite constructions, Fig. 580) and Heterometrus gravimanus inside burrows
re
in open terrain. The mothers had 30 to 60 cm long oblique burrows usually once curved from which several narrow burrows
created by their juveniles separated under surface. Every burrow of juveniles has a proper exit 15 to 90 cm from the main exit of
the mother´s burrow. On the locality we recorded night temperature 32.8 ºC– 26.5 ºC (minimum temperature) and night humidity
varied between 51% and 79%. Figure 581. Locality 15CF, Sri Lanka, North Central Province, Polonnaruwa District, near
Kaudulla National Park, 08°08'40.6"N 080°51'04"E, 101 m a.s.l. During a day 23.IV.2015 we recorded Buthoscorpio
sarasinorum (under stones or woods), Isometrus thwaitesi (under dry bark of standing trees), Reddyanus besucheti (under
stones), and Chaerilus ceylonensis (under stones). Acknowledgments The mothers had 30 to 60 cm long oblique burrows usually once curved from which several narrow burrow
created by their juveniles separated under surface. Every burrow of juveniles has a proper exit 15 to 90 cm from the main exit o
the mother´s burrow. On the locality we recorded night temperature 32.8 ºC– 26.5 ºC (minimum temperature) and night humidit
varied between 51% and 79%. Figure 581. Locality 15CF, Sri Lanka, North Central Province, Polonnaruwa District, nea
Kaudulla National Park, 08°08'40.6"N 080°51'04"E, 101 m a.s.l. During a day 23.IV.2015 we recorded Buthoscorpi
sarasinorum (under stones or woods), Isometrus thwaitesi (under dry bark of standing trees), Reddyanus besucheti (unde
)
d
h
l
l
(
d
) Figures 579–581: Figures 579–580. Locality 15CE, Sri Lanka, North Central Province, Polonnaruwa District, Giritale,
08°01'26.0"N 080°54'37.2"E, 233 m a.s.l. During night collecting on 22.–23.IV.2015 in the hotel garden (UV detection) we
corded Heterometrus swammerdami (inside the termite constructions, Fig. 580) and Heterometrus gravimanus inside burrows
re
in open terrain. The mothers had 30 to 60 cm long oblique burrows usually once curved from which several narrow burrows
created by their juveniles separated under surface. Every burrow of juveniles has a proper exit 15 to 90 cm from the main exit of
the mother´s burrow. On the locality we recorded night temperature 32.8 ºC– 26.5 ºC (minimum temperature) and night humidity
varied between 51% and 79%. Figure 581. Locality 15CF, Sri Lanka, North Central Province, Polonnaruwa District, near
Kaudulla National Park, 08°08'40.6"N 080°51'04"E, 101 m a.s.l. During a day 23.IV.2015 we recorded Buthoscorpio
sarasinorum (under stones or woods), Isometrus thwaitesi (under dry bark of standing trees), Reddyanus besucheti (under
stones), and Chaerilus ceylonensis (under stones). Kovařík et al.: Scorpions of Sri Lanka 121 t
k
i
dhu Road, 08°48'26.3"N 080°10'26"E,
tion)
g
Figures 582–584: Figures 582–583. Locality 15CG, Sri Lank
Resort, 07°59'25.8"N 080°43'24.6"E, 280 m a.s.l. During nigh
(UV detection) we recorded Buthoscorpio sarasinorum (running
among leaves) and Reddyanus loebli (hidden under scales of bar
On the locality we recorded night temperature 28.4 ºC– 24 ºC (m
and 87%. Figure 584. Locality 15CH, Sri Lanka, Northern Prov
90 m a.s.l. Acknowledgments During night collecting on 24.–25.IV.2015 (UV detec
sympatrically (running on branches and trunks of trees also sitin
a, Central Province, Matale District, Habarana, Wananiwahana
collecting on 23.–24.IV.2015 in Wananiwahana resort garden
on the land among leaves), Reddyanus besucheti (on the land
s of standing tree trunks, see the blue arrows on the Fig. 583). inimum temperature) and night humidity varied between 55%
nce, Mannar District, Ma
we recorded Isometrus maculatus and Isometrus thwaitesii
on leaves one to four metres high) Reddyanus ceylonensis sp t
k
i
dhu Road, 08°48'26.3"N 080°10'26"E,
tion)
g
a
Figures 582–584: Figures 582–583. Locality 15CG, Sri Lank
Resort, 07°59'25.8"N 080°43'24.6"E, 280 m a.s.l. During nigh
(UV detection) we recorded Buthoscorpio sarasinorum (running
among leaves) and Reddyanus loebli (hidden under scales of bar
On the locality we recorded night temperature 28.4 ºC– 24 ºC (m
and 87%. Figure 584. Locality 15CH, Sri Lanka, Northern Prov
90 m a.s.l. During night collecting on 24.–25.IV.2015 (UV detec
sympatrically (running on branches and trunks of trees, also sitin
n. (on the land among leaves), Reddyanus loebli (hidden under sc
a, Central Province, Matale District, Habarana, Wananiwahana
collecting on 23.–24.IV.2015 in Wananiwahana resort garden
on the land among leaves), Reddyanus besucheti (on the land
s of standing tree trunks, see the blue arrows on the Fig. 583). inimum temperature) and night humidity varied between 55%
nce, Mannar District, Ma
we recorded Isometrus maculatus and Isometrus thwaitesii
on leaves one to four metres high), Reddyanus ceylonensis sp. les of barks of standing tree trunks). t
k
i
dhu Road, 08°48'26.3"N 080°10'26"E,
tion)
g
a
Figures 582–584: Figures 582–583. Locality 15CG, Sri Lank
Resort, 07°59'25.8"N 080°43'24.6"E, 280 m a.s.l. During nigh
(UV detection) we recorded Buthoscorpio sarasinorum (running
among leaves) and Reddyanus loebli (hidden under scales of bar
On the locality we recorded night temperature 28.4 ºC– 24 ºC (m
and 87%. Figure 584. Locality 15CH, Sri Lanka, Northern Prov
90 m a.s.l. During night collecting on 24.–25.IV.2015 (UV detec
sympatrically (running on branches and trunks of trees, also sitin
n. (on the land among leaves), Reddyanus loebli (hidden under sc
a, Central Province, Matale District, Habarana, Wananiwahana
collecting on 23.–24.IV.2015 in Wananiwahana resort garden
on the land among leaves), Reddyanus besucheti (on the land
s of standing tree trunks, see the blue arrows on the Fig. 583). Acknowledgments Locality 15CJ, Sri Lanka, Northern Pro
During night collecting on 26.–27.IV.2015 (UV detection) we re
leaves or in their vicinity), Heterometrus swammerdami (inside
Heterometrus gravimanus inside burrows in open terrain (Fig. 58
twice curved. A female shortly after maturity ecdysis was in the
together with three juveniles which had their own narrow burrow
after the third ecdysis had a proper burrow 30 cm long oblique
locality we recorded night temperature 31.6 ºC– 27.2 ºC (mini
h
D
i
; the type locality), Reddyanus loebli
he
u
c
t
a
m
87%. ern Province, Mannar District, Marichchukkaddi env, border of
uring night collecting on 25.–26.IV.2015 (UV detection) we
cally (running on branches and trunks of trees, also sitting on
on the land among leaves
locality we recorded temperature between 37.6 ºC (maximum
re) shortly before sunrise. Humidity varied between 67% and
vince, Jaffna District, 09°42'51.6"N 080°04'44.8"E, 19 m a.s.l. orded Lychas srilankensis (on the land usually under branches,
he termite constructions or inside heaps of garden "waste") and
7). The females had 30 to 60 cm long oblique burrows once or
burrow together with a male; another female was in the burrow
s separated under surface from the mother´s burrow; a juvenile
nd once curved. There also lives Isometrus maculatus. On the
um temperature) and night humidity varied between 60% and Figures 585–587: Figure 585. Locality 15CI, Sri Lanka, Nort
Wilpattu National Park, 08°33'32.3"N 079°56'51"E, 7 m a.s.l. recorded Isometrus maculatus and Isometrus thwaitesii sympatr
leaves one to four metres high), Reddyanus ceylonensis sp. n. (
(hidden under scales of bark of standing tree trunks). Near to t
daytime temperature and 26.2 ºC (minimum nighttime temperat
81%. Figures 586–587. Locality 15CJ, Sri Lanka, Northern Pro
During night collecting on 26.–27.IV.2015 (UV detection) we re
leaves or in their vicinity), Heterometrus swammerdami (inside
Heterometrus gravimanus inside burrows in open terrain (Fig. 58
twice curved. A female shortly after maturity ecdysis was in the
together with three juveniles which had their own narrow burrow
after the third ecdysis had a proper burrow 30 cm long oblique
locality we recorded night temperature 31.6 ºC– 27.2 ºC (mini
h
D
i
; the type locality), Reddyanus loebli
he
u
c
t
a
m
87%. Acknowledgments inimum temperature) and night humidity varied between 55%
nce, Mannar District, Ma
we recorded Isometrus maculatus and Isometrus thwaitesii
on leaves one to four metres high), Reddyanus ceylonensis sp. les of barks of standing tree trunks). 122 Euscorpius — 2016, No. 220 Figures 585–587: Figure 585. Locality 15CI, Sri Lanka, Nort
Wilpattu National Park, 08°33'32.3"N 079°56'51"E, 7 m a.s.l. recorded Isometrus maculatus and Isometrus thwaitesii sympatr
leaves one to four metres high), Reddyanus ceylonensis sp. n. (
(hidden under scales of bark of standing tree trunks). Near to t
daytime temperature and 26.2 ºC (minimum nighttime temperat
81%. Figures 586–587. Locality 15CJ, Sri Lanka, Northern Pro
During night collecting on 26.–27.IV.2015 (UV detection) we re
leaves or in their vicinity), Heterometrus swammerdami (inside
Heterometrus gravimanus inside burrows in open terrain (Fig. 58
twice curved. A female shortly after maturity ecdysis was in the
together with three juveniles which had their own narrow burrow
after the third ecdysis had a proper burrow 30 cm long oblique
locality we recorded night temperature 31.6 ºC– 27.2 ºC (mini
h
D
i
; the type locality), Reddyanus loebli
he
u
c
t
a
m
87%. ern Province, Mannar District, Marichchukkaddi env, border of
uring night collecting on 25.–26.IV.2015 (UV detection) we
cally (running on branches and trunks of trees, also sitting on
on the land among leaves
locality we recorded temperature between 37.6 ºC (maximum
re) shortly before sunrise. Humidity varied between 67% and
vince, Jaffna District, 09°42'51.6"N 080°04'44.8"E, 19 m a.s.l. orded Lychas srilankensis (on the land usually under branches,
he termite constructions or inside heaps of garden "waste") and
7). The females had 30 to 60 cm long oblique burrows once or
burrow together with a male; another female was in the burrow
s separated under surface from the mother´s burrow; a juvenile
nd once curved. There also lives Isometrus maculatus. On the
um temperature) and night humidity varied between 60% and Figures 585–587: Figure 585. Locality 15CI, Sri Lanka, Nort
Wilpattu National Park, 08°33'32.3"N 079°56'51"E, 7 m a.s.l. recorded Isometrus maculatus and Isometrus thwaitesii sympatr
leaves one to four metres high), Reddyanus ceylonensis sp. n. (
(hidden under scales of bark of standing tree trunks). Near to t
daytime temperature and 26.2 ºC (minimum nighttime temperat
81%. Figures 586–587. Figures 585–587: Figure 585. Locality 15CI, Sri Lanka, Nort
Wilpattu National Park, 08°33'32.3"N 079°56'51"E, 7 m a.s.l.
recorded Isometrus maculatus and Isometrus thwaitesii sympatr
leaves one to four metres high), Reddyanus ceylonensis sp. n. (
(hidden under scales of bark of standing tree trunks). Near to t
daytime temperature and 26.2 ºC (minimum nighttime temperat
81%. Figures 586–587. Locality 15CJ, Sri Lanka, Northern Pro
During night collecting on 26.–27.IV.2015 (UV detection) we re
leaves or in their vicinity), Heterometrus swammerdami (inside
Heterometrus gravimanus inside burrows in open terrain (Fig. 58
twice curved. A female shortly after maturity ecdysis was in the
together with three juveniles which had their own narrow burrow
after the third ecdysis had a proper burrow 30 cm long oblique
locality we recorded night temperature 31.6 ºC– 27.2 ºC (mini
h
D
i
; the type locality), Reddyanus loebli
he
u
c
t
a
m
87%.
ern Province, Mannar District, Marichchukkaddi env, border of
uring night collecting on 25.–26.IV.2015 (UV detection) we
cally (running on branches and trunks of trees, also sitting on
on the land among leaves
locality we recorded temperature between 37.6 ºC (maximum
re) shortly before sunrise. Humidity varied between 67% and
vince, Jaffna District, 09°42'51.6"N 080°04'44.8"E, 19 m a.s.l.
orded Lychas srilankensis (on the land usually under branches,
he termite constructions or inside heaps of garden "waste") and
7). The females had 30 to 60 cm long oblique burrows once or
burrow together with a male; another female was in the burrow
s separated under surface from the mother´s burrow; a juvenile
nd once curved. There also lives Isometrus maculatus. On the
um temperature) and night humidity varied between 60% and Acknowledgments ern Province, Mannar District, Marichchukkaddi env, border of
uring night collecting on 25.–26.IV.2015 (UV detection) we
cally (running on branches and trunks of trees, also sitting on
on the land among leaves
locality we recorded temperature between 37.6 ºC (maximum
re) shortly before sunrise. Humidity varied between 67% and
vince, Jaffna District, 09°42'51.6"N 080°04'44.8"E, 19 m a.s.l. orded Lychas srilankensis (on the land usually under branches,
he termite constructions or inside heaps of garden "waste") and
7). The females had 30 to 60 cm long oblique burrows once or
burrow together with a male; another female was in the burrow
s separated under surface from the mother´s burrow; a juvenile
nd once curved. There also lives Isometrus maculatus. On the
um temperature) and night humidity varied between 60% and Kovařík et al.: Scorpions of Sri Lanka 123 s
h
n
th
i
7.4 ºC– 24.5 ºC (minimum temperature) and night humidity varied between 64% and 87%. Figures 588–589: Figure 588. Locality 15CK, Sri Lanka, Nor
m a.s.l. During day 27.IV.2015 we recorded Hottentotta tamulu
589. Locality 15CL, Sri Lanka, North Central Province, Anurad
a.s.l. During night collecting on 27.–28.IV.2015 (UV detectio
trunks of trees, also sitting on leaves), Reddyanus besucheti (on
f bark of standing tree trunks) and Heterometrus swammerdam
thern Province, Jaffna District, 09°49'15.4"N 080°08'41.6"E, 19
under stones or woods and also in vicinity of houses. Figure
apura District, Mihintale, 08°20'51.8"N 080°30'27.7"E, 156 m
) we recorded Isometrus thwaitesii (running on branches and
e ground among leaves), Reddyanus loebli (hidden under scales
(inside burrows). On the locality we recorded night temperature
o
2 s
h
n
th
i
7.4 ºC– 24.5 ºC (minimum temperature) and night humidity varied between 64% and 87%. Figures 588–589: Figure 588. Locality 15CK, Sri Lanka, Nor
m a.s.l. During day 27.IV.2015 we recorded Hottentotta tamulu
589. Locality 15CL, Sri Lanka, North Central Province, Anurad
a.s.l. During night collecting on 27.–28.IV.2015 (UV detectio
trunks of trees, also sitting on leaves), Reddyanus besucheti (on
f bark of standing tree trunks) and Heterometrus swammerdam
thern Province, Jaffna District, 09°49'15.4"N 080°08'41.6"E, 19
under stones or woods and also in vicinity of houses. Figure
apura District, Mihintale, 08°20'51.8"N 080°30'27.7"E, 156 m
) we recorded Isometrus thwaitesii (running on branches and
e ground among leaves), Reddyanus loebli (hidden under scales
(inside burrows). On the locality we recorded night temperature
o
2 Euscorpius — 2016, No. Acknowledgments 220 124 Figures 590–591: Figure 590. Locality 15CM, Sri Lanka, Central Province, Matale District, Pallegama, 07°32'49.5"N
080°47'50"E, 434 m a.s.l. During a day 28.IV.2015 we recorded Reddyanus loebli (under bark). Figure 591. Locality 15CN, Sri
Lanka, North Central Province, Puttalam District, Eluwankulam, 08°12'35.1"N 079°51'32"E, 52 m a.s.l. During night collecting
on 28.IV.2015 (UV detection) between 9.00 and 10.00 p.m. we recorded Charmus laneus (running on the ground among leaves),
Lychas srilankensis (on the ground under branches or leaves or in their vicinity), Reddyanus ceylonensis sp. n. (on the ground
and inside old moulder stump), Reddyanus loebli (hidden under scales of bark of standing tree trunks) and Heterometrus
swammerdami (inside burrows). On the locality we recorded between 9.00 and 10.00 p.m. temperature 28.5 ºC– 25.9 ºC and
humidity between 61% and 78%. Figures 590–591: Figure 590. Locality 15CM, Sri Lanka, Central Province, Matale District, Pallegama, 07°32'49.5"N
080°47'50"E, 434 m a.s.l. During a day 28.IV.2015 we recorded Reddyanus loebli (under bark). Figure 591. Locality 15CN, Sri
Lanka, North Central Province, Puttalam District, Eluwankulam, 08°12'35.1"N 079°51'32"E, 52 m a.s.l. During night collecting
on 28.IV.2015 (UV detection) between 9.00 and 10.00 p.m. we recorded Charmus laneus (running on the ground among leaves),
Lychas srilankensis (on the ground under branches or leaves or in their vicinity), Reddyanus ceylonensis sp. n. (on the ground
and inside old moulder stump), Reddyanus loebli (hidden under scales of bark of standing tree trunks) and Heterometrus
swammerdami (inside burrows). On the locality we recorded between 9.00 and 10.00 p.m. temperature 28.5 ºC– 25.9 ºC and
humidity between 61% and 78%. Figures 590–591: Figure 590. Locality 15CM, Sri Lanka, Central Province, Matale District, Pallegama, 07°32'49.5"N
080°47'50"E, 434 m a.s.l. During a day 28.IV.2015 we recorded Reddyanus loebli (under bark). Figure 591. Locality 15CN, Sri
Lanka, North Central Province, Puttalam District, Eluwankulam, 08°12'35.1"N 079°51'32"E, 52 m a.s.l. During night collecting
on 28.IV.2015 (UV detection) between 9.00 and 10.00 p.m. we recorded Charmus laneus (running on the ground among leaves),
Lychas srilankensis (on the ground under branches or leaves or in their vicinity), Reddyanus ceylonensis sp. n. (on the ground
and inside old moulder stump), Reddyanus loebli (hidden under scales of bark of standing tree trunks) and Heterometrus
swammerdami (inside burrows). On the locality we recorded between 9.00 and 10.00 p.m. temperature 28.5 ºC– 25.9 ºC and
humidity between 61% and 78%. Acknowledgments Kovařík et al.: Scorpions of Sri Lanka 125 Figures 592–594: Figure 592. Locality 15CO, Sri Lanka, North Central Province, Puttalam District, Eluwankulam
08°17'15"N 079°50'38.7"E, 38 m a.s.l. During night collecting from 10.30 p.m. on 28.IV.2015 to 4.00 a.m. on 29.IV.2015 (UV
detection) we recorded Charmus laneus (running on the ground among leaves), Isometrus thwaitesii (running on branches o
bushes, also sitting on leaves), Lychas srilankensis (on the ground under branches, leaves or in their vicinity), Reddyanus
ceylonensis sp. n. (on the ground), Chaerilus ceylonensis (inside oblique usually not curved burrows 15 to 30 cm long) and
Heterometrus swammerdami (inside long burrows). On the locality we recorded between 10.00 p.m. and 4.00 a.m. temperature
25.9 ºC and humidity between 58% and 70%. On the locality we recorded 6 scorpion species and genera of all three Sri Lankan
cality 15CP, Sri Lanka, Southern Province, Matara District, Kekanadura village, 05°58'28.2"N
80°36'20.5"E, 40 m a.s.l. During night collecting on 30.IV.2015 (UV detection) we recorded Isometrus maculatus (inside a
families. Figures 593–594. Lo
0
house, the species occupy the roof of the house, Fig. 593) and Heterometrus serratus (inside almost straight burrows in a ground
wall) On the locality we recorded night temperature 28 4 ºC 25 2 ºC (minimum temperature) and night humidity varied between Figures 592–594: Figure 592. Locality 15CO, Sri Lanka, North Central Province, Puttalam District, Eluwankulam,
08°17'15"N 079°50'38.7"E, 38 m a.s.l. During night collecting from 10.30 p.m. on 28.IV.2015 to 4.00 a.m. on 29.IV.2015 (UV
detection) we recorded Charmus laneus (running on the ground among leaves), Isometrus thwaitesii (running on branches of
bushes, also sitting on leaves), Lychas srilankensis (on the ground under branches, leaves or in their vicinity), Reddyanus
ceylonensis sp. n. (on the ground), Chaerilus ceylonensis (inside oblique usually not curved burrows 15 to 30 cm long) and
Heterometrus swammerdami (inside long burrows). On the locality we recorded between 10.00 p.m. and 4.00 a.m. temperature
25.9 ºC and humidity between 58% and 70%. On the locality we recorded 6 scorpion species and genera of all three Sri Lankan
cality 15CP, Sri Lanka, Southern Province, Matara District, Kekanadura village, 05°58'28.2"N
80°36'20.5"E, 40 m a.s.l. During night collecting on 30.IV.2015 (UV detection) we recorded Isometrus maculatus (inside a
families. Figures 593–594. Lo
0
house, the species occupy the roof of the house, Fig. 593) and Heterometrus serratus (inside almost straight burrows in a ground
wall). Acknowledgments On the locality we recorded night temperature 28.4 ºC– 25.2 ºC (minimum temperature) and night humidity varied between
63% and 89%. Figures 592–594: Figure 592. Locality 15CO, Sri Lanka, North Central Province, Puttalam District, Eluwankulam,
08°17'15"N 079°50'38.7"E, 38 m a.s.l. During night collecting from 10.30 p.m. on 28.IV.2015 to 4.00 a.m. on 29.IV.2015 (UV
detection) we recorded Charmus laneus (running on the ground among leaves), Isometrus thwaitesii (running on branches of
bushes, also sitting on leaves), Lychas srilankensis (on the ground under branches, leaves or in their vicinity), Reddyanus
ceylonensis sp. n. (on the ground), Chaerilus ceylonensis (inside oblique usually not curved burrows 15 to 30 cm long) and
Heterometrus swammerdami (inside long burrows). On the locality we recorded between 10.00 p.m. and 4.00 a.m. temperature
25.9 ºC and humidity between 58% and 70%. On the locality we recorded 6 scorpion species and genera of all three Sri Lankan
cality 15CP, Sri Lanka, Southern Province, Matara District, Kekanadura village, 05°58'28.2"N
80°36'20.5"E, 40 m a.s.l. During night collecting on 30.IV.2015 (UV detection) we recorded Isometrus maculatus (inside a
families. Figures 593–594. Lo
0
house, the species occupy the roof of the house, Fig. 593) and Heterometrus serratus (inside almost straight burrows in a ground
wall). On the locality we recorded night temperature 28.4 ºC– 25.2 ºC (minimum temperature) and night humidity varied between
63% and 89%. Euscorpius — 2016, No. 220 126 Euscorpius — 2016, No. 220
126
Figures 595–596: Figure 595. Locality 15CQ, Sri Lanka, Uva Province, Monaragala District, Monaragala, 06°52'30.7"N
ide oblique burrows). On the locality we recorded a
mperature of 33.1 ºC at 5.00 p.m., which gradually dropped to 24.6 ºC (minimum temperature) before sunrise. Humidity during
081°21'17"E, 288 m a.s.l. During night collecting on 2.–3.V.2015 (UV detection) we recorded Reddyanus loebli (under scales of
bark and under bark) and Heterometrus serratus (females with juveniles ins
te
the night varied between 61% and 84%. Figure 596. Locality 15CR, Sri Lanka, Eastern Province, Ampara District, Lahugala
Kitulana National Park, 06°52'46"N 081°43'21.8"E, 40 m a.s.l. During night collecting on 3.–4.V.2015 (10.00 p.m. to 01.00 a.m.)
we recorded Isometrus maculatus (running on branches, also sitting on leaves on trees), Reddyanus basilicus (on the ground
among leaves) and Reddyanus loebli (hidden under scales of bark of standing tree trunks). On the locality we recorded between
10.00 p.m. on 3.V. and 01.00 a.m. Acknowledgments During night collecting on 4.V.2015 (between 10.00 and 11.30 p.m.) we recorded Buthoscorpio
sarasinorum (running on the ground among leaves and escaping to borrows), Reddyanus basilicus (on the ground among leaves),
and Reddyanus loebli (hidden under scales of bark of standing tree trunks). Near to the locality we recorded maximum day
.00 p.m.) during rain we recorded Buthoscorpio sarasinorum (running during
temperature 39 ºC and 27 ºC night temperature at 10.00 p.m.; humidity 68%. Figure 598. Locality 15CT, Sri Lanka, Central
Province, Kandy District, Tree centre Wildlif Trust Sri Lanka “Rantambe”, 07°12'22.1"N 080°57'20.7"E, 171 m a.s.l. During
ight collecting on 5.V.2015 (between 11.00 and 12
n
rain on the ground among leaves) and Reddyanus loebli (hidden under scales of bark of standing tree trunks). On the locality we
recorded night temperature 28.4 ºC– 24.8 ºC (minimum temperature) and night humidity varied between 61% and 83%. Figures 597–598: Figure 597. Locality 15CS, Sri Lanka, Eastern Province, Ampara District, Ampara env., 07°20'01.3"N
081°41'57.1"E, 56 m a.s.l. During night collecting on 4.V.2015 (between 10.00 and 11.30 p.m.) we recorded Buthoscorpio
sarasinorum (running on the ground among leaves and escaping to borrows), Reddyanus basilicus (on the ground among leaves),
and Reddyanus loebli (hidden under scales of bark of standing tree trunks). Near to the locality we recorded maximum day
.00 p.m.) during rain we recorded Buthoscorpio sarasinorum (running during
temperature 39 ºC and 27 ºC night temperature at 10.00 p.m.; humidity 68%. Figure 598. Locality 15CT, Sri Lanka, Central
Province, Kandy District, Tree centre Wildlif Trust Sri Lanka “Rantambe”, 07°12'22.1"N 080°57'20.7"E, 171 m a.s.l. During
ight collecting on 5.V.2015 (between 11.00 and 12
n
rain on the ground among leaves) and Reddyanus loebli (hidden under scales of bark of standing tree trunks). On the locality we
recorded night temperature 28.4 ºC– 24.8 ºC (minimum temperature) and night humidity varied between 61% and 83%. Figures 597–598: Figure 597. Locality 15CS, Sri Lanka, Eastern Province, Ampara District, Ampara env., 07°20'01.3"N
081°41'57.1"E, 56 m a.s.l. During night collecting on 4.V.2015 (between 10.00 and 11.30 p.m.) we recorded Buthoscorpio
sarasinorum (running on the ground among leaves and escaping to borrows), Reddyanus basilicus (on the ground among leaves),
and Reddyanus loebli (hidden under scales of bark of standing tree trunks). Acknowledgments on 4.V.2015 temperature 28 ºC, which gradually dropped to 26.9 ºC and humidity 73%. Figures 595–596: Figure 595. Locality 15CQ, Sri Lanka, Uva Province, Monaragala District, Monaragala, 06°52'30.7"N
ide oblique burrows). On the locality we recorded a
mperature of 33.1 ºC at 5.00 p.m., which gradually dropped to 24.6 ºC (minimum temperature) before sunrise. Humidity during
081°21'17"E, 288 m a.s.l. During night collecting on 2.–3.V.2015 (UV detection) we recorded Reddyanus loebli (under scales of
bark and under bark) and Heterometrus serratus (females with juveniles ins
te
the night varied between 61% and 84%. Figure 596. Locality 15CR, Sri Lanka, Eastern Province, Ampara District, Lahugala
Kitulana National Park, 06°52'46"N 081°43'21.8"E, 40 m a.s.l. During night collecting on 3.–4.V.2015 (10.00 p.m. to 01.00 a.m.)
we recorded Isometrus maculatus (running on branches, also sitting on leaves on trees), Reddyanus basilicus (on the ground
among leaves) and Reddyanus loebli (hidden under scales of bark of standing tree trunks). On the locality we recorded between
10.00 p.m. on 3.V. and 01.00 a.m. on 4.V.2015 temperature 28 ºC, which gradually dropped to 26.9 ºC and humidity 73%. Figures 595–596: Figure 595. Locality 15CQ, Sri Lanka, Uva Province, Monaragala District, Monaragala, 06°52'30.7"N
ide oblique burrows). On the locality we recorded a
mperature of 33.1 ºC at 5.00 p.m., which gradually dropped to 24.6 ºC (minimum temperature) before sunrise. Humidity during
081°21'17"E, 288 m a.s.l. During night collecting on 2.–3.V.2015 (UV detection) we recorded Reddyanus loebli (under scales of
bark and under bark) and Heterometrus serratus (females with juveniles ins
te
the night varied between 61% and 84%. Figure 596. Locality 15CR, Sri Lanka, Eastern Province, Ampara District, Lahugala
Kitulana National Park, 06°52'46"N 081°43'21.8"E, 40 m a.s.l. During night collecting on 3.–4.V.2015 (10.00 p.m. to 01.00 a.m.)
we recorded Isometrus maculatus (running on branches, also sitting on leaves on trees), Reddyanus basilicus (on the ground
among leaves) and Reddyanus loebli (hidden under scales of bark of standing tree trunks). On the locality we recorded between
10.00 p.m. on 3.V. and 01.00 a.m. on 4.V.2015 temperature 28 ºC, which gradually dropped to 26.9 ºC and humidity 73%. Kovařík et al.: Scorpions of Sri Lanka Kovařík et al.: Scorpions of Sri Lanka 127 Figures 597–598: Figure 597. Locality 15CS, Sri Lanka, Eastern Province, Ampara District, Ampara env., 07°20'01.3"N
081°41'57.1"E, 56 m a.s.l. Euscorpius — 2016, No. 220 are scorpions evolving a new sensory organ? Re-
vista Ibérica de Aracnología, 8: 69–72. script. The specimens were collected and exported under
Permit No. WL/3/2/79/14 from Department of Wildlife
Conservation, Sri Lanka. The cytogenetic analysis was
supported by a grant received from the Ministry of Ed-
ucation, Youth and Sports of the Czech Republic No. SVV 260208/2015. We thank Janet Becaloni (BMNH),
Peter Schwendinger and Lionel Monod (MHNG), and
Jason Dunlop (ZMHB) for their kind loans of spec-
imens. Partial funding received from the International
Research Centre (InRC) of University of Peradeniya, Sri
Lanka for field survey was gratefully acknowledged. Further, we thank two anonymous reviewers for their
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keley, Berkeley, California. 319 pp. (unpublished). 133 Kovařík et al.: Scorpions of Sri Lanka Bulletin du Muséum National d’Histoire Naturelle
Paris, 8: 835–850. VACHON, M. 1963. De l'utilité, en systématique, d'une
nomenclature des dents des chélicères chez les scor-
pions. Bulletin du Museum national d'histoire nat-
urelle (Paris), ser. 2, 35: 161–66. VACHON, M. & R. STOCKMANN. 1968. Contribution
à l’étude des Scorpions africains appartenant au
genre Buthotus Vachon 1949 et étude de la
variabilité. Monitore Zoologico Italiano (N. S.), 2
(Supplemento): 81–149. VACHON, M. 1969. Grosphus griveaudi, nouvelle
espèce de scorpion Buthidae malgache. Bulletin du
Muséum National d’ Histoire Naturelle, 41(2): 476–
483. VAZ, G.G. 2000. Age of relict coral reef from the
continental shelf off Karaikal, Bay of Bengal: evi-
dence of last glacial maximum. Current Science,
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appartenant au genre Isometrus H. et E. (Buthidae) á
propos de l`espéce Isometrus maculatus (Geer) hab-
itant Îlé de Pâques. Cahiers Pacifique, 16: 169–180. VERONIKA, K., K. AKILAN, A. MURUGAN-
ANTHAN & T. ESWARAMOHAN. 2013. Di-
versity and identification key to the species of
Scorpions (Scorpiones: Arachnida) from Jaffna Pen-
insula, Sri Lanka. Journal of Entomology and
Zoology Studies, 1(5): 70–77. VACHON, M. 1974. Etude des caractères utilisés pour
classer les familles et les genres de Scorpions
(Arachnides). 1. La trichobothriotaxie en Arach-
nologie. Sigles trichobothriaux et types de tri-
chobothriotaxie chez les scorpions. Bulletin du Mus-
éum National d’Histoire Naturelle, Paris, (3), 140
(Zool. 104), mai-juin 1973: 857–958. WERNER, F. 1934. Scorpiones, Pedipalpi. In H. G. Bronns Klassen und Ordnungen des Tierreichs. Akademische Verlaggesellschaft, Leipzig. 5(IV) 8
(Scorpiones pp. 1–316): 1–490. VACHON, M. 1982. Les scorpions de Sri Lanka (Re-
cherches sur les scorpions appartenant ou déposés
au Muséum d`Histoire naturelle de Genéve III.). Revue suisse de Zoologie, 89: 77–114. WERNER, F. 1936. Neu-Eingänge von Skorpionen im
Zoologischen Museum in Hamburg. Festschrift zum
60. Geburtstage von Professor Dr. Embrik Strand,
Riga, 2: 171–193. VACHON, M. 1986. Étude de la denture des doigts des
pédipalpes chez les scorpions du genre Lychas.
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CHARACTERISTICS OF THE COMPONENT COMPOSITION OF THE BODY OF REPRESENTATIVES OF GAMING SPORTS AND SINGLE COMBAT
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№4 (2022) DOI http://dx.doi.org/10.26739/2181-9300-2022-4 Мавлянов Фарход Шавкатович
тиббиёт фандар доктори, Самарқанд давлат тиббиёт
университети болалар жарроҳлиги кафедраси доценти
ORCID ID: 0000-0003-2650-4445 2 Главный редактор: Yuldashev Botir Akhmatovich
Candidate of Medical Sciences, Associate Professor of
Pediatrics, Neonatology and Propaedeutics of Pediatrics,
Samarkand State Medical University No. 2. ORCID ID: 0000-0003-2442-1523
Eshkobilov Tura Juraevich
candidate of medical Sciences, associate Professor
of the Department of Forensic medicine and pathological
anatomy of the Samarkand state medical University
ORCID ID: 0000-0003-3914-7221
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Responsible for publication:
Abzalova Shaxnoza Rustamovna
Candidate of Medical Sciences, Associate Professor,
Tashkent Pediatric Medical Institute. ORCID ID: 0000-0002-0066-3547 Главный редактор: cXWS]i[a``n\]a^^XV[r 3 3 T[a_XW[i[`SUSS_S^[ǜeYfc`S^[¬Yfc`S^T[a_XW[i[`n[bcS]e[][¬
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Chief Editor:
Rizaev Jasur Alimjanovich
MD, DSc, Professor of Dental Medicine,
Rector of the Samarkand State Medical University
ORCID ID: 0000-0001-5468-9403
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Aripova Tamara Uktamovna
Director of the Institute of Immunology and Human Genomics -
Doctor of Medical Sciences, Professor, Academician of the
Academy of Sciences of the Republic of Uzbekistan
Jin Young Choi
Professor Department of Oral and Maxillofacial
Surgery School of Dentistry Dental Hospital
Seoul National University, President of the
Korean Society of Maxillofacial Aesthetic Surgery
Gulyamov Surat Saidvalievich
Doctor of Medical Sciences, Professor Tashkent Pediatric
Medical Institute Vice-Rector for Research and Innovation. ORCID ID: 0000-0002-9444-4555
Abdullaeva Nargiza Nurmamatovna
Doctor of Medical Sciences, Professor, Vice-Rector
Samarkand State Medical University, Chief Physician of
the 1st Clinic ORCID ID: 0000-0002-7529-4248
Khudoyarova Dildora Rakhimovna
Doctor of Medical Sciences, Associate Professor,
Head of the Department of Obstetrics and Gynecology,
Samarkand State Medical University No.1
ORCID ID: 0000-0001-5770-2255
Rabbimova Dilfuza Tashtemirovna
Candidate of Medical Sciences, Associate Professor,
Head of the Department of Propaedeutics of Pediatrics,
Samarkand State Medical University. ORCID ID:0000-0003-4229-6017
Oripov Firdavs Suratovich
Doctor of Medical Sciences, Associate Professor,
Head of the Department of Histology, Cytology and
Embryology of Samarkand State Medical University. ORCID ID: 0000-0002-0615-0144
Yarmukhamedova Saodat Khabibovna
Candidate of Medical Sciences, Associate Professor,
Head of the Department of Propaedeutics of Internal
Medicine, Samarkand State Medical University. ORCID ID: 0000-0001-5975-1261
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№3 (2022) DOI http://dx.doi.org/10.26739/2181-9300-2022-3
Deputy Chief Editor:
Ziyadullaev Shukhrat Khudayberdievich
Doctor of Medical Sciences, Vice-Rector for scientific work
and Innovation, Samarkand State Medical University
ORCID ID: 0000-0002-9309-3933
Responsible secretary:
Samieva Gulnoza Utkurovna
doctor of Medical Sciences, Associate Professor,
Samarkand State Medical University
ORCID ID: 0000-0002-6142-7054
Akbarov Mirshavkat Mirolimovich
Doctor of Medical Sciences,
Republican Specialized Center of Surgery
named after academician V.Vakhidov
Saidamir Saidov
Doctor of Medical Sciences,
Tashkent Pharmaceutical Institute,
ORCID ID: 0000-0002-6616-5428
Turaev Feruz Fatkhullaevich
MD, DSc, Department of Acquired Heart Diseases,
V.Vakhidov Republican Specialized Center Surgery
ORCID ID: 0000-0002-6778-6920
Khudanov Bakhtinur Oybutaevich
Associate professor of Tashkent State Dental Institute,
Ministry of Innovative Development
of the Republic of Uzbekistan
Babadjanov Oybek Abdujabbarovich
Doctor of sciences in medicine, Tashkent Pediatric
Medical Institute, Department of Dermatovenerology,
pediatric dermatovenerology and AIDS
ORCID ID: 0000-0002-3022-916X
Terebaev Bilim Aldamuratovich
Candidate of Medical Sciences, Associate Professor,
Tashkent Pediatric Medical Institute,
Faculty of Children Department of Surgery. ORCID ID: 0000-0002-5409-4327. ɖɮ ɸɮɖ Page Maker: Khurshid Mirzakhmedov 4 4 T[a_XW[i[`SUSS_S^[ǜeYfc`S^[¬Yfc`S^T[a_XW[i[`n[bcS]e[][¬
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OBSTETRICS AND GYNECOLOGY
1. Zebiniso A. Nasirova, Malika Ya. Aliyeva
THE ROLE OF HORMONE REPLACEMENT THERAPY IN PATIENTS
WITH PREMATURE OVARIAN INSUFFICIENCY……………………………………….10
2. Sitora Turopova, Zebiniso A. Nasirova
MODERN ASPECTS OF ABDOMINAL DELIVERY (Literature review)…………………16
3. Parvina Rasulova, Zebiniso A. Nasirova
MODERN ASPECTS OF PATHOLOGICAL WEIGHT GAIN DURING
PREGNANCY (Literature review)…………………………………………………………...22
4. Shahlo A.Rustamova, Nargiza Kh. Vafokulova
INDICATIONS FOR CAESAREAN SECTION IN PREGNANT WOMEN
IN SAMARKAND REGION AND ITS IMPACT ON THE INTESTINAL
MICROFLORA IN NEWBORNS……………………………………………………………29
ALLERGOLOGY AND IMMUNOLOGY
5. Hamida R. Ibrakhimova, Ruslan R. Nurullaev
DESCRIPTION OF THE STATUS OF CYTOKINES IN ADULTS AND CHILDREN
WITH A PRONOUNCED ALLERGIC BACKGROUND OF PARASITIC DISEASES……37
6. Feruza A. Mustafayeva
RESULTS OF THE STUDY OF WOMEN'S IMMUNE SYSTEM IN
INFECTIOUS DISEASES OF SMALL BELLY ORGANS....................................................43
7. Zilola A. Rajabova, Nazokatkhon Sh. Abdullaeva, Kodirzhon T. Boboev, Timur R. Alimov
PRIMARY IMMUNODEFICIENCY: MODERN CONCEPTS, STATE OF
THE PROBLEM AND PROSPECTS………………………………………………………..50
8. Mashrab Yusupov, Zhasur A.Rizaev, Shukhrat X. Ziyadullaev
THE VALUE OF CYTOKINES IN CHILDREN WITH ESCHERICHIOSIS.......................58
9. Ziyaviddin Z. Khakimov, Alisher K. Rakhmanov, Nodira B. Bekova
INFLUENCE OF CALCIUM CHANNEL ANTAGONISTS ON
THE DEVELOPMENT OF ADJUVANT ARTHRITIS IN RATS…………………………..64
10. Yuliana Yu. Assesorova
BETA-THALASSEMIA: THE STATE OF THE PROBLEM AND PROSPECTS
(REVIEW)……………………………………………………………………………………72
PEDIATRIC SURGERY
11. Sardor J. Kamolov, Farkhod Sh. Mavlyanov
DIAGNOSIS AND TREATMENT OF EMERGENCY ABDOMINAL
PATHOLOGY AT THE PRESENT STAGE…………………………………………………80
12. Madamin M. Madazimov, Ziyoviddin D. Isomiddinov, Muhammadyahyo G. Teshaboev
STUDY OF THE LONG-TERM RESULTS OF THE TREATMENT
OF SCARED DEFORMITIES IN THE LARGE JOINTS OF THE
LEG AFTER BURN IN CHILDREN…………………………………………………………85
MORPHOLOGY
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OBSTETRICS AND GYNECOLOGY ALLERGOLOGY AND IMMUNOLOGY
5. Hamida R. Ibrakhimova, Ruslan R. Nurullaev
DESCRIPTION OF THE STATUS OF CYTOKINES IN ADULTS AND CHILDREN
WITH A PRONOUNCED ALLERGIC BACKGROUND OF PARASITIC DISEASES……37
6. Feruza A. Mustafayeva
RESULTS OF THE STUDY OF WOMEN'S IMMUNE SYSTEM IN
INFECTIOUS DISEASES OF SMALL BELLY ORGANS....................................................43
7. Zilola A. Rajabova, Nazokatkhon Sh. Abdullaeva, Kodirzhon T. Boboev, Timur R. Alimov
PRIMARY IMMUNODEFICIENCY: MODERN CONCEPTS, STATE OF
THE PROBLEM AND PROSPECTS………………………………………………………..50
8. Mashrab Yusupov, Zhasur A.Rizaev, Shukhrat X. Ziyadullaev
THE VALUE OF CYTOKINES IN CHILDREN WITH ESCHERICHIOSIS.......................58
9. Ziyaviddin Z. Khakimov, Alisher K. Rakhmanov, Nodira B. Bekova
INFLUENCE OF CALCIUM CHANNEL ANTAGONISTS ON
THE DEVELOPMENT OF ADJUVANT ARTHRITIS IN RATS…………………………..64
10. Yuliana Yu. Assesorova
BETA-THALASSEMIA: THE STATE OF THE PROBLEM AND PROSPECTS
(REVIEW)……………………………………………………………………………………72
PEDIATRIC SURGERY
11. Sardor J. Kamolov, Farkhod Sh. Mavlyanov
DIAGNOSIS AND TREATMENT OF EMERGENCY ABDOMINAL
PATHOLOGY AT THE PRESENT STAGE…………………………………………………80
12. Madamin M. Madazimov, Ziyoviddin D. Isomiddinov, Muhammadyahyo G. Teshaboev
STUDY OF THE LONG-TERM RESULTS OF THE TREATMENT
OF SCARED DEFORMITIES IN THE LARGE JOINTS OF THE
LEG AFTER BURN IN CHILDREN…………………………………………………………85
MORPHOLOGY
13. Nigora Kh. Asadova
THE EFFECT OF RADIATION ON THE MORPHOFUNCTIONAL
STATE OF THE THYMUS STRUCTURE IN THREE MONTH OLD
WHITE MONGREL RATS......................................................................................................90 5 W i
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14. Payzilla Urinbаyev, Sherzod Eranov, Tura Eshkobilov, Nurali Eranov
MORPHOLOGICAL SUBSTANTIATION OF CAPSULOPLASTY OF THE ANNULAR
LIGAMENT IN OLD ANTERIOR-MEDIAL DISLOCATIONS OF THE RADIAL HEAD IN
CHILDREN…………………………………………………………………………………..96
15. Farida M. Khamidova, Jasur M. Ismoilov
STAGES OF DEVELOPMENT AND MORPHOFUNCTIONAL CHARACTERISTICS OF
BRONCH GLANDS IN CHILDREN WITH PRENATAL ONTOGENESIS AND LUNG
PATHOLOGY………………………………………………………………………………104
NARCOLOGY
16. Bobir T. Turaev
INFLUENCE OF SOCIAL FACTORS ON INDIVIDUALS WITH DOMESTIC ALCOHOL
PROBLEM DURING THE COVID-19 PANDEMIC............................................................113
NEUROLOGY
17. Marguba Sh. Ismatova
PHYSIOLOGICAL FEATURES OF A CORPUS CALLOSUM…………………………..118
18. Shoxrux Sh. Fatxullaev, Mukaddaskhon A. Khamrakulova
METHODS OF FUNCTIONAL INVESTIGATION OF VIBRATION DISEASE IN MINING
INDUSTRY WORKERS……………………………………………………………………122
ONCOLOGY
19. Jurabek A. Abdurakhmonov, Nodir M. Rahimov, Shakhnoza Sh. Shakhanova
MODERN VIEW ON ASCITE IN OVARIAN CANCER………………………………….130
20. Shakhnoza Niyozova, Sergey Kamishov
TARGETED THERAPY IN THE TREATMENT OF PATIENTS
METASTATIC COLORECTAL CANCER………………………………………………...140
21. Djamila Sh. Polatova, Ahmad Yu. Madaminov, Nodir M. Rahimov
SIGNIFICANCE OF EXPRESSION OF PD-L1 AND P53 PROTEINS IN HUMAN
PAPILLOMAVIRUS-ASSOCIATED
OROPHARYNGEAL
SQUAMOUS
CELL
CARCINOMA………………………………………………………………………………144
22. Nodir M. Rahimov, Shakhnoza Sh. Shakhanova, Alisher A. Khakimov, Tatyana Yu. Kalyuta, Marina G. Velikanova, Alexander Korolev
EFFICIENCY OF RADIOTHERAPY TREATMENT IN PATIENTS WITH BONE
METASTASES OF PROSTATE CANCER AND RENAL-CELL CANCER.....................152
OTORHINOLARYNGOLOGY
23. Muhammad A. OBSTETRICS AND GYNECOLOGY Bekmurodov, Gayrat U.Lutfullayev
PATHOGENESIS AND TREATMENT FEATURES OF NASAL BLEEDING………….160 14. Payzilla Urinbаyev, Sherzod Eranov, Tura Eshkobilov, Nurali Eranov
MORPHOLOGICAL SUBSTANTIATION OF CAPSULOPLASTY OF THE ANNULAR
LIGAMENT IN OLD ANTERIOR-MEDIAL DISLOCATIONS OF THE RADIAL HEAD IN
CHILDREN…………………………………………………………………………………..96
15. Farida M. Khamidova, Jasur M. Ismoilov
STAGES OF DEVELOPMENT AND MORPHOFUNCTIONAL CHARACTERISTICS OF
BRONCH GLANDS IN CHILDREN WITH PRENATAL ONTOGENESIS AND LUNG
PATHOLOGY………………………………………………………………………………104 14. Payzilla Urinbаyev, Sherzod Eranov, Tura Eshkobilov, Nurali Eranov
MORPHOLOGICAL SUBSTANTIATION OF CAPSULOPLASTY OF THE ANNULAR
LIGAMENT IN OLD ANTERIOR-MEDIAL DISLOCATIONS OF THE RADIAL HEAD IN
CHILDREN…………………………………………………………………………………..96
1
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16. Bobir T. Turaev
INFLUENCE OF SOCIAL FACTORS ON INDIVIDUALS WITH DOMESTIC ALCOHOL
PROBLEM DURING THE COVID-19 PANDEMIC............................................................113
NEUROLOGY
17. Marguba Sh. Ismatova
PHYSIOLOGICAL FEATURES OF A CORPUS CALLOSUM…………………………..118
18. Shoxrux Sh. Fatxullaev, Mukaddaskhon A. Khamrakulova
METHODS OF FUNCTIONAL INVESTIGATION OF VIBRATION DISEASE IN MINING
INDUSTRY WORKERS……………………………………………………………………122
ONCOLOGY
19. Jurabek A. Abdurakhmonov, Nodir M. Rahimov, Shakhnoza Sh. Shakhanova
MODERN VIEW ON ASCITE IN OVARIAN CANCER………………………………….130
20. Shakhnoza Niyozova, Sergey Kamishov
TARGETED THERAPY IN THE TREATMENT OF PATIENTS
METASTATIC COLORECTAL CANCER………………………………………………...140
21. Djamila Sh. Polatova, Ahmad Yu. Madaminov, Nodir M. Rahimov
SIGNIFICANCE OF EXPRESSION OF PD-L1 AND P53 PROTEINS IN HUMAN
PAPILLOMAVIRUS-ASSOCIATED
OROPHARYNGEAL
SQUAMOUS
CELL
CARCINOMA………………………………………………………………………………144
22. Nodir M. Rahimov, Shakhnoza Sh. Shakhanova, Alisher A. Khakimov, Tatyana Yu. Kalyuta, Marina G. Velikanova, Alexander Korolev
EFFICIENCY OF RADIOTHERAPY TREATMENT IN PATIENTS WITH BONE INDUSTRY WORKERS……………………………………………………………………122
ONCOLOGY
19. Jurabek A. Abdurakhmonov, Nodir M. Rahimov, Shakhnoza Sh. Shakhanova
MODERN VIEW ON ASCITE IN OVARIAN CANCER………………………………….130
20. Shakhnoza Niyozova, Sergey Kamishov
TARGETED THERAPY IN THE TREATMENT OF PATIENTS
METASTATIC COLORECTAL CANCER………………………………………………...140
21. Djamila Sh. Polatova, Ahmad Yu. Madaminov, Nodir M. Rahimov
SIGNIFICANCE OF EXPRESSION OF PD-L1 AND P53 PROTEINS IN HUMAN
PAPILLOMAVIRUS-ASSOCIATED
OROPHARYNGEAL
SQUAMOUS
CELL
CARCINOMA………………………………………………………………………………144
22. Nodir M. Rahimov, Shakhnoza Sh. Shakhanova, Alisher A. Khakimov, Tatyana Yu. Kalyuta, Marina G. Velikanova, Alexander Korolev
EFFICIENCY OF RADIOTHERAPY TREATMENT IN PATIENTS WITH BONE
METASTASES OF PROSTATE CANCER AND RENAL-CELL CANCER.....................152
OTORHINOLARYNGOLOGY
23. Muhammad A. Bekmurodov, Gayrat U.Lutfullayev
PATHOGENESIS AND TREATMENT FEATURES OF NASAL BLEEDING………….160
OPHTHALMOLOGY
24. Dilfuza Z. Jalalova, Abdumalik A. Hadjimetov
EVALUATION OF MARKERS OF ENDOTHELIAL FUNCTION IN TEAR FLUID IN
PATIENTS WITH ARTERIAL HYPERTENSIONANNOTATION……………………….169
25. Khalidjon M. Kamilov, Nigora N. Gaybullaeva
OPTIMIZATION OF METHODS FOR EARLY DIAGNOSIS OF OPEN-ANGLE
GLAUCOMA IN PRIMARY HEALTHCARE……………………………………………..174 39.
Dildora A. Rustamova, Jasur Alimdjanovich Rizaev
THE STUDY OF MARKERS OF ENDOTHELIAL DYSFUNCTION IN PATIENTS WITH
SYSTEMIC VASCULITIS AFTER CORONAVIRUS INFECTION………………………270
40.
Nodira Sh. Nazarova, Lola T. Mirzakulova
EVALUATION
OF
THE
EFFICIENCY
OF
MUCOPOLYSACCHARIDES
IN
NORMALIZING METABOLIC PROCESSES IN PERIODON TISSUES………………...277 39. Dildora A. Rustamova, Jasur Alimdjanovich Rizaev
THE STUDY OF MARKERS OF ENDOTHELIAL DYSFUNCTION IN PATIENTS WITH
SYSTEMIC VASCULITIS AFTER CORONAVIRUS INFECTION………………………270
40. Nodira Sh. Nazarova, Lola T. Mirzakulova
EVALUATION
OF
THE
EFFICIENCY
OF
MUCOPOLYSACCHARIDES
IN
NORMALIZING METABOLIC PROCESSES IN PERIODON TISSUES………………...277
FORENSIC MEDICAL EXAMINATION
41. Sayit I. Indiaminov, Aziza E. Davranova
THE SEVERITY OF HEALTH CAUSE IN CHILDREN WITH BLUNT INJURIES OF THE
EYEBALL AND ITS ADDITIONS…………………………………………………………287
42. Sayit I. Indiaminov, Khasan N. Abdumuminov
DEFECTS OF THE STRUCTURE OF THE CHEST AND ABDOMEN IN CYCLISTS
DURING ROAD TRAFFIC ACCIDENTS………………………………………………….295
43. Iskandar B. Shopulatov, Sayit I. Indiaminov
FORENSIC AND SOME CLINICAL ASPECTS OF WRIST BONE FRACTURES………304
THERAPY
44. Farida V. Khudoikulova, Zilola F. Mavlyanova
NON-ALCOHOLIC FATTY LIVER DISEASE, MODERN VIEWS……………………..310
45. Eleonora N. Tashkenbaeva, Abdumalik I. Mukhiddinov, Gulnora A. Abdieva, Dilrabo D. Xaydarova, Barchinoy M. Togayeva
CLINICAL FEATURES OF THE COURSE AND DEVELOPMENT OF ARTERIAL
HYPERTENSION WITH THE RISK OF CARDIOVASCULAR COMPLICATIONS IN
COVID-19…………………………………………………………………………………...318
46. Abdumalik I. Mukhiddinov, Eleonora N. Tashkenbaeva, Gulnora A. Abdieva, Dilrabo D. Xaydarova, Barchinoy M. Togayeva
FEATURES OF THE CLINICAL COURSE AND MODERN DIAGNOSIS OF
HYPERTENSION IN COMORBIDITY WITH CHRONIC OBSTRUCTIVE PULMONARY
DISEASE IN PATIENTS WITH COVID-19………………………………………………..326
47. Gulchehra R. Yuldasheva, Dilfuza A. Inoyatova
FEATURES OF THE COURSE OF CHRONIC PANCREATITIS WITH METABOLIC
SYNDROME………………………………………………………………………………..333
TRAUMATOLOGY AND ORTHOPEDICS
48. Sadulla Ibragimov, Nurali Eranov, Sherzod Eranov
KNEE ARTHROSCOPY AND RESULTS OF ARTHROSCOPIC MENISCECTOMY….338
49. Sharof M. Davirov, Payzulla U. Urinboyev
LENGTHENING OF THE TIBIA WITH EXTENSIVE BONE DEFECTS USING THE
ILIZAROV APPARATUS USING A NEW DISTRACTION DEVICE…………………...343
UROLOGY
50. Iskandar S. Allazov
THE OPTIMUM OPTION FOR ACCESS TO THE SCROTOUM ORGANS DURING
SCROTOTOMY…………………………………………………………………………….353
51
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t 45. Eleonora N. Tashkenbaeva, Abdumalik I. Mukhiddinov, Gulnora A. Abdieva, Dilrabo D. Xaydarova, Barchinoy M. Togayeva
CLINICAL FEATURES OF THE COURSE AND DEVELOPMENT OF ARTERIAL
HYPERTENSION WITH THE RISK OF CARDIOVASCULAR COMPLICATIONS IN
COVID-19…………………………………………………………………………………...318 46. Abdumalik I. Mukhiddinov, Eleonora N. Tashkenbaeva, Gulnora A. Abdieva, Dilrabo D. Xaydarova, Barchinoy M. Togayeva
FEATURES OF THE CLINICAL COURSE AND MODERN DIAGNOSIS OF
HYPERTENSION IN COMORBIDITY WITH CHRONIC OBSTRUCTIVE PULMONARY
DISEASE IN PATIENTS WITH COVID-19………………………………………………..326 46. Abdumalik I. Mukhiddinov, Eleonora N. Tashkenbaeva, Gulnora A. Abdieva, Dilrabo D. Xaydarova, Barchinoy M. Togayeva
FEATURES OF THE CLINICAL COURSE AND MODERN DIAGNOSIS OF
HYPERTENSION IN COMORBIDITY WITH CHRONIC OBSTRUCTIVE PULMONARY
DISEASE IN PATIENTS WITH COVID-19………………………………………………..326 OPHTHALMOLOGY 6 T[a_XW[i[`SUSS_S^[ǜeYfc`S^[¬Yfc`S^T[a_XW[i[`n[bcS]e[][¬
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26. Nodira Yangieva, Feruza Mirbabaeva
ANALYSIS OF INDICATORS OF PRIMARY AND GENERAL INCIDENCE OF AGE-
RELATED MACULAR DEGENERATION IN THE REPUBLIC OF UZBEKISTAN FOR
THE PERIOD 2010-2019…………………………………………………………………...180
27. Botir U. Tokhtaev, Amin A. Yusupov, Temur T. Saidov
THE ROLE AND PLACE OF LASER METHODS IN THE COMPLEX TREATMENT OF
OPEN-CORUS GLAUCOMA………………………………………………………………186
PEDIATRICS
28. Rustam Kh. Sharipov, Nodira A. Rasulova
ASSESSMENT OF FACTORS FOR THE DEVELOPMENT OF RICKETS AND THE
CONSEQUENCES OF PERINATAL DAMAGE TO THE NERVOUS SYSTEM IN YOUNG
CHILDREN…………………………………………………………………………………193
29. Nodira A. Khamidova
INTERRELATION BETWEEN THE NERVOUS AND IMMUNE SYSTEMS IN
CHILDREN WITH RHEUMATOID ARTHRITIS (literature review).................................200
REHABILITOLOGY AND SPORTS MEDICINE
30. Mohinur I. Ismatova
ANTHROPOMETRIC CHANGES IN SPECIFICITY IN ATHLETES ENGAGED IN
RHYTHMIC GYMNASTICS……………………………………………………………….210
31. Yokutkhon Kamalova
CHARACTERISTICS OF THE COMPONENT COMPOSITION OF THE BODY OF
REPRESENTATIVES OF GAMING SPORTS AND SINGLE COMBAT………………..214
32. Sardor M. Makhmudov, Olga A. Kim
ASSESSMENT OF THE NUTRITIONAL STATUS OF YOUNG PEOPLE ON THE BASIS
OF BIOPEDANCEMETRY……………………………………………………………...…219
33. Maftuna Z. Ravshanova
EARLY REHABILITATION OF ATHLETES WITH ANKLE JOINT INJURY BY
VARIOUS RECOVERY METHODS………………………………………………………225
34. Zilola F. Mavlyanova, Malika Sh. Ibragimova, Zhakhongir B. Tokhtiev
STUDY OF MORPHOFUNCTIONAL STATUS AND ITS FEATURES IN ATHLETES
ENGAGED IN KURASH…………………………………………………………………...232
STOMATOLOGY
35. Afzal S. Abdullaev, Aziz S. Kubayev, Jasur A. Rizaev
EXCITABILITY THRESHOLD IN NEURITIS OF THE LOWER
ALVEOLAR NERVE……………………………………………………………………….238
36. Mekhriniso K. Kamalova, Nigina A. Sadullayeva
A MODERN APPROACH TO SURGICAL TREATMENT OF FRACTURES OF THE
ZYMATOMAXANDILLARY COMPLEX………………………………………………...246
37. Jasur A. Rizaev, Irina R. Aghababyan
ASSOCIATION
OF
PERIODONTAL
DISEASES
WITH
ACUTE
CORONARY
SYNDROME………………………………………………………………………………..252
38. Jasur A. Rizayev, Malika Sh. Akhrorova
ASSESSMENT OF CHANGES IN THE ORAL CAVITY AND MUCOSAL FLOOR IN
PATIENTS WITH COVID-19………………………………………………………………263 7 7 W i
¬
W i
¬
|
39. Dildora A. Rustamova, Jasur Alimdjanovich Rizaev
THE STUDY OF MARKERS OF ENDOTHELIAL DYSFUNCTION IN PATIENTS WITH
SYSTEMIC VASCULITIS AFTER CORONAVIRUS INFECTION………………………270
40. Nodira Sh. Nazarova, Lola T. Mirzakulova
EVALUATION
OF
THE
EFFICIENCY
OF
MUCOPOLYSACCHARIDES
IN
NORMALIZING METABOLIC PROCESSES IN PERIODON TISSUES………………...277 TRAUMATOLOGY AND ORTHOPEDICS 48. Sadulla Ibragimov, Nurali Eranov, Sherzod Eranov
KNEE ARTHROSCOPY AND RESULTS OF ARTHROSCOPIC MENISCECTOMY….338
49. Sharof M. Davirov, Payzulla U. Urinboyev
LENGTHENING OF THE TIBIA WITH EXTENSIVE BONE DEFECTS USING THE
ILIZAROV APPARATUS USING A NEW DISTRACTION DEVICE…………………...343
UROLOGY
50. Iskandar S. Allazov
THE OPTIMUM OPTION FOR ACCESS TO THE SCROTOUM ORGANS DURING
SCROTOTOMY…………………………………………………………………………….353
51. Khasan S. Allazov, Yusuf N. Iskandarov, Iskandar S. Allazov, Firdavs M.Tuxtayev
EPITSISTOKUTANEOSTOMIYA........................................................................................361 8 52. Raykhana R. Sakhatalieva, Razhabboy I. Isroilov, Mavlyuda A. Mamatalieva
LEVEL OF EXPRESSION OF ANTIAPOPTOSIS PROTEIN BCL-2 IN BLADDER
LEUKOPLAKIA……………………………………………………………………………366 SURGERY 53. Ismoil A. Arziev
SURGICAL TREATMENT OF BILE PERITONITIS AS A COMPLICATION OF ACUTE
DESTRUCTIVE CHOLECYSTITIS......................................................................................372 53. Ismoil A. Arziev
SURGICAL TREATMENT OF BILE PERITONITIS AS A COMPLICATION OF ACUTE
DESTRUCTIVE CHOLECYSTITIS......................................................................................372
54. Akhmadjon S. Babajanov, Alisher F. Zayniev, Jurabek I. Alimov
THE CURRENT STATE OF THE PROBLEM OF DIAGNOSIS OF THYROID NODULES
(literature review)……………………………………………………………………………379
55. Zafar B. Kurbaniyazov, Kosim E. Rakhmanov, Sanjar A.Anarboev, Furkat O. Mizamov
EXPERIMENTAL - MORPHOLOGICAL AND CLINICAL SUBSTANTIATION
OF CHEMOTHERAPY IN THE PREVENTION OF RECURRENT
LIVER ECHINOCOCCOSIS.................................................................................................387
56. Saydinjon B. Makhmudov, Akhmadjon S. Babajanov, Ulugbek A. Sherbekov, Diyor Sh. Abdurakhmanov
SELECTION CRITERIA FOR HERNIOALLO- AND ABDOMINOPLASTY BASED ON
THE RESULTS OF HERNIOABDOMINOMETRY……………………………………….395
57. Gayrat E. Mirzabaev, Dilshod M. Khakimov, Akram K. Botirov, Akhmadillo Z. Otakuziev, Zhokhongir A. Botirov
PULMONARY EMBOLISM AND THE ROLE OF THE BLOOD CLOTTING SYSTEM IN
ACUTE CALCULOUS CHOLECYSTITIS ON THE BACKGROUND OF OBESITY…..401
58. Dilshod M. Khakimov, Gayrat E. Mirzabaev, Akram K. Botirov, Akhmadillo
Z.Otakuziev, Zhokhongir A. Botirov
SURGICAL TACTICS IN ACUTE CALCULOUS CHOLECYSTITIS ON THE
BACKGROUND OF OBESITY, TAKING INTO ACCOUNT THE COAGULATION AND
ANTISERUM SYSTEM OF THE BLOOD…………………………………………………408
59. Bakhtiyor Z. Khamdamov, Ilkhom B. Khamdamov, Alisher B. Khamdamov,
Abdukhamit S. Toirov, Akhmadjon S. Babajanov
LASER PHOTODYNAMIC THERAPY AS A METHOD OF TREATMENT OF RESIDUAL
CAVITY AFTER LIVER ECHINOCOCCECTOMY………………………………………416
60. Abdurakhim A. Avazov, Ishnazar B. Mustafakulov, Yokubjon Э. Khursanov, Zilola A. Dzhuraeva
METHODS OF EARLY SURGICAL TREATMENT OF BURNS………………………..423
61. Ishnazar B. Mustafakulov, Khushvakt A. Umedov, Abduraim A. Avazov, Zilola A. Jurayeva
«DAMAGE CONTROL» TACTICS IN SURGERY OF COMBINED ABDOMINAL
TRAUMA…………………………………………………………………………………...428
ОРИГИНАЛЬНАЯ СТАТЬЯ
62. Rizaev A. Jhasur, Makhmonov S. Lutfulla, Gadaev G. Abdugaffor, Turakulov I. Rustam
ASSESSMENT OF EXTERNAL FACTORS INVOLVED IN PREDICTION OF IRON
DEFICIENCY ANEMIA ASSOCIATED WITH HELICOBACTER PYLORI....................436 54. Akhmadjon S. Babajanov, Alisher F. Zayniev, Jurabek I. Alimov
THE CURRENT STATE OF THE PROBLEM OF DIAGNOSIS OF THYROID NODULES
(literature review)……………………………………………………………………………379 54. Akhmadjon S. Babajanov, Alisher F. Zayniev, Jurabek I. Alimov
THE CURRENT STATE OF THE PROBLEM OF DIAGNOSIS OF THYROID NODULES
(literature review)……………………………………………………………………………379 55. Zafar B. Kurbaniyazov, Kosim E. Rakhmanov, Sanjar A.Anarboev, Furkat O. Mizamov
EXPERIMENTAL - MORPHOLOGICAL AND CLINICAL SUBSTANTIATION
OF CHEMOTHERAPY IN THE PREVENTION OF RECURRENT
LIVER ECHINOCOCCOSIS.................................................................................................387 56. Saydinjon B. Makhmudov, Akhmadjon S. Babajanov, Ulugbek A. Sherbekov, Diyor Sh. Abdurakhmanov
SELECTION CRITERIA FOR HERNIOALLO- AND ABDOMINOPLASTY BASED ON
THE RESULTS OF HERNIOABDOMINOMETRY……………………………………….395 THE RESULTS OF HERNIOABDOMINOMETRY……………………………………….395
57. Gayrat E. Mirzabaev, Dilshod M. Khakimov, Akram K. Botirov, Akhmadillo Z. Otakuziev, Zhokhongir A. SURGERY Botirov
PULMONARY EMBOLISM AND THE ROLE OF THE BLOOD CLOTTING SYSTEM IN
ACUTE CALCULOUS CHOLECYSTITIS ON THE BACKGROUND OF OBESITY…..401 57. Gayrat E. Mirzabaev, Dilshod M. Khakimov, Akram K. Botirov, Akhmadillo Z. Otakuziev, Zhokhongir A. Botirov
PULMONARY EMBOLISM AND THE ROLE OF THE BLOOD CLOTTING SYSTEM IN
ACUTE CALCULOUS CHOLECYSTITIS ON THE BACKGROUND OF OBESITY…..401 58. Dilshod M. Khakimov, Gayrat E. Mirzabaev, Akram K. Botirov, Akhmadillo
Z.Otakuziev, Zhokhongir A. Botirov
SURGICAL TACTICS IN ACUTE CALCULOUS CHOLECYSTITIS ON THE
BACKGROUND OF OBESITY, TAKING INTO ACCOUNT THE COAGULATION AND
ANTISERUM SYSTEM OF THE BLOOD…………………………………………………408 59. Bakhtiyor Z. Khamdamov, Ilkhom B. Khamdamov, Alisher B. Khamdamov,
Abdukhamit S. Toirov, Akhmadjon S. Babajanov
LASER PHOTODYNAMIC THERAPY AS A METHOD OF TREATMENT OF RESIDUAL
CAVITY AFTER LIVER ECHINOCOCCECTOMY………………………………………416
60. Abdurakhim A. Avazov, Ishnazar B. Mustafakulov, Yokubjon Э. Khursanov, Zilola A. Dzhuraeva
METHODS OF EARLY SURGICAL TREATMENT OF BURNS………………………..423
61. Ishnazar B. Mustafakulov, Khushvakt A. Umedov, Abduraim A. Avazov, Zilola A. Jurayeva
«DAMAGE CONTROL» TACTICS IN SURGERY OF COMBINED ABDOMINAL
TRAUMA…………………………………………………………………………………...428
ОРИГИНАЛЬНАЯ СТАТЬЯ
62. Rizaev A. Jhasur, Makhmonov S. Lutfulla, Gadaev G. Abdugaffor, Turakulov I. Rustam
ASSESSMENT OF EXTERNAL FACTORS INVOLVED IN PREDICTION OF IRON
DEFICIENCY ANEMIA ASSOCIATED WITH HELICOBACTER PYLORI....................436 ЎЙИН СПОРТЛАРИ ВА ЖАНГ САНЪАТЛАРИ ВАКИЛЛАРИНИНГ ТАРКИБИЙ
ҚИСМЛАРИНИНГ ХУСУСИЯТЛАРИ For citation: Kamalova Yokutkhon Akhmadzhanovna. Characteristics of the component
composition of the body of representatives of gaming sports and single combat. Journal of
Biomedicine and Practice. 2022, vol. 7, issue 4, pp.214-218 КАМАЛОВА Ёқутхон Ахмаджановна
Самарқанд Давлат тиббиёт университети КАМАЛОВА Ёқутхон Ахмаджановна
Самарқанд Давлат тиббиёт университети ОРИГИНАЛЬНАЯ СТАТЬЯ 62. Rizaev A. Jhasur, Makhmonov S. Lutfulla, Gadaev G. Abdugaffor, Turakulov I. Rustam
ASSESSMENT OF EXTERNAL FACTORS INVOLVED IN PREDICTION OF IRON
DEFICIENCY ANEMIA ASSOCIATED WITH HELICOBACTER PYLORI....................436 9 T[a_XW[i[`SUSS_S^[ǜeYfc`S^[¬Yfc`S^T[a_XW[i[`n[bcS]e[][¬
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Цель: изучить возрастные особенности физического развития спортсменов, занимающихся
футболом и индивидуальной борьбой, морфофункциональные и биоимпедансометрические
особенности их организма. Методы: Для определения параметров компонентного состава использовали анализатор
биоимпеданса Inbodi 230 (Южная Корея). Определялись следующие параметры: масса тела,
общая мышечная масса (ОММ), масса жировой ткани тела, общий уровень жидкости в
организме (ОУЖ ), индекс массы тела (ИМТ), процентное содержание жира в организме
(ПСЖ), объем талии/бедра (Т/Б) ) и основной метаболический индекс. Полученные результаты: Наиболее высокие показатели выявлены у детей младшего
школьного возраста, занимающихся футболом: у них средняя длина тела 150 см, масса тела -
45,6 кг, окружность талии - 58 см, окружность бедер - 78 см, относительно низкое физическое
развитие у детей, занимающихся футболом. в борьбе: длина тела - 128,0-145,0 см, масса тела
- 32-45 кг, окружность талии - 58,5-63,3 см, окружность бедер - 68,3-75,6 см. Выводы. Выявлена статистически значимая корреляция между длиной тела, мышечно-
скелетной массой, клеточной массой, жировой массой и индексом массы тела. Наши
результаты подтверждают, что обследование спортсменов методом биоимпеданса можно
провести в короткие сроки и качественно. р
р
р
Ключевые слова: футбол, единоборства, спортсмены, биоимпедансный анализатор,
морфологические и функциональные показатели. KAMALOVA Yokutkhon
Samarkand State Medical University KИРИШ:
Жисмоний
ривожланишни
баҳолаш
морфологик
ва
функционал
кўрсаткичларни (тана оғирлиги, бўй, бел ва сон айланаси, тана массаси кўрсаткичлари)
ўлчашни ўз ичига олади [1]. Тананинг морфофункционал ҳолатини тўлиқ ўрганиш спортчига
спорт фаолиятининг организмга бериладиган жисмоний юклама миқдорини озайтиришга ва
шу орқали спортчи ҳаёт фаолиятини узайтиришга асос бўлиб хизмат қилади [2]. АННОТАЦИЯ Ц
Maқсад: Спортнинг футбол ва якка кураш билан шуғулланувчи спортчиларини жисмоний
ривожланишининг
ёш
хусусиятларини,
улар
танасининг
морфофункционал
ва
биоимпедансометрик хусусиятларини ўрганиш Maтериал ва методлар: Компонент таркибининг параметрларини аниқлаш учун “Inbody
230”(Жанубий Корея) биоимпеданс анализатори ишлатилди. Қуйидаги параметрлар
аниқланди: тана вазни, умумий мушак массаси (УММ), танадаги ёг тўқималарининг массаси,
умумий тана суюқлик даражаси (УСД), тана массаси индекси (ТМИ), тана ёғининг фоизи
(ТЁФ), бел/сон (Б/С) ва асосий метаболизм индекси Натижалар: Футбол билан шуғулланадиган бошланғич мактаб ёшидаги болаларда энг юқори
кўрсаткичлар аниқланди: уларда ўрта ҳисобда тана узунлиги 150 см, тана вазни - 45,6 кг, бел
айланаси 58 см, сон айланаси 78 см, спортнинг кураш тури билан шуғулланадиган болаларда
нисбатан жисмоний ривожланишнинг паст кўрсаткичлари аниқланди: тана узунлиги - 128,0-
145,0 см, тана вазни - 32-45 кг, бел айланаси 58,5-63,3 см, сон айланаси -68,3-75,6 см га тенг
бўлди Xулоса. Bizning natijalarimiz тана узунлиги, мушак-скелет массаси, ҳужайралар массаси ва ёғ
массалари билан тана массаси индекси ўртасидаги статистик жиҳатдан муҳим боғлиқликни
аниқлади. Биоимпеданс анализатори орқали спортчиларни текширувдан ўтказиш қисқа
муддатда, сифатли амалга оширилиши мумкин. Kaлит сўзлар: футбол, жанг санъати, спортчилар, биоимпеданс анализатори, морфологик ва
функционал параметрлар. КАМАЛОВА Ёкутхон Ахмаджановна
Самаркандский Государственный медицинский университет
ХАРАКТЕРИСТИКА КОМПОНЕНТНОГО СОСТАВА ТЕЛА ПРЕДСТАВИТЕЛЕЙ
ИГРОВОГО СПОРТА И ЕДИНОБОРСТВА 214 CHARACTERISTICS OF THE COMPONENT COMPOSITION OF THE BODY OF
REPRESENTATIVES OF GAMING SPORTS AND SINGLE COMBAT ANNOTATION Objective: To study the age characteristics of the physical development of athletes involved in
football and individual wrestling, the morphofunctional and bioimpedansometric features of their
bodies Methods: Bioimpedance analyzer Inbody 230 (South Korea) was used to determine the parameters
of the component composition. The following parameters were determined: body weight, total muscle
mass (ТMM), body fat tissue mass, total body fluid level (TBF), body mass index (BMI), body fat
percentage (BFP), waist / thigh (W / Th), and basic metabolic index. p
g (
)
g (
)
Results: The highest rates were found in children of primary school age involved in football: they
have an average body length of 150 cm, body weight - 45.6 kg, waist circumference - 58 cm, hip
circumference - 78 cm, relatively low physical development in children involved in football . in
wrestling: body length - 128.0-145.0 cm, body weight - 32-45 kg, waist circumference - 58.5-63.3
cm, hip circumference - 68.3-75.6 cm. Conclusions. A statistically significant correlation was found between body length, musculoskeletal
mass, cell mass, fat mass and body mass index. Our results confirm that the examination of athletes
by the bioimpedance method can be carried out in a short time and with high quality. Key words: football, wrestling, athletes, bioimpedance analyzer, morphological and functional
indicators. KИРИШ:
Жисмоний
ривожланишни
баҳолаш
морфологик
ва
функционал
кўрсаткичларни (тана оғирлиги, бўй, бел ва сон айланаси, тана массаси кўрсаткичлари)
ўлчашни ўз ичига олади [1]. Тананинг морфофункционал ҳолатини тўлиқ ўрганиш спортчига
спорт фаолиятининг организмга бериладиган жисмоний юклама миқдорини озайтиришга ва
шу орқали спортчи ҳаёт фаолиятини узайтиришга асос бўлиб хизмат қилади [2]. 215 SS_S^[ǜeYfc`S^[¬Yfc`S^T[a_XW[i[`n[bcS]e[][¬
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ɖɮɹɹ №4 | 2022 №4 | 2022 ТАДҚИҚОТ МАҚСАДИ: Спортнинг футбол ва якка кураш билан шуғулланувчи
спортчиларини жисмоний ривожланишининг ёш хусусиятларини, улар танасининг
морфофункционал ва биоимпедансометрик хусусиятларини ўрганиш [1]. ТАДҚИҚОТ МАҚСАДИ: Спортнинг футбол ва якка кураш билан шуғулланувчи
спортчиларини жисмоний ривожланишининг ёш хусусиятларини, улар танасининг
морфофункционал ва биоимпедансометрик хусусиятларини ўрганиш [1]. рф фу
р
у у
р
ўр
[ ]
МАТЕРИАЛЛАР ВА ТАДҚИҚОТ УСУЛЛАРИ: Тадқиқотда 58 та спортчи (35 та
спортчи футбол билан ва 23та спортчи якка кураш билан шуғулланаётган) 8 ёшдан – 15 ёшгача
бўлган спортчи иштирок этишди. Барча спортчилар 2 гуруҳга бўлинган: 1 гуруҳ -8 -11 ёш, 2
гуруҳ - 12-15 ёш. у у
Сўров дастури қуйидаги кўрсаткичларни ўз ичига киритди: тана узунлиги; тананинг
кўндаланг ўлчамлари; тананинг айлана ўлчамлари, тери-ёғ қатламларининг қалинлиги; тана
массаси. CHARACTERISTICS OF THE COMPONENT COMPOSITION OF THE BODY OF
REPRESENTATIVES OF GAMING SPORTS AND SINGLE COMBAT Компонент таркибининг параметрларини аниқлаш учун “Инбоди 230”(Жанубий
Корея) биоимпеданс анализатори ишлатилди. Қуйидаги параметрлар аниқланди: тана вазни, умумий мушак массаси (УММ),
танадаги ёг тўқималарининг массаси, умумий тана суюқлик даражаси (УСД), тана массаси
индекси (ТМИ), тана ёғининг фоизи (ТЁФ), бел/сон (Б/С) ва асосий метаболизм индекси. Биоимпедансометрия спортчиларнинг жисмоний ривожланиш даражасини ҳар томонлама
баҳолаш учун ишлатилиши мумкин [2]. Биоимпедансометрия таҳлилини ўтказувчи Inbody мосламаси. Биоимпеданс таҳлили
асосан корелляция принципига асосланиб организм кўрсатгичларини тадқиқ этади. Ушбу
принципга асосланиб ҳисобланиладиган УММ, МЎН, ТҚМТ каби кўрсатгичлар спортчини
жисмоний ривожланганлик даражасини кўрсатади. Шу билан бир қаторда ортиқча тана
массасини биоимпедансдаги ТМИ кўрсатгичлари орқали билиб олиш мумкин. ТМИ
кўрсатгичи бўйича спортчиларни тана массаси кам, нормал масса, нормадан ортиқ каби
гуруҳларга бўлиб ўрганиш мумкин [3]. ТАДҚИҚОТ НАТИЖАЛАРИ: Ёш даврлари бўйича олинган маълумотларни
таққослаш, шунингдек, турли спорт турларининг болалар ва ўсмирлар организмига таъсирини
аниқлаш катта қизиқиш уйғотади. Кўриб чиқиладиган морфологик кўрсаткичлар орасида тана узунлиги, тана оғирлиги,
бел ва сон айланаси, тана массаси индекслари ва умумий тана ҳажми бўйича бел /сон, тананинг
умумий ҳажми тўғрисидаги маълумотларни ифодалайди [4]. Кўриниб турибдики, футбол билан шуғулланадиган бошланғич мактаб ёшидаги
болаларда энг юқори кўрсаткичлар аниқланди: уларда ўрта ҳисобда тана узунлиги 150 см, тана
вазни - 45,6 кг, бел айланаси 58 см, сон айланаси 78 см, спортнинг кураш тури билан
шуғулланадиган болаларда нисбатан жисмоний ривожланишнинг паст кўрсаткичлари
аниқланди: тана узунлиги - 128,0-145,0 см, тана вазни - 32-45 кг, бел айланаси 58,5-63,3 см,
сон айланаси -68,3-75,6 см га тенг бўлди. Бу кўрсаткичлар жадвалда келтирилган. 1 жадвал. 1 жадвал. Бошланғич мактаб ёшидаги спортчиларнинг антропометрик кўрсаткичлари
таҳлили
Кўрсаткичлар
Спортчилар - бошланғич
мактаб ёшидаги
футболчилар
Спортчилар - бошланғич
мактаб ёшидаги
курашчилар
Тана узунлиги
150 кг
128,0 – 145,0 кг
Тана массаси
45,6 см
32 -45 кг
Бел айланаси
58 см
58,5 – 63,3 см
Сон айланаси
78 см
68,3 – 75,6 см д
шланғич мактаб ёшидаги спортчиларнинг антропометрик кўрсаткичлари
таҳлили 216 T[a_XW[i[`SUSS_S^[ǜeYfc`S^[¬Yfc`S^T[a_XW[i[`n[bcS]e[][¬
ɮ ɸ ɖɹ
ɮ
ɖɮɹɹ 2 жадвал. CHARACTERISTICS OF THE COMPONENT COMPOSITION OF THE BODY OF
REPRESENTATIVES OF GAMING SPORTS AND SINGLE COMBAT Ўсмир ёшдаги спортчиларнинг антропометрик кўрсаткичлари таҳлили
Кўрсаткичлар
Спортчилар - ўсмир
футболчилар
Спортчилар - ўсмирлик
давридаги курашчилар
Тана узунлиги
160, 2 см
155,4 см
Тана массаси
54,1 кг
47,3 кг
Бел айланаси
65, 2 см
60,4 см
Сон айланаси
82, 5 см
76,7 см 2 жадва
Ўсмир ёшдаги спортчиларнинг антропометрик кўрсаткичлари таҳлили Ўсмирлик даврида спорт билан шуғулланадиган футболчиларнинг жисмоний ривожланиш
кўрсаткичлари юқори бўлиб, уларнинг ўртача тана узунлиги 160,2 см, тана вазни - 54,1 кг, бел
айланаси 65,2 см, сон айланаси 82,5 см, курашчиларнинг тана узунлиги - 155,4 см, тана вазни
- 47,3 кг, бел айланаси 60,4 см, сон айланаси 76,7 см.га тенг. Ўсмирлик даврида спорт билан шуғулланадиган футболчиларнинг жисмоний ривожланиш
кўрсаткичлари юқори бўлиб, уларнинг ўртача тана узунлиги 160,2 см, тана вазни - 54,1 кг, бел
айланаси 65,2 см, сон айланаси 82,5 см, курашчиларнинг тана узунлиги - 155,4 см, тана вазни
- 47,3 кг, бел айланаси 60,4 см, сон айланаси 76,7 см.га тенг. Маълумки, ҳажми ва ўсиш суръати бир томондан, ирсий омиллар билан тартибга солинган
бўлса, иккинчи томондан, улар атроф-муҳит таъсирида ривожланади. Футбол билан шуғулланадиган ўғил болалар ва ёшлар баланд бўйли ва лептоморф тана
тузилиши билан ажралиб туради. Ёш курашчилар футбол билан шуғулланадиганларга
қараганда бўйи пастроқ, кўкрак қафасининг катталиги, шунингдек, брахиморф тана тузилиши
билан ажралиб туради [6]. ур
р
у у
- 47,3 кг, бел айланаси 60,4 см, сон айланаси 76,7 см.га тенг. Маълумки, ҳажми ва ўсиш суръати бир томондан, ирсий омиллар билан тартибга солинган
бўлса, иккинчи томондан, улар атроф-муҳит таъсирида ривожланади. Футбол билан шуғулланадиган ўғил болалар ва ёшлар баланд бўйли ва лептоморф тана
тузилиши билан ажралиб туради. Ёш курашчилар футбол билан шуғулланадиганларга
қараганда бўйи пастроқ, кўкрак қафасининг катталиги, шунингдек, брахиморф тана тузилиши Маълумки, ҳажми ва ўсиш суръати бир томондан, ирсий омиллар билан тартибга солинган
бўлса, иккинчи томондан, улар атроф-муҳит таъсирида ривожланади. Футбол билан шуғулланадиган ўғил болалар ва ёшлар баланд бўйли ва лептоморф тана
тузилиши билан ажралиб туради. Ёш курашчилар футбол билан шуғулланадиганларга
қараганда бўйи пастроқ, кўкрак қафасининг катталиги, шунингдек, брахиморф тана тузилиши
билан ажралиб туради [6]. ХУЛОСА:
Шундай
қилиб,
болаларнинг
жисмоний
ривожланишининг
ёш
хусусиятларини билиш, у ёки бу кўрсаткичнинг мажбурий ёки бир томонлама
ривожланишини истисно қилган ҳолда, болаларнинг уйғун жисмоний ривожланиши
масаласига яхлит ёндашиш имконини беради. Тана узунлиги,мушак-скелет массаси,
ҳужайралар массаси ва ёғ массалари билан тана массаси индекси ўртасидаги статистик
жиҳатдан муҳим боғлиқлик аниқланди; Биоимпеданс орқали спортчиларни текширувдан ўтказиш қисқа муддатда, сифатли амалга
оширилади. Спортчининг ўзи ҳам олинган натижаларни 81,4 % ҳолларда таҳлил қила олади. CHARACTERISTICS OF THE COMPONENT COMPOSITION OF THE BODY OF
REPRESENTATIVES OF GAMING SPORTS AND SINGLE COMBAT Натижаларга асосан мураббий спортчининг қайси тана кўрсатгичи билан ишлаши
мумкинлиги тўғрисида хулоса қила олади. Текширувдан ўтказиш спортчи учун хеч қандай
ноқулайликлар келтириб чиқармайди [7]. Шуни таъкидлаш керакки, спорт болалар ва ёшларнинг тана тузилиши ва саломатлик
кўрсаткичларини шакллантиришга ижобий таъсир кўрсатади. Мониторинг режимида ёш
спортчиларда организмнинг таркибий қисмларини ўрганиш уларнинг функционал ҳолатини,
жисмоний ривожланишини, шунингдек, машғулот пайтида қўлланиладиган жисмоний
фаолиятнинг етарлилигини баҳолаш учун муҳим аҳамиятга эга [10]. Спортчилар танасининг таркибий қисмларининг таҳлил қилинган хусусиятларидан
спорт натижаларини прогнози учун ва турли спорт турларини танлаш учун ҳам фойдаланиш
керак. 1.
Абдусаломова М., Равшанова М. Особенности реабилитационных мероприятий при
болях в пояснице у спортсменов, занимающихся спортом со скоростными и силовыми
качествами //InterConf. – 2020. 2.
Васильев Г.Ф. Актуальные вопросы методического и психологического обеспечения
подготовки спортсменов-единоборцев / Г.Ф. Васильев, О.В. Тиунова // Теория и
практика физ. культуры. – 2017. – № 2. – С. 20–22. 1.
Абдусаломова М., Равшанова М. Особенности реабилитационных мероприятий при
болях в пояснице у спортсменов, занимающихся спортом со скоростными и силовыми
качествами //InterConf. – 2020.
2.
Васильев Г.Ф. Актуальные вопросы методического и психологического обеспечения
подготовки спортсменов-единоборцев / Г.Ф. Васильев, О.В. Тиунова // Теория и
практика физ. культуры. – 2017. – № 2. – С. 20–22. REFERENCES / СНОСКИ/ИҚТИБОСЛАР: 1. Абдусаломова М., Равшанова М. Особенности реабилитационных мероприятий при
болях в пояснице у спортсменов, занимающихся спортом со скоростными и силовыми
качествами //InterConf. – 2020. 2. Васильев Г.Ф. Актуальные вопросы методического и психологического обеспечения
подготовки спортсменов-единоборцев / Г.Ф. Васильев, О.В. Тиунова // Теория и
практика физ. культуры. – 2017. – № 2. – С. 20–22. 217 T[a_XW[i[`SUSS_S^[ǜeYfc`S^[¬Yfc`S^T[a_XW[i[`n[bcS]e[][¬
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ɖɮɹɹ №4 | 2022
ISSN 2181-9300
DOI Journal 10.26739/2181-9300
T[a_XW[i[`SUSS_S^[ǜeYfc`S^[
!Y[^W da`
Yfc`S^T[a_XW[i[`n[bcS]e[][
ea_!`a_Xc
ɮ ɸ ɖɹ
ɮ
ɖɮɹɹ
!
W i
¬
W i W i
¬
W i ISSN 2181-9300
DOI Journal 10.26739/2181-9300 T[a_XW[i[`SUSS_S^[ǜeYfc`S^[¬Yfc`S^T[a_XW[i[`n[bcS]e[][¬
ɮ ɸ ɖɹ
ɮ
ɖɮɹɹ T[a_XW[i[`SUSS_S^[ǜeYfc`S^[¬Yfc`S^T[a_XW[i[`n[bcS]e[][¬
ɮ ɸ ɖɹ
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ɖɮɹɹ №4 | 2022 3. Ермакова И.В. Оценка компонентов массы тела у детей 10-11 лет с помощью
биоимпедансного анализа / И.В. Ермакова, Т.И. Бурая, Н.Б. Сельверова // Новые
исследования. – 2011. – № 4. – С. 61-69 4. Методы определения физического развития и функционального состояния юных
спортсменов/Алимов А.В., Камилова Р.Т., Исакова Л.И. [и др.]: Учебная программа. –
Ташкент 2013, 56с. 5. Mavlyanova Z. F. et al. Correlation of neurological and nutritive status in children with cerebral
paralysis //European Journal of Molecular & Clinical Medicine. – 2020. – Т. 7. – №. 2. – С. 2020. 6. Мавлянов
И.Р.,
Усманходжаева
А.А.,
Мавлянова
З.Ф. особенности
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nerve neuritis rehabilitation //InterConf. – 2021. 9. Камалова Ё. Особенности физической реабилитации остеохондроза поясничного отдела
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Смирнова Г.А., Андриянов А.И., Кравченко Е.В., Коновалова И.А. Вестник Российской
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11. Nemov R.S. Psychology. Book 1: General Foundations of Psychology/R.S. Nemov.- 5th
edition. - M.: VLADOS, 2010. -687s. 3. Samygin S.I. Psychology/S.I. 12. Makarova G.A. "Sports Medicine" Moscow. 2003. p. 479 218 T[a_XW[i[`SUSS_S^[ǜeYfc`S^[
!Y[^W da`
Yfc`S^T[a_XW[i[`n[bcS]e[][
ea_!`a_Xc
ɮ ɸ ɖɹ
ɮ
ɖɮɹɹ
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English
| null |
Circulating proteomic patterns in AF related left atrial remodeling indicate involvement of coagulation and complement cascade
|
bioRxiv (Cold Spring Harbor Laboratory)
| 2,018
|
cc-by
| 5,480
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Background Left atrial (LA) electro-anatomical remodeling and diameter increase in atrial fibrillation (AF)
indicates disease progression and is associated with poor therapeutic success. Further-
more, AF leads to a hypercoagulable state, which in turn promotes the development of a
substrate for AF and disease progression in the experimental setting. The aim of this study
was to identify pathways associated with LA remodeling in AF patients using untargeted pro-
teomics approach. Editor: Pablo Garcia de Frutos, Institut
d’Investigacions Biomediques de Barcelona, SPAIN Received: May 16, 2018
Accepted: November 12, 2018
Published: November 29, 2018 Received: May 16, 2018
Accepted: November 12, 2018
Published: November 29, 2018 Methods Peripheral blood samples of 48 patients (62±10 years, 63% males, 59% persistent AF)
undergoing AF catheter ablation were collected before ablation. 23 patients with left atrial
low voltage areas (LVA), defined as <0.5 mV, and 25 patients without LVA were matched for
age, gender and CHA2DS2-VASc score. Untargeted proteome analysis was performed
using LC-ESI-Tandem mass spectrometry in a label free intensity based workflow. Signifi-
cantly different abundant proteins were identified and used for pathway analysis and pro-
tein-protein interaction analysis. Copyright: © 2018 Kornej et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files. Funding: This project was supported by Peter-
Osypka Grant 2015 (German Society of
Cardiology) for JK and Volkswagen Foundation
Germany through the Lichtenberg professorship
program for PB and DH (# 84901). The funders
had no role in study design, data collection and OPEN ACCESS Citation: Kornej J, Bu¨ttner P, Hammer E,
Engelmann B, Dinov B, Sommer P, et al. (2018)
Circulating proteomic patterns in AF related left
atrial remodeling indicate involvement of
coagulation and complement cascade. PLoS ONE
13(11): e0198461. https://doi.org/10.1371/journal. pone.0198461 Jelena Kornej1☯*, Petra Bu¨ttnerID1☯, Elke Hammer2,3, Beatrice Engelmann2,
Borislav Dinov1, Philipp Sommer1, Daniela Husser1, Gerhard Hindricks1, Uwe Vo¨lker2,3,
Andreas Bollmann1 Jelena Kornej1☯*, Petra Bu¨ttnerID1☯, Elke Hammer2,3, Beatrice Engelmann2,
Borislav Dinov1, Philipp Sommer1, Daniela Husser1, Gerhard Hindricks1, Uwe Vo¨lker2,3,
Andreas Bollmann1 Jelena Kornej1☯*, Petra Bu¨ttnerID1☯, Elke Hammer2,3, Beatrice Engelmann2,
Borislav Dinov1, Philipp Sommer1, Daniela Husser1, Gerhard Hindricks1, Uwe Vo¨lker2,3,
Andreas Bollmann1 1 Department of Electrophysiology, Heart Center Leipzig, University of Leipzig, Leipzig, Germany,
2 Interfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald, Greifswald,
Germany, 3 DZHK (German Center for Cardiovascular Research), partner site Greifswald, Greifswald,
Germany a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 * jelena.kornej@gmx.de RESEARCH ARTICLE ☯These authors contributed equally to this work.
* jelena.kornej@gmx.de y
* jelena.kornej@gmx.de Proteome screening in AF related atrial remodeling analysis, decision to publish, or preparation of the
manuscript. This pilot proteomic study identified plasma protein candidates associated with electro-ana-
tomical remodeling in AF and pointed towards an imbalance in coagulation and complement
pathway, tissue remodeling and inflammation. Competing interests: The authors have declared
that no competing interests exist. Competing interests: The authors have declared
that no competing interests exist. Introduction Atrial fibrillation (AF) is the most common sustained arrhythmia occurring in approximately
2% of the general population [1]. It is a progressive disease as an evolution from paroxysmal to
persistent AF (AF type) is frequently observed. This is related to advanced structural and elec-
trical left atrial remodeling. Structural remodeling might be suggested by electrocardiography
as increased left atrial diameter (LAD). Evidence for electro-anatomical remodeling is cur-
rently detectable only invasively during catheter ablation in up to 20–25% of AF patients [2]
and is represented by low voltage areas (LVA), e.g. pathologically low electrical signals with
amplitudes less than 0.5 mV. Previous studies suggested that LVA correspond to areas of
fibrotic and electrically silent myocardium [3]. Advanced LA remodeling is associated with
worse rhythm outcomes after catheter ablation [2,4,5] and as consequence is associated with
higher rates of repeated catheter ablations. One of the most serious complications in AF patients related to hypercoagulability and
impaired blood flow is an ischemic stroke [6,7] also leading to increased hospitalization and
cost-intensive treatment.[1] The increased risk for thromboembolic events is attributable to
alterations in blood flow combined with anatomical and structural defects resulting in the ful-
filment of Virchow’s triad for thrombogenesis and a hypercoagulable state in AF patients [8]. Interestingly, recent experimental studies indicated that hypercoagulability promotes the
development of an electro-anatomical substrate and AF progression [9]. In the current study we applied a novel approach, where we performed an untargeted prote-
ome screening of high and medium abundant circulating blood proteins to identify differen-
tially expressed proteins in AF patients with and without LVA and with LAD or < 44mm. Furthermore, the proteins were used to characterize potential pathophysiological pathways
associated with LA remodeling in AF patients. Results Analysis covered 280 non-redundant circulating plasma proteins. The presence of LVA cor-
related with 30 differentially abundant proteins of coagulation and complement cascade
(q<0.05). 1 / 10 PLOS ONE | https://doi.org/10.1371/journal.pone.0198461
November 29, 2018 Conclusions analysis, decision to publish, or preparation of the
manuscript. analysis, decision to publish, or preparation of the
manuscript. PLOS ONE | https://doi.org/10.1371/journal.pone.0198461
November 29, 2018 Study population The study population comprised 48 patients with symptomatic AF who underwent catheter
ablation at Heart Center Leipzig, Germany. Twenty-three patients with LVA and 25 without
LVA respectively were matched according to age, gender and CHA2DS2-VASc score. The
study was approved by the local Ethical Committee (Medical Faculty, University Leipzig) and
patients provided written informed consent for participation. Paroxysmal and persistent AF
were defined according to current guidelines [10]. Paroxysmal AF self-terminated within 7
days after onset. Persistent AF lasted longer than 7 days or required drugs or direct current
cardioversion for termination. In all patients, transthoracic and transesophageal echocardiog-
raphy were performed prior to the ablation. LAD was measured in parasternal long axis view
in M-Mode. All class I or III antiarrhythmic medications with exception of amiodarone were
discontinued for at least 5 half-lives before the AF ablation. PLOS ONE | https://doi.org/10.1371/journal.pone.0198461
November 29, 2018 2 / 10 Proteome screening in AF related atrial remodeling Blood samples Blood samples were obtained in EDTA test tubes in fasting state prior ablation procedure from
femoral vein and processed within one hour after collection. Blood plasma was prepared
(1000 × g for 10 min at 20˚C) and aliquots were stored at -70˚C for subsequent analyses. Electro-anatomical mapping and definition of substrate The electro-anatomical mapping was performed in sinus rhythm. In patients presenting with
atrial fibrillation, the arrhythmia was terminated by electrical cardioversion and the mapping
was further performed in sinus rhythm. End-point of the catheter ablation was isolation of the
PV with proof of both exit and entrance block. The electro-anatomical voltage maps of the LA
excluding the pulmonary veins were created using multielectrode spiral catheter with inter-
electrode distance 2-5-2 or ablation catheter with a 3.5 mm electrode tip and contact measure-
ment properties (SmartTouch Thermocool and TactiCath, St Jude Medical (SJM), Saint Paul,
MN, USA) as mapping catheter. Electro-anatomical mapping was performed using 3-D elec-
tro-anatomical mapping systems (Carto, Biosence Webster, Diamond Bar, CA, USA or EnSite
Precision, SJM). Electrograms with amplitudes over 0.5 mV were defined as normal potentials,
and signals with amplitude under 0.5 mV as low-voltage potentials. In areas with low-voltage
amplitudes we aimed at sufficient mapping points density (>200 points). Points with insuffi-
cient catheter-to-tissue contact or inside ablation lines were excluded. At the end of the procedure, an attempt to induce AF or left atrial macro-reentry tachycar-
dia (LAMRT) was performed using a standardized protocol (burst stimulation with 300, 250,
200 ms from coronary sinus). According to the underlying LVA and inducible LAMRT addi-
tional ablation lines were applied. PLOS ONE | https://doi.org/10.1371/journal.pone.0198461
November 29, 2018 Proteomic analysis Samples were prepared, measured by mass spectrometry and data analyzed according to a
modified protocol described earlier [11]. Briefly, plasma samples were subjected to immunoaf-
finity subtraction of six high abundant proteins using the Multiple Affinity Removal System
(MARS6) (Agilent Technologies, Wilmington, DE, USA). Protein in the flow through fraction
was precipitated using trichloroacetic acid (TCA) at a final concentration of 15% (v/v). Precip-
itates were reconstituted in urea/thiourea (8M/2M) and protein was determined using a Brad-
ford assay (Bio-Rad, Hercules, CA, USA). Four μg protein lysate were reduced and alkylated
and digested with trypsin (Promega, Madison, WI, USA) with a ratio of 1:25 at 37˚C overnight. Peptide purification was performed with μC18 ZipTip (Millipore Cooperation, Billerica, MA,
USA) as described before [12]. LC-ESI tandem mass spectrometry was carried out on a
nanoAquity UPLC (Waters)—LTQ Orbitrap Velos MS (Thermo Scientific Electron, Bremen,
Germany) configuration. High precision precursor scans were recorded in the Orbitrap and
MS/MS data were recorded in data-dependent mode for the Top-20 peaks in the ion trap. Raw MS data were processed using the Refiner MS 10.0 and Analyst 10.0 module (Gene-
data, Basel, Switzerland). Identification was performed against a human FASTA-formatted
database containing 20,155 unique sequence entries (reviewed human database, release of
2016/06) with MASCOT (v2.3.2, Matrix Science, London, UK) as search engine. Carbamido-
methylation of cysteine was set as fixed modification and oxidation of methionine as variable
modification. Enzyme specificity was selected to trypsin with using 10 ppm MS tolerance and
0.6 Da MS/MS tolerances. Peptide FDR was set to 1% of protein groups identified. Peptides
sharing protein group identifications within the data set were not considered for quantitative
analyses. Only proteins identified by at least one unique or razor peptide were considered for
further analysis (S1 and S3 Tables for further information). PLOS ONE | https://doi.org/10.1371/journal.pone.0198461
November 29, 2018 3 / 10 Proteome screening in AF related atrial remodeling Relative quantification protein values were exported from Refiner as summed intensities of
Hi3 peptides per protein. Preprocessing steps before statistical analysis included removal of
proteins with more than 50% missing values per group, which diminished the initially identi-
fied 280 to 237 proteins, and log10 transformation of data. All methods were performed in accordance with the relevant guidelines and regulations. Statistical analysis Data are presented as mean and standard deviation (SD) if normally distributed or as median
[interquartile range] for skewed continuous variables and as proportions for categorical vari-
ables. Continuous variables were tested for normal distribution using the Kolmogorov-Smir-
nov test. The differences between continuous values were assessed using an unpaired t-test or
the Mann–Whitney, and a chi-square test for categorical variables. A p-value <0.05 was con-
sidered statistically significant, and all analyses were performed with SPSS statistical software
version 23. Proteomic data significance tests were corrected for multiple testing using Benja-
mini-Hochberg false discovery rate and significance cut-off set to q<0.05. We detected differentially expressed proteins by comparing patients with LVA vs. without
LVA (23 vs 25 patients) and patients with LAD<44mm (n = 20) and LAD44mm (n = 28). Categorization of proteins in particular physiological pathways was done using WebGestalt
[13] accessing databases of Kyoto Encyclopedia of Genes and Genomes (KEGG) [14] and gene
ontology (GO) [15]. Over-enrichment p-values are not reported as the applied screening
method only detects highly abundant plasma proteins and an unbiased over-enrichment anal-
ysis of the plasma wide proteome is not possible. Results Untargeted proteomic analysis was performed in 48 AF patients matched for age, gender and
CHA2DS2-VASc score). The clinical characteristics of study population are presented in Table 1. There were no differences in age, gender, AF type, BMI, renal function or CHA2DS2-VASc score. However, patients with LVA had significantly larger LAD than patients without (p = 0.01). PLOS ONE | https://doi.org/10.1371/journal.pone.0198461
November 29, 2018 Proteome screening in AF related atrial remodeling Table 1. Clinical characteristics of study population. Low voltage areas
p-value
No (n = 25)
Yes (n = 23)
Age, y
66 ± 10
67 ± 11
0.074
Males, %
64
65
0.930
Persistent AF, %
68
74
0.653
BMI, kg/m2
30 ± 5
30 ± 4
0.657
eGFR, ml/min/1.73m2
72 ± 14
66 ± 20
0.218
CHA2DS2-VASc score
2.8 ± 1.3
3.3 ± 1.6
0.240
LA Diameter, mm
42 ± 6
47 ± 7
0.010
EF, %
56 ± 7
52 ± 12
0.201
Medication, %
Betablockers
80
86
0.562
ACE inhibitors
52
77
0.072
Statins
44
59
0.320
Antiarrhythmics
32
39
0.140
Anticoagulation
0.520
Heparin
12
4
VKA
28
48
NOAC
48
44
None
8
-
Other
4
4
https://doi org/10 1371/journal pone 0198461 t001 Discussion This study used an untargeted proteomic approach to discover high and medium abundant pro-
teins related to left-atrial remodeling in AF patients. Significant differences in protein profiles in
patients with and without LVA were detected. The pathway analysis suggests that alterations in
the complement and coagulation cascade, inflammatory processes, and tissue remodeling are
associated with LVA and LAD. We therefore assume that the observed alterations are more spe-
cific for LVA than for increased left diameter although both phenotypes are interrelated. This is in
line with previous findings [4] and of great clinical importance. However, because of relatively
small cohort, proteomic differences in LAD phenotype did not reach significance after FDR. Proteomic analysis Our protein analysis covered a dynamic range of 4–5 magnitudes as estimated by published
concentrations of the highest abundant protein in our data set (apolipoprotein A1: 2000–
3000μg/μl) and one of the lowest abundant proteins S100A9 protein (0.1–0.01 μg/μl). This analysis covered 1.985 unique peptides representing 280 non-redundant proteins (see S1
Table). Among the 237 proteins being quantified in at least 50% of the patients in each group, 30
proteins displayed different levels (t-test (FDR), p<0.05) in patients presenting with LVA in com-
parison to the control group (Table 2). L-selectin and peptidase inhibitor 16 had higher abun-
dances in the control group with fold changes > 1.5. Ten proteins were identified to be >1.5-fold
more abundant in the LVA group. Tetratricopeptide repeat protein 39A and SWI/SNF complex
subunit SMARCC1 were more than 2-fold enriched in plasma from this group. We annotated all 30 candidate proteins to physiological pathways using WebGestalt [16] to
get an insight into physiological impact. F9, CFH, CFP, C1QB, and C1Q were assigned to
GO:0072376 Protein activation cascade. CASP8, PI16, SERPINA4, SMARCC1, and SERPINA7
were assigned to GO:0045861 Negative regulation of proteolysis. CFH, CFP, C1QB, and C1QC
were assigned to GO:0006959 Humoral immune response. F9, CFH, PLG, C1QB, and C1QC
were assigned to KEGG:hsa04610 Complement and coagulation cascades. When comparing LAD<44mm and LAD>44mm 26 proteins were found differentially
expressed in t-test but did not pass multiple testing corrections (Table 2 and S2 Table). 4 / 10 PLOS ONE | https://doi.org/10.1371/journal.pone.0198461
November 29, 2018 PLOS ONE | https://doi.org/10.1371/journal.pone.0198461
November 29, 2018 Proteome screening in AF related atrial remodeling Table 2. Significantly differentially expressed circulating plasma proteins in AF patients with- compared to patients without LVA. UP–unique peptides of the pro-
tein identified in proteome screening, P–t-test p-value, P(FDR)–p-value with FDR, FC–fold change with LVA vs. without LVA, negative fold change indicates lower con-
centrations in the LVA group. An asterisk marks proteins that were also differentially abundant in patients with LAD<44mm vs. LAD44mm (significance in t-test but
f ll
i
FDR) Table 2. Significantly differentially expressed circulating plasma proteins in AF patients with- compared to patients without LVA. UP–unique peptides of the pro-
tein identified in proteome screening, P–t-test p-value, P(FDR)–p-value with FDR, FC–fold change with LVA vs. without LVA, negative fold change indicates lower con-
centrations in the LVA group. An asterisk marks proteins that were also differentially abundant in patients with LAD<44mm vs. LAD44mm (significance in t-test but
not following FDR). centrations in the LVA group. An asterisk marks proteins that were also differentially abundant in patients with LAD<44mm vs. LAD44mm (significance in t test but
not following FDR). Protein names patients, could therefore attribute to the hypercoagulation state often observed in AF. F9 is
part of the intrinsic coagulation cascade, is activated through factor 11, and then cleaves factor
10 which facilitates prothrombin cleavage. Prothrombin was found to be lower in LVA patient
plasma but did not pass FDR significance testing (S1 Table). Altogether, our data indicate an
imbalance of coagulation and complement cascade activation, homeostasis, and regulation in
AF patients with LVA and generate hypothesis regarding whether progression of electro-ana-
tomical remodeling is cause or consequence to those alterations. patients, could therefore attribute to the hypercoagulation state often observed in AF. F9 is
part of the intrinsic coagulation cascade, is activated through factor 11, and then cleaves factor
10 which facilitates prothrombin cleavage. Prothrombin was found to be lower in LVA patient
plasma but did not pass FDR significance testing (S1 Table). Altogether, our data indicate an
imbalance of coagulation and complement cascade activation, homeostasis, and regulation in
AF patients with LVA and generate hypothesis regarding whether progression of electro-ana-
tomical remodeling is cause or consequence to those alterations. Complement and coagulation cascade and electro-anatomical remodeling As initially stated, hypercoagulability associated with an increased risk for stroke, is a common
finding in AF patients [6]. In our study, we observed differential abundance of proteins from
the complement and coagulation system. Classical complement cascade is started by activation
of multimolecular C1-complex which consists of C1q, C1r, and C1s. We found C1q-B and
C1q-C significantly lower in the patients with LVA, whereas the third C1q subcomplex C1q-A
was detected but unchanged. C1qr as well as C1qs were also found to be lower in LVA patient
plasma but did not pass FDR significance testing (S1 Table). Decreased C1-complex activity
was found to associate with impaired immune response and auto-immune reactions [16]. CFH and properdin were also less abundant in the patients with LVA. Both are complement
system regulators which identify and protect the host from complement also during pathologi-
cal states. An imbalance might therefore contribute to tissue damage [17,18]. Plasminogen typically is highly abundant in blood and if catalyzed into plasmin is fibrino-
lytic. Downregulation of plasminogen, which might explain the lower levels observed in LVA 5 / 10 Protein names
UP
P
P (FDR)
FC
Complement C1q subcomponent subunit B
4
0.005
0.044
-1.16
Complement C1q subcomponent subunit C
4
0.005
0.044
-1.16
Caspase-8
1
0.005
0.044
1.92
Complement factor H
40
0.000
0.018
-1.25
Properdin
3
0.005
0.044
-1.23
Cortactin-binding protein 2
1
0.003
0.039
1.76
Dynein heavy chain 12 axonemal
1
0.002
0.025
1.61
Coagulation factor IX
6
0.003
0.035
-1.35
Vitamin D-binding protein
24
0.001
0.018
-1.22
Hepatocyte growth factor activator
6
0.001
0.018
-1.27
Putative heat shock 70 kDa protein 7
1
0.001
0.018
1.77
Kin of IRRE-like protein 3
1
0.005
0.044
1.47
Phosphatidylcholine-sterol acyltransferase
5
0.001
0.018
-1.25
Mannosyl-oligosaccharide 12-alpha-mannosidase
1
0.001
0.018
1.54
Endonuclease 8-like 3
1
0.004
0.041
1.32
Nicotinamide/nicotinic acid mononucleotide adenylyltransferase 3
1
0.001
0.018
1.33
Protocadherin-15
1
0.006
0.046
-1.20
Peptidase inhibitor 16
1
0.001
0.020
-1.69
Plasminogen
22
0.001
0.018
-1.19
Ras-related GTP-binding protein B
1
0.003
0.039
1.44
L-selectin
2
0.000
0.018
-1.75
Kallistatin
8
0.001
0.018
-1.35
Thyroxine-binding globulin
11
0.001
0.018
-1.29
SWI/SNF complex subunit SMARCC1
1
0.001
0.018
2.49
Serine palmitoyltransferase 2
1
0.002
0.025
1.89
Transcription elongation factor SPT6
1
0.000
0.018
1.83
Protein-glutamine gamma-glutamyltransferase 6
1
0.005
0.044
1.41
E3 ubiquitin-protein ligase TRIM33
1
0.006
0.046
1.39
Tetratricopeptide repeat protein 39A
1
0.001
0.018
2.04
Zinc finger protein 30 homolog
1
0.001
0.018
1.75
https://doi.org/10.1371/journal.pone.0198461.t002 Fibrosis, inflammation, and electro-anatomical remodeling LVA is supposed to be an electro-anatomical remodeling with fibrotic character. Fibrosis is
characterized by increased production of ECM, cell death, cell trans-differentiation e.g. myofi-
broblast activation and infiltration of inflammatory cells [19]. Several proteins found altered
by our approach are known to contribute to those pathological processes. The highest plasma
level decrease in the LVA group was found for cell adhesion molecule L-selectin. L-selectin is PLOS ONE | https://doi.org/10.1371/journal.pone.0198461
November 29, 2018 6 / 10 Proteome screening in AF related atrial remodeling expressed on a variety of leukocytes, but is best explored in naïve T-lymphocytes where its
shedding from the cell membrane in the primary lymph nodes occurs during their activation
and has implications for subsequent cell fate. Shedding from the surface is thought to mobilize
intracellular cytotoxic granules leading to the release of lytic enzymes towards target cells [20]. Interestingly, L-selectin was also proposed to be a receptor for the earlier discussed CFH, thus
connecting immune function and complement system [21]. Furthermore, L-selectin was
found to be down regulated through shedding by atrial natriuretic peptide (ANP) leading to
decreased neutrophil infiltration [22]. Recently we found that plasma ANP levels are signifi-
cantly higher and therewith predictive for LVA in AF patients [23] what is well in line with the
literature reports and the now observed lower levels of L-selectin. Furthermore, we observed altered levels of proteins that are annotated to GO:0045861 Neg-
ative regulation of proteolysis whereas proteolysis is indispensable in fibrosis and inflamma-
tion [24]. Peptidase inhibitor 16 (PI16), which was lower in the LVA group, was assigned to
this group. PI16 might contribute to electro-anatomical remodeling as the protein is known to
be regulated by sheer stress, to inhibit ECM-remodeling matrix metallo-proteinases, and is
thought to maintain anti-inflammatory and non-activated cell homeostasis [25]. In context of
heart failure, PI16 is supposed to inhibit myocyte hypertrophy [25]. Caspase-8, another pro-
teolytic enzyme, is involved in controlled extrinsic apoptosis induced via death receptor. The
observed increase in the LVA patient plasma might represent apoptotic activity. Higher circu-
lating levels of caspase-8 were found to associate with increased incidence of cardiovascular
events [26]. Several proteins found at higher abundance in LVA patients are involved in tran-
scriptional processes or chromatin remodeling respectively, namely SWI/SNF complex sub-
unit SMARCC1 (SMARCC1), transcription elongation factor SPT6 (SUPT6H), and zinc
finger protein 30 homologue (ZFP30). Fibrosis, inflammation, and electro-anatomical remodeling Chromatin remodeling was already identified as prom-
ising tool for intervention in fibrotic and inflammatory disease (YANG, targeting). SMARCC1
showed the highest observed difference between the experimental groups in our screening. SMARCC1 is component of a chromatin remodeling complex which is needed to induce tran-
scription e.g. in neurogenesis [27]. ZFP30 encodes a transcription factor which was found to
be involved in differentiation processes and inflammatory response [28]. SUPT6H alters
nuclear histone status to allow for a transcriptionally competent chromatin state [29] especially
important for expression of genes in developmental and differentiation e.g. myogenesis [30]. The second most altered protein in our study was tetratricopeptide repeat protein 39A
(TTC39A) whose function is widely unknown. Interestingly, an interaction of TTC39A with
P2RY12, a receptor involved in platelet aggregation, regarded to be a potential target of throm-
boembolism treatment, was supposed [31]. Altogether, we suppose that the observed markers associate with altered cell proliferation
rates or cell differentiation processes. PLOS ONE | https://doi.org/10.1371/journal.pone.0198461
November 29, 2018 Conclusions This pilot proteomic study identified plasma protein candidates associated with electro-ana-
tomical remodeling in AF and pointed towards an involvement of coagulation and comple-
ment pathway, inflammation, and tissue remodeling. Further studies are underway to replicate
and apply these findings. Proteome screening in AF related atrial remodeling physiological pathways. We used pathway annotation tools to get insights into the physiologi-
cal pathways the identified candidates are assigned to. In general, the source of the detected proteins is beyond the scope of this study and causal asso-
ciation with atrial LVA cannot be proved. Further larger studies are needed to address this aspect. Rhythm outcomes, although clinically relevant, were not analyzed for the following reasons:
Arrhythmia recurrences depend on different factors (operator experience, ablation catheters
and systems used, post-ablation and follow-up strategies, length of Holter ECG). Therefore we
expect that the analysis of this factor is possible in larger prospective studies with continuous
monitoring covered by e.g. implantable loop recorders. Supporting information S1 Table. Proteins detected in proteomic approach comparing 23 patients with LVA and
25 patients without LVA. UP–number of unique peptides belonging to the protein which
were detected in the screening, p-value—from T-Test comparing LVA and controls, p-value
(FDR)–Benjamini Hochberg false discovery rate, FC–fold change in protein levels. Proteins in
bold were found significantly different in LVA group compared to controls. (DOC) S2 Table. Plasma proteins found differentially abundant when comparing patients with
LAD>44mm compared to patients with LAD<44mm. P-value–derived from T-Test, p-value
(FDR)–T-Test data with Benjamini Hochberg false discovery rate, FC–fold change in protein levels. (DOC) S3 Table. Peptide data of the proteins significantly different between patient subgroups
with and without LVA, UP/RP = unique/ razor peptides, Modifications—
CM = Carbamidomethyl, O = Oxidation. (DOC) S3 Table. Peptide data of the proteins significantly different between patient subgroups
with and without LVA, UP/RP = unique/ razor peptides, Modifications—
CM = Carbamidomethyl, O = Oxidation. (DOC) Limitations Several proteins that were discussed typically act inside cells or are bound to them. We inter-
pret this to be indicative for shedding, secretion, endothelial damage (e.g. in consequence of
increased atrial sheer stress) or cell death (e.g. fibrosis-related decline of cardiomyocytes). Although, sample processing aimed to limit cell contamination of the plasma used in this
study the contamination with cell debris is unlikely, nevertheless, it cannot be excluded. The used technology only detected proteins with high and medium abundance in plasma;
typically, 250–350 proteins with concentrations in a range of μg to mg per ml. Lower concen-
trations were not detected whereas we cannot estimate the quantity of natriuretic peptides,
growth factors or cytokines. This limitation also impedes over-enrichment analysis of PLOS ONE | https://doi.org/10.1371/journal.pone.0198461
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of inflammation. 2017; 2017:2570154. https://doi.org/10.1155/2017/2570154 PMID: 28642633 25. Hazell GGJ, Peachey AMG, Teasdale JE, Sala-Newby GB, Angelini GD, Newby AC, et al. PI16 is a
shear stress and inflammation-regulated inhibitor of MMP2. Scientific reports. 2016; 6:39553. https://
doi.org/10.1038/srep39553 PMID: 27996045 26. Xue L, Borne´ Y, Mattisson IY, Wigren M, Melander O, Ohro-Melander M, et al. FADD, Caspase-3, and
Caspase-8 and Incidence of Coronary Events. Arteriosclerosis, thrombosis, and vascular biology. 2017; 37:983–989. https://doi.org/10.1161/ATVBAHA.117.308995 PMID: 28302628 27. Bachmann C, Nguyen H, Rosenbusch J, Pham L, Rabe T, Patwa M, et al. mSWI/SNF (BAF) Com-
plexes Are Indispensable for the Neurogenesis and Development of Embryonic Olfactory Epithelium. PLoS genetics. 2016; 12:e1006274. https://doi.org/10.1371/journal.pgen.1006274 PMID: 27611684 28. Rutledge H, Aylor DL, Carpenter DE, Peck BC, Chines P, Ostrowski LE, et al. Genetic regulation of
Zfp30, CXCL1, and neutrophilic inflammation in murine lung. Genetics. 2014; 198:735–745. https://doi. org/10.1534/genetics.114.168138 PMID: 25114278 29. Sarma K, Reinberg D. Histone variants meet their match. Nature reviews. Molecular cell biology. 2005;
6:139–149. https://doi.org/10.1038/nrm1567 PMID: 15688000 30. Wang AH, Zare H, Mousavi K, Wang C, Moravec CE, Sirotkin HI, et al. The histone chaperone Spt6
coordinates histone H3K27 demethylation and myogenesis. The EMBO journal. 2013; 32:1075–1086. https://doi.org/10.1038/emboj.2013.54 PMID: 23503590 31. Huttlin EL, Ting L, Bruckner RJ, Gebreab F, Gygi MP, Szpyt J, et al. The BioPlex Network: A Systematic
Exploration of the Human Interactome. Cell. 2015; 162:425–440. https://doi.org/10.1016/j.cell.2015.06. 043 PMID: 26186194 10 / 10
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Excerpta Medica
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Unhealthy Lifestyle and Gut Dysbiosis: A Better Understanding of the Effects of Poor Diet and Nicotine on the Intestinal Microbiome
|
Frontiers in endocrinology
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Petia Kovatcheva-Datchary,
University of Würzburg, Germany Petia Kovatcheva-Datchary,
University of Würzburg, Germany University of Würzburg, Germany
Reviewed by:
Raylene A. Reimer,
University of Calgary, Canada
Marie-Christine Simon,
University of Bonn, Germany
*Correspondence:
Theodore C. Friedman
theodorefriedman@cdrewu.edu
†Present address:
Jason E. Martinez
Lewis Katz School of Medicine,
Temple University,
Philadelphia, PA, United States Reviewed by:
Raylene A. Reimer,
University of Calgary, Canada
Marie-Christine Simon,
University of Bonn, Germany The study of the intestinal or gut microbiome is a newer field that is rapidly gaining
attention. Bidirectional communication between gut microbes and the host can impact
numerous biological systems regulating immunity and metabolism to either promote or
negatively impact the host’s health. Habitual routines, dietary choices, socioeconomic
status, education, host genetics, medical care and environmental factors can all
contribute to the composition of an individual’s microbiome. A key environmental factor
that may cause negative outcomes is the consumption of nicotine products. The effects of
nicotine on the host can be exacerbated by poor dietary choices and together can impact
the composition of the gut microbiota to promote the development of metabolic disease
including non-alcoholic fatty liver disease. This review explores the contribution of nicotine,
poor dietary choices and other unhealthy lifestyle factors to gut dysbiosis. *Correspondence:
Theodore C. Friedman
theodorefriedman@cdrewu.edu †Present address:
Jason E. Martinez
Lewis Katz School of Medicine,
Temple University,
Philadelphia, PA, United States Specialty section:
This article was submitted to
Gut Endocrinology,
a section of the journal
Frontiers in Endocrinology
Received: 11 February 2021
Accepted: 17 May 2021
Published: 08 June 2021 Specialty section:
This article was submitted to
Gut Endocrinology,
a section of the journal
Frontiers in Endocrinology Received: 11 February 2021
Accepted: 17 May 2021
Published: 08 June 2021 REVIEW
published: 08 June 2021
doi: 10.3389/fendo.2021.667066 REVIEW
published: 08 June 2021
doi: 10.3389/fendo.2021.667066 Keywords: gut microbiome, gut microbiota, gut dysbiosis, nicotine, obesity, high-fat diet, nonalcoholic fatty liver
disease, e-cigarette 1 Department of Internal Medicine, Charles R. Drew University of Medicine and Science, Los Angeles, CA, United States,
2 Department of Medicine, David Geffen School of Medicine at University of California, Los Angeles, CA, United States,
3 Vatche and Tamar Manoukian Division of Digestive Diseases, Department of Medicine, David Geffen School of Medicine at
University of California, Los Angeles (UCLA), Los Angeles, CA, United States, 4 David Geffen School of Medicine at University of
California, UCLA Microbiome Center, Los Angeles, CA, United States, 5 Division of Gastroenterology, Hepatology and Parenteral
Nutrition, Veterans Administration Greater Los Angeles Healthcare System, Los Angeles, CA, United States Citation: The microbiome has become a rising topic for the
lay public and may hold the key to information capable of reducing
co-morbidities and preventing disease (1). Of note, some
companies market to the public the ability to analyze the
microbiome of an individual to “provide precision nutrition for
metabolic disease” (5). More research is needed, especially on the
cross-communicationbetweenthemicrobiomeandhostinorderto
understand the impact of the microbiome on human disease (3). The use of germ-free (GF) animals has provided evidence that
specific microbiota influences the immune system differently. In
1885, Pasteur first proposed the generation of animals deprived
of microorganisms to explore the relationship between microbes
and their host (22). GF animals display significant defects in the
development of primary (thymus and bone marrow) and
secondary (lymph nodes and spleen) lymphoid organs and are
associated with a decreased frequency of CD4+ and CD8+
intestinal T cell subsets. GF mice also have reduced numbers
of intraepithelial lymphocytes that express the abT cell receptor
(TCR) (22, 23). Specific bacterial populations have been linked to
specific T-cell effector subset development. For example, T helper
17 [Th17] cells, potent sources of interleukin-17 (IL-17), play a
critical role in the clearance of pathogens and the maintenance of
the mucosal barrier integrity (22). GF animals have an absence of
Th17 (22) and thus, Th17 development is believed to be dependent
on the intestinal microbiota (22). Additionally, recent studies have
shown that the adhesion of certain microbes [e.g., segmented
filamentous bacteria (SFB), Citrobacter rodentium, and
Escherichia coli O157] is necessary to trigger a Th17 cell response
in intestinal epithelial cells (24). This was shown by colonizing SFB
in adult GF mice versus standard mouse microbiota (22, 24, 25). Colonization with SFB conferred enhanced protection compared
with GF animals after infection with the bacterial pathogen
citrobacter rodentium, a direct outcome of Th17-cell enrichment
in these animal’s intestinal microbiomes (25). Interestingly, an
exaggerated Th17 response is believed to promote autoimmune
arthritis (25, 26), such that microbial signaling and host immune
response requires a fine balance. Often, the terms “microbiome” and “microbiota” are used
interchangeably. In an effort to define the terms more precisely
(6) microbiota will be used to define the assemblage of
microorganisms present in a defined environment, whereas (7),
mostly the colon, but also the upper gastrointestinal tract (8), saliva
(9), middle meatus (10), bronchial wash (11), sputum (12),
subgingival (13), and throat (14). Citation: Martinez JE, Kahana DD,
Ghuman S, Wilson HP, Wilson J,
Kim SCJ, Lagishetty V, Jacobs JP,
Sinha-Hikim AP and Friedman TC
(2021) Unhealthy Lifestyle and Gut
Dysbiosis: A Better Understanding of
the Effects of Poor Diet and Nicotine on
the Intestinal Microbiome. Front. Endocrinol. 12:667066. doi: 10.3389/fendo.2021.667066 There is considerable variation in microbiome composition and function across individuals. This
interindividual variability and plasticity of the intestinal microbiota has hindered efforts to identify a
“healthy” microbiota. Diversity and microbial stability are often used as key indicators of gut health
because of their inverse association with chronic disease and metabolic dysfunction (1). Reduced
microbial diversity has been shown to be associated with various disease states (1). Instability of the
gut microbiome can be caused by many factors, including infection, diet, exercise, sleep pattern,
exposure to antibiotics, and various co-morbidities. Gut dysbiosis is a broad term (2) that can be
defined as the imbalance of gut microbiota associated with an unhealthy outcome. Dysbiosis There is considerable variation in microbiome composition and function across individuals. This
interindividual variability and plasticity of the intestinal microbiota has hindered efforts to identify a
“healthy” microbiota. Diversity and microbial stability are often used as key indicators of gut health
because of their inverse association with chronic disease and metabolic dysfunction (1). Reduced
microbial diversity has been shown to be associated with various disease states (1). Instability of the
gut microbiome can be caused by many factors, including infection, diet, exercise, sleep pattern,
exposure to antibiotics, and various co-morbidities. Gut dysbiosis is a broad term (2) that can be
defined as the imbalance of gut microbiota associated with an unhealthy outcome. Dysbiosis June 2021 | Volume 12 | Article 667066 Frontiers in Endocrinology | www.frontiersin.org Gut Dysbiosis, Nicotine, and Intestinal Microbiome Martinez et al. involves the loss of beneficial microbial input or signal and an
expansion of pathogenic microbes (pathobionts). Dysbiosis is
thought to trigger pro-inflammatory effects and immune
dysregulation associated with various disease states, including
non-alcoholic steatohepatitis (NASH) (3). molecules that interact with the host environment. The
microbiome, in turn, regulates the development of the immune
system (22). For example, a healthy microbiome has an anti-
inflammatory function by inhibiting histone deacetylases in
regulatory T cells (Tregs) through G-protein coupled receptors
(GPCRs) (19). In 2014, the International Scientific Association for Probiotics
and Prebiotics (ISAPP) provided evidence-based
recommendations of strain-specific probiotics for defined
conditions (4). IMPACT OF MICROBIOTA ON HEALTH A unique bidirectional interaction between the mucosal immune
system and the gut microbiota allows for the avoidance of an
inappropriate immune response towards nonpathogenic microbes
while suppressing pathogenic microbes (pathobionts) (27). For
example, Bifidobacterium and lactic acid bacteria have been shown
to secrete factors that hinder inflammation, presumably via the
downregulation of interleukin-8 secretion, NF-kB dependent gene
expression, and macrophage-attracting chemokine production (28). Furthermore, Bifidobacterium and lactic acid bacteria are associated
with the upregulation of anti-inflammatory Treg cell gene expression
(29). Some studies suggest that microbial-derived SCFAs may be
contributingtothemodulationofhostimmuneresponsesdirectlyvia
G-protein-coupled receptors and epigenetic mechanisms, such as
methylation activity within the promoter regions of certain genes
(30, 31). The gut is known to harbor a unique and dynamic microbiome as it
is exposed to constant external stimuli, including diet, infectious
agents, antibiotics and xenobiotics (15–17). Digestible and non-
digestible carbohydrates, proteins, fats, polyphenols, prebiotics and
probiotics can induce shifts in the microbiota and elicit effects on
host immunologic and metabolic markers (18). There appears to be
a close relationship between the gut microbiome, health, and diet,
suggesting that improvements in health can be modulated via diet
through the microbiota. Citation: Microbiome will refer to the
entire habitat of a host region with its surrounding environmental
conditions, including all microorganisms, bacteria, archaea, lower
and higher eukaryotes, viruses, and their genomes (6). As will be discussed in the body of this review, fiber-depleted
diets create a condition ripe for dysbiosis. Moreover, nicotine, the
exposure to which may be increasing with the rise of electronic
cigarettes (e-cigarettes), has been shown to accentuate the effect of
diet and potentially disrupt the microbiome and promote disease. Microbiome, Immune Dysfunction,
and Inflammation The microbiome serves many important functions. In healthy
individuals, it confers protection from pathogenic organisms that
cause infection. For example, the microbiota produce short-
chain fatty acids (SCFAs) via fermentation of complex plant
carbohydrates, providing an energy source for colonocytes to
maintain full differentiation and regeneration (19). Furthermore,
the microbiota synthesizes essential vitamins and amino acids,
regulate fat metabolism (20, 21), and produce various small Dysbiosis, as defined above, is believed to contribute to the
development of various immune-mediated conditions, including
inflammatory bowel disease (IBD) (21), rheumatoid arthritis
(32), type 1 diabetes mellitus (33), multiple sclerosis (34), and
systemic lupus erythematosus (SLE) (35), among many others. IBD, which comprises of Crohn’s disease and ulcerative colitis, is June 2021 | Volume 12 | Article 667066 Frontiers in Endocrinology | www.frontiersin.org 2 Gut Dysbiosis, Nicotine, and Intestinal Microbiome Martinez et al. a chronic inflammatory disease that is increasing in prevalence
worldwide (36) and has been proposed to arise from an
inappropriate mucosal inflammatory response to dysbiosis; this
response is believed to be a result of genetic susceptibility and
environmental exposure (37). IBD patients have decreased
microbial diversity compared with healthy controls, alongside
alterations in both composition and function of the intestinal
microbiome (38–43). Jacobs and colleagues investigated if
unaffected siblings and parents of individuals with IBD carry a
pre-disease microbial risk state due to shared genetic and
environmental factors. By studying the microbiome and
metabolome of pediatric IBD patients and their unaffected
first-degree relatives (43), they were able to identify a high
correlation between fecal microbial and metabolomics profiles
and disease status. Their research proposed that in families at
risk for IBD, healthy individuals possess an intestinal microbial/
metabolomic state with increased susceptibility to IBD (43). This
finding highlights the ability of microbes to increase
susceptibility to inflammatory disease via the production of
bioactive metabolites, which affect immune activity and
epithelial function (43). low levels of Bifidobacterium, a key bacterial group, is notably
linked to obesity, particularly in children (56). Furthermore, a
gut microbiome that is largely dominated by Firmicutes showed
altered methylation in gene promoters linked to obesity and
cardiovascular disease (57). Some bacterial species, such as
Lactobacillus spp., can be obesogenic or anti-obesogenic,
depending on the specific strain; others have the potential to
alleviate obesity-associated metabolic complications (58, 59). Exercise and the Intestinal Microbiome Exercise and the Intestinal Microbiome
Exercise has received much praise for its ability to regulate
weight, insulin sensitivity, metabolic activity and contribute to
overall improvement in health. There is growing evidence to
support the role of exercise in regulating human intestinal
microbiota (63–73). Emerging research shows that exercise
training independently altered the composition and function of
the gut microbiota (74–78). Matsumoto et al. originally found
that 5 weeks of exercise training in animals resulted in an
increased production of the short chain fatty acid butyrate, a
product of the bacterial fermentation of dietary fiber by bacteria
such as Bifidobacteria (79). Matsumoto et al. also found that
exercise training in mice increased the relative abundance of
butyrate-producing taxa (75, 80). Butyrate is the primary fuel for
colonocytes and has been shown to increase colonic epithelial
cell proliferation, regulate host immune system and gene
expression, and promote the integrity of the gut barrier (74,
81, 82). Conflicting evidence still exists for exercise and the
intestinal microbiome; for example, some rodent studies found
that exercise reduced the ratio of Firmicutes to Bacteroidetes (77,
80, 83, 84), while others found that exercise increased the ratio
(75, 76, 85). These discrepancies may be influenced by the kind
and degree of exercise (e.g., in mice, voluntary wheel running or
forced treadmill running), the contingencies of the diet, age of
the animal, species/strain, and method of research. y
Obesity, which confers an increased risk for numerous diseases,
including hypercholesterolemia, hypertension, type 2 diabetes,
cancer, non-alcoholic fatty liver disease (NAFLD),
atherosclerosis, cardiovascular disease, and stroke (44, 45), is
associated with gut dysbiosis (46). Intestinal microbiota
influence the digestion, absorption, metabolism, and storage of
ingested nutrients with profound effects on host physiology (46). Environmental and dietary factors can yield a microbiome that
modulates host metabolism to promote obesity (46, 47). Advancements in studying the role of a high-fat diet (HFD)
and Western diet (WD) on the microbiome has provided
insights into the mechanisms of how gut dysbiosis leads to
detrimental metabolic changes and why many individuals who
consume a HFD or WD develop gut dysbiosis. Studies of lean and genetically obese (ob/ob) mice (48, 49) and
(fa/fa) rats (50) have revealed differences in their metabotypes
[i.e., metabolic phenotypes (51)]. Lean and obese gut
microbiomes are characterized by different representation of
members of the Bacteroidetes, Firmicutes and Actinobacteria
phyla of bacteria. Microbiome, Immune Dysfunction,
and Inflammation Part of the mechanism of action is via the interaction between
the gut microbiota, host immunity, and gut barrier function
(60, 61). An abundance of Akkermansia muciniphila, has been linked
to a healthy metabolic profile, with greater improvement in
obesity-associated metabolic parameters (plasma triglycerides,
body fat distribution, and insulin tolerance) for individuals with
obesity following dietary intervention (62). These findings
highlight the critical role of the gut microbiota in maintaining
the metabolic integrity of the host, from energy harvest to
metabolic activity. But energy intake is also balanced with
energy expenditure, which segues to the topic of exercise and
its impact on the microbiome. Frontiers in Endocrinology | www.frontiersin.org June 2021 | Volume 12 | Article 667066 Exercise and the Intestinal Microbiome One intriguing discovery that follows from
these studies is the link between gut microbiomes and host
energy harvest and homeostasis (52). Some individuals may
harbor microbiota that are more efficient at energy harvest
than others; for example, some types of bacteria may be better
at processing carbohydrates than others. Other types of bacteria
may be adept at manipulating host genes and metabolism in
order to store energy, turn off satiety signals, or upregulate
inflammatory pathways (52). In professional rugby players, Clarke et al. found that the
intestinal microbiota showed an increase in alpha diversity
(variance within the sample), with a higher relative abundance
of 40 different bacterial taxa compared to lean sedentary controls
(63); these athletes had a lower abundance of lactobacillus and
bacteroides species than the lean sedentary group (63). Bressa et
al. found that women who performed at least 3 hours of exercise
per week had a greater abundance of Faecalibacterium
prausnizii, Roseburia hominis , and A. muciniphilia compared
to sedentary controls (64). A. muciniphila has been associated A higher baseline ratio of Firmicutes to Bacteroidetes ratio is
seen in individuals with obesity, and for these subjects, a
reduction in caloric intake resulted in a lower Firmicutes to
Bacteroidetes ratio (53, 54). However, Magne et al. (55) reported
that it was difficult to associate the Firmicutes/Bacteroidetes ratio
with a determined health status or as a hallmark of obesity. Yet, June 2021 | Volume 12 | Article 667066 3 Gut Dysbiosis, Nicotine, and Intestinal Microbiome Martinez et al. in neural tissue and implicated in PD, misfolds and forms clumps
in neural tissue in response to gut dysbiosis (97–99). One
plausible hypothesis is that the innervation of the GI tract is
easily damaged (96, 97) and that ENS injury is caused by an
unknown PD pathogen that may present as a-syn pathology. Several clinical studies revealed that PD patients displayed a-syn
aggregates in the enteroendocrine system (96, 97) and that these
aggregates are related to damage of enteric neurons and
associated with GI tract dysfunction. This type of protein
aggregate accumulation affects both the myenteric and
submucosal plexuses of the gut in PD patients and is gradually
distributed from the most distal point of the esophagus to the
rectum (96). Moreover, gut dysbiosis is believed to result in
upregulated inflammatory pathways that may trigger the
initiation of synucleinopathy (100–102). The Composition of the Intestinal
Microbiome Revolves Heavily Around Diet The Composition of the Intestinal
Microbiome Revolves Heavily Around Diet Dietary factors are often potent modulators of microbiota
composition and function. Transient, diet-induced alterations
occur independently of body weight and adiposity and are
detectable in humans within 24 to 48 hours after dietary intake
(103). A micronutrient-dense, high-fiber diet with sufficient
water intake and high-quality protein, along with avoidance of
common Western dietary components, such as saturated and
trans-fat, simple sugar, refined flour, high-fructose corn syrup,
and other processed foods, is believed to have a protective effect
regarding intestinal dysbiosis (104). Particularly important for the health of the microbiome are
carbohydrates (CHO) that are indigestible yet metabolically
available to microbes within the intestines. Termed
“microbiota-accessible carbohydrates” (MACs) (105), these
include fermentable fibers and non-digestible polysaccharides
found in resistant starch foods, such as those originating from
plants (106). Intestinal microbes contain several hundred-fold
more CHO-degrading enzymes than what is produced by human
enterocytes; this enables the microbes to digest MACs for their
primary source of energy (46). IBS pathophysiology is not fully understood, but for many
patients, an element of visceral hyperalgesia is implicated and
alterations in the bidirectional communication of the gut-brain
axis may cause an exaggerated pain response to an otherwise
normal digestive process (94). Labus and colleagues identified a
correlation between brain architecture and the gut microbiota in
a distinct subgroup of IBS patients, suggesting that gut
microbiota and their metabolites may influence specific brain
structures. The authors concluded that a microbe-gut-brain axis
plays an important role in the pathophysiology of disrupted
sensory processing in IBS (95). The importance of MACs on microbiota composition and
function is documented in multiple studies. In one illustrative
mouse experiment, a diet low in MACs resulted in a decrease in
numerous taxa and a loss of diversity across several generations
of offspring that were not recovered after reintroduction of
MACs (105, 107). In humans, a low-MAC diet results in poor
production of intestinal microbiota-generated SCFA, which are
known to reduce inflammation through a variety of mechanisms
(104). Decreased SCFA production and increased mucus
foraging by the microbiota demonstrate consequences to low
MAC intake (46). However, the intake of excessive calories to Emerging evidence also suggests that gut microbiota play an
important role in several neurological conditions, such as
Parkinson’s disease (PD), Alzheimer’s disease, and multiple
sclerosis (96). Brain-Gut-Microbiome Interactions Communication between the gut microbiota and the brain is an
important area of research. A vital bidirectional signaling system
between the gastrointestinal tract and the brain helps maintain
metabolic homeostasis and is regulated via neural (central and
enteric nervous systems, CNS and ENS, respectively),
immunological, and hormonal systems (87). The perturbation
of these systems through external factors, such as diet or
antimicrobial use, leads to alterations within stress-response
mechanisms, behavior, and neurologic health (88, 89). A well-documented, clinical association of an aberrant gut-
brain axis is the manifestation of stress-related symptoms, such
as anxiety or depression, which can lead to constipation or
diarrhea and is sometimes diagnosed as irritable bowel
syndrome (IBS) (90). In animal models, Neufield and
colleagues illustrated that colonization of GF mice with specific
pathogen-free microbiota decreased anxiety-like behavior in a
well-validated maze model of anxiolytic action (91). The study
also showed changes in the murine neurochemistry, with
upregulated expression of brain-derived neurotrophic factor
(BDNF) mRNA in the hippocampal dentate gyrus (91). BDNF
expression is believed to be critical for supporting synaptic
plasticity and neuronal differentiation and survival. Stressful
exposures can reduce the expression of BDNF, thereby
theoretically affecting cognitive and emotional health (92). Bistoletti and colleagues demonstrated the effect of broad-
spectrum antibiotics in transiently decreasing BDNF levels and
increasing anxiety behaviors in juvenile mice (93). Together,
these studies provide important evidence that the brain and
behavior, specifically anxiety, can be influenced by the
microbiota through the gut-brain axis. Exercise and the Intestinal Microbiome If a dysfunctional gut
contributes to PD, then there lies a strong possibility that the gut
affects the brain in a host of ways, including neurological and
psychiatric disease. with lower body mass index (BMI) (62) and improved metabolic
health in other studies, whereas F. prausnitzii and R. hominis are
known to be butyrate producers (86). Overall, there is strong
indication that exercise can benefit the intestinal microbiome by
i m p r o v i n g
m i c r o b i a l
d i v e r s i t y
a n d
i n c r e a s i n g
butyrate production. Frontiers in Endocrinology | www.frontiersin.org June 2021 | Volume 12 | Article 667066 The Composition of the Intestinal
Microbiome Revolves Heavily Around Diet Several studies have observed that PD patients
may have gastrointestinal disorders before displaying motor
symptoms, and suggested that gut dysbiosis may be implicated,
but the specific link is not clearly understood (96). Studies in rats
have demonstrated that alpha-synuclein (a-syn), a protein found June 2021 | Volume 12 | Article 667066 June 2021 | Volume 12 | Article 667066 4 Gut Dysbiosis, Nicotine, and Intestinal Microbiome Martinez et al. diversity of Bifidobacterium and Eubacterium species, as well as
increased Enterobacteria and Bacteroides (115–117). One
mechanism by which gut microbes mediate the negative
metabolic consequences of a HFD is through translocation of
lipopolysaccharide (LPS), also known as endotoxin, a cell-wall
component of Gram-negative bacteria. Increases in circulating
LPS can occur after a high-fat meal, with exacerbated effects in
individuals with obesity (126). Once in circulation, LPS elicits a
potent inflammatory response via Toll-like receptor-4 (TLR-4)
signaling, which has been implicated in the development of
cardiovascular and metabolic disease (45, 127). Other
functional differences include the association between the WD
and an increase in the production of cancer-promoting
nitrosamines (128, 129); this is likely related to the high
quantity and poor quality of animal protein in the WD,
especially processed meat. obtain an increase in MAC cannot be recommended due to the
consequence of a caloric surplus (108). Instead, balancing caloric
intake based on basal metabolic rate and total daily energy
expenditure, alongside consumption of micro-nutrient dense,
high-fiber, well-balanced foods, may be a better approach for
optimizing gut microbial and human health (109). A high-protein diet (HPD) is another approach for
potentially negating the harmful effects of a Western diet
(WD). In a study done by Wang and colleagues, rats fed a WD
for 12 weeks showed an increase in body weight and fat mass. When the rats were switched to a HPD for 6 weeks, the rats had
reduced fat mass without significant weight loss, a retention of
muscle mass, normalized blood glucose levels, and decreased
feeding after intraperitoneal injection of cholecystokinin (CCK)
compared to rats with diet-induced obesity treated with CCK
(110). The authors concluded that a HPD may be useful in
promoting fat loss, restoring glucose homeostasis, and improving
CCK sensitivity, as well as maintaining muscle mass during
periods of caloric restriction. The Composition of the Intestinal
Microbiome Revolves Heavily Around Diet Furthermore, since the HPD-fed rats
showed an enrichment of 114 operational taxonomic units (OTUs)
and depletion of 188 OTUs, it was concluded that the microbiome
was involved with the measured metabolic alterations. An example
of the significant microbial difference is the positive association
between A. muciniphila and Phascolarctobacterium with decreased
fat mass in the HPD-fed rats compared to WD-fed rats (110). A. muciniphila was identified to correlate with fat loss and may
represent a secondary mechanism for the beneficial effects of
HPD (110). Furthermore, the study showed that WD-fed rats had
increased cytokine expression in the hypothalamus and dorsal
medulla, which was unchanged after switching to HPD (110). The MD, largely acknowledged as a healthier diet than the
WD, is characterized by intake of a beneficial fatty acid profile
rich in mono- and polyunsaturated fatty acids, nondigestible
fibrous plant sources and other low glycemic carbohydrates, and
high levels of polyphenols, along with other antioxidants and
micronutrients (119). Several studies have identified that a
typical MD carries a lower risk of obesity, results in a better
lipid profile, and lowers inflammation. From a microbial
perspective, these characteristics were associated with increases
in Prevotella, Lactobacillus, and Bifidobacterium, and decreases
in Clostridium (130–135). Diets enriched in fiber and fermentable, plant-based foods
include vegan and vegetarian diets. These two diets were shown
to promote significantly lower counts of Bacteroides and
Bifidobacterium species (p < 0.001), compared to an unrestricted
control diet (136). One study determined that differences in the
intestinal microbiomes of subjects consuming an omnivorous diet
versus subjects consuming a vegan or vegetarian diet showed
significantly lower stool pH than controls (137). This is likely due
totheformationofSCFA,likebutyricacid,aswellaslacticacidfrom
Lactobacillus bacteria. A lower stool pH is believed to confer an
element of colonization resistance against pathogens. Kaptan and colleagues found consumption of a low-calorie diet
by adolescent rats led to an increase in microbial diversity, adult
neurogenesis, BDNF levels, and improved cognition (111). Conversely, mice fed a HFD exhibited gut dysbiosis, decreased
synaptic plasticity, and increased anxiety-like behaviors (112–114). Overall, diet has an important effect on the microbiome and
its ability to communicate amongst different systems in the body. The studies noted above on MACs, WD, HPD, low-calorie diets,
and HFD, and their effects on the microbiome, highlight the
importance of maintaining a healthy gut microbiome through
various dietary interventions. The Composition of the Intestinal
Microbiome Revolves Heavily Around Diet A fairly new diet that was initially recommended for those with
celiac disease (CD) but has now gained popularity by the general
population is the gluten-free diet (GFD). In patients with CD, GFD
is intended to reduce the effects of an autoimmune response against
deamidated gliadin (a component of gluten). However, in one
study, Sanz et al. enrolled 10 healthy subjects to consume a GFD
for 30 days and noted an associated decrease in beneficial
populations of bacteria (Bifidobacterium and Lactobacillus), while
potentially increasing unhealthy populations of bacteria; this was
hypothesized to be caused by a reduced polysaccharide intake
associated by GFD (121). In addition, the total number of
Enterobacteriaceae and E. coli increased, theoretically increasing
the risk for opportunistic pathogens (121). A different study on the
effects of short-term GFD showed increases in Clostridiaceae and
Victivallaceae, and reductions in Ruminococcus bromii,
Veillonellaceae and Roseburia faecis (122). Veillonellaceae is
considered to be a pro-inflammatory family of bacteria. The
clinical consequence of GFD in non-celiac individuals is largely Frontiers in Endocrinology | www.frontiersin.org Metabolic Health Impact of Various Diets
on the Microbiome Many diets emphasize the utility of a specific macronutrient (e.g.,
high-protein or low-fat) or the avoidance of a specific ingredient
(e.g., dairy- or gluten-free). Several well-known diets have been
studied for their ability tomodulateintestinal microbiota,including
WD (high animal fat/protein) (115–117), Mediterranean (MD)
(high-fiber,high-monounsaturatedfat,antioxidant-rich,andlowin
red meat) (118–120), vegetarian, vegan, and gluten-free (121–125). The WD, which is high in animal protein and saturated fat
and low in fiber, is usually low in MACs and has been shown to
lead to a reduction in microbial diversity and altered
functionality of the intestinal microbiota compared to control
diets. Many studies document that a WD caused decreased Many diets emphasize the utility of a specific macronutrient (e.g.,
high-protein or low-fat) or the avoidance of a specific ingredient
(e.g., dairy- or gluten-free). Several well-known diets have been
studied for their ability tomodulateintestinal microbiota,including
WD (high animal fat/protein) (115–117), Mediterranean (MD)
(high-fiber,high-monounsaturatedfat,antioxidant-rich,andlowin
red meat) (118–120), vegetarian, vegan, and gluten-free (121–125). The WD, which is high in animal protein and saturated fat
and low in fiber, is usually low in MACs and has been shown to
lead to a reduction in microbial diversity and altered
functionality of the intestinal microbiota compared to control
diets. Many studies document that a WD caused decreased June 2021 | Volume 12 | Article 667066 Frontiers in Endocrinology | www.frontiersin.org 5 Gut Dysbiosis, Nicotine, and Intestinal Microbiome Martinez et al. Nolan-Kenney and colleagues compared the composition of
intestinal microbiomes in smokers vs. non-smokers by
collecting stool samples in a cross-sectional study of 249
participants selected from the Health Effects of Arsenic
Longitudinal Study (HEALS) in Bangladesh (147). They
examined the associations between the status and the intensity
of smoking with the relative abundance and presence of
individual bacterial taxon, from phylum to genus (147). In
current/active smokers, they found that the relative abundance
of bacterial taxa among the Eryispelotrichi-to-Catenibacterium
lineage was significantly higher compared to non-smokers (147). They calculated a 1.91 odds ratio (OR) (95% confidence interval
[CI] = 1.36 to 2.69) for the genus Catenibacterium when
comparing the mean relative abundance in current smokers
with that in subjects who never smoked, and a 1.89 OR (95%
CI = 1.39 to 2.56) for the family Erysipelotrichaceae, order
Erysipelotrichale, and class Erysipelotrichi (false discovery rate-
adjusted p-values=0.0008 to 0.01) (147). Metabolic Health Impact of Various Diets
on the Microbiome Moreover, for each of
these bacterial taxa, a nicotine/smoking dose-response
association was observed, with increasing mean relative
abundance of specific taxa as packs per day of cigarettes
increased. In addition, the presence of Alphaproteobacteria was
significantly greater (OR = 4.85, false discovery rate-adjusted p-
values = 0.04) in current smokers vs. non-smokers (147). The
data are consistent with other studies that associate smoking and
its intensity with a change in the intestinal microbial
composition (Table 1), suggesting that cigarette smoking plays
a significant role in gut dysbiosis, especially as the level of
tobacco exposure increases. unknown and therefore GFD cannot be recommended for the
general population based on the available data. Intermittent fasting, another recently popular diet, led to
changes in the microbiome as well as improvement in
metabolic parameters (138). Eight weeks of intermittent fasting
revealed that the community structure of the intestinal
microbiota was not significantly changed overall, but there
were changes in the abundance of Ruminococcaceae at the
family level and Roseburia at the genus level. This was
accompanied by an increased production of SCFA, decreased
circulating levels of lipopolysaccharide (LPS) and inflammatory
cytokines, ameliorated markers of oxidative stress, improved
vasodilatory parameters, and reduced subject body fat mass
(138). There is great need for further research on the health
benefits of fasting and the role it plays in autophagy and cellular
regeneration, especially in the liver. EFFECT OF NICOTINE ON MICROBIOME
AND INTERACTIONS WITH DIET Smoking cigarettes has an impact on gut health, including
changes in the microbiome that can affect overall health. Nicotine, the psychoactive component of tobacco, binds to
nicotinic acetylcholine receptors (nAChR), such as the a4/b2
receptor, and low-affinity receptors, such as a7 in the CNS and
peripheral tissues (139, 140). Nicotine, when given with a HFD,
leads to hepatic and muscle steatosis that is thought to be due, at
least in part, to increased abdominal fat lipolysis (141–143). In an
animal study, we found that the a7nAChR agonist PNU-282987
protects against nicotine and HFD–induced hepatic steatosis in
genetically obese mice (144). In this mouse model, smoke-
exposed mice showed an alteration in colonic bacterial activity
and community structure, with an increase of Lachnospiraceae
sp (145). Frontiers in Endocrinology | www.frontiersin.org Electronic Cigarettes (E-Cigarettes)
and Public Health E-cigarette use is a public health crisis that is sweeping the
United States; this epidemic involves not only adults, but also
teens. E-cigarettes came to the markets in the mid-2000s and
were advertised as ‘safer’ alternatives to conventional cigarettes
and an effective way to stop smoking (152). However, e-cigarettes
are much less regulated than traditional cigarettes, leading to
extremely variable nicotine levels, with some reaching levels
above combustible cigarettes (153). Many studies have shown
detrimental effects of e-cigarette use including on the liver, heart
and lung (144, 145, 154–161). In a mouse model, we have found
that e-cigarette use is linked to cardiovascular and hepatic
diseases (144, 155, 159). Our laboratory is studying the effects
of e-cigarettes on mouse microbiota and we will be reporting our
results in the near future. In human microbiome studies, tobacco smokers displayed a
higher relativeabundance of Prevotella, lower relative abundanceof
Bacteroides, and a lower Shannon diversity (a measurement of
diversity) compared to controls (146). Biedermann and colleagues
described a decrease of Bacteroides as well as alterations in the
abundance of Alphaproteobacteria and Betaproteobacteria
following the cessation of smoking (147). Indeed, smoking
cessation induced profound changes in the gut microbiome, with
an increase of Firmicutes and Actinobacteria and a decrease of
Bacteroidetes and Proteobacteria at the phylum level; smoking
cessation also induced an increase in microbial diversity (148). Importantly, the intestinal microbial composition of smokers and
non-smokers were different when fed identical meals to avoid the
influences of dietary factors (149). Other human studies have
confirmed that smoking is associated with variances not only in
the intestinal microbiome, but also the upper gastrointestinal tract
(8), saliva (9), middle meatus (10), bronchial wash (11), sputum
(12), subgingival (13), and throat (14). ThestudybyStewartandcolleagues(146)alsofoundthattobacco
smoking had a significant effect on the bacterial profiles when
compared to e-cigarette users. The most significant associations
were an increased relative abundance of Prevotella (P=0.006) and
decreased Bacteroides (P =0.036) in the stool of tobacco smokers
versus e-cigarette users. In contrast, no significant difference was
found in the alpha diversity, beta-diversity (variability in community
composition) or taxonomic relative abundances between e-cigarette
users and controls. Therefore, the authors concluded that the use of
e-cigarette users may represent a safer alternative compared to Several studies investigated the intestinal microbiomes of
smokers vs. non-smokers, but these involved mostly Crohn’s
disease patients or had a small sample size (148, 150, 151). June 2021 | Volume 12 | Article 667066 n = 37
(18 adults with COPD, 8
smokers with no airways
disease, and 11 healthy
individuals) Electronic Cigarettes (E-Cigarettes)
and Public Health June 2021 | Volume 12 | Article 667066 Frontiers in Endocrinology | www.frontiersin.org 6 Gut Dysbiosis, Nicotine, and Intestinal Microbiome Martinez et al. TABLE 1 | Human research studies conducted on the effect of smoking or nicotine on
Study
Study Purpose
Sample Size
(146)
To evaluate the effect of electronic cigarettes
(EC) or tobacco smoking in oral and gut
microbiota. n = 30
(10 EC users, 10 tobacco
users, 10 controls)
(147)
To compare the gut microbiome of smokers
versus nonsmokers. n = 249
(148)
To assess the changes in the intestinal
microbiome associated with smoking
cessation. n = 20
(10 subjects in the
experimental group; 5
continuing smoker control
subjects; 5 non-smoker
control subjects)
(149)
To identify the association between human
intestinal microbiota (HIM) and smoking habits
via data mining analysis. n = 92
(8)
To assess the relationship between tobacco
use and changes in the upper gastrointestinal
microbiome. n = 278
(46.8% current smokers,
12.6% former smokers,
40.6% never smokers)
(9)
To investigate the association of cigarette
smoking with the oral microbiome. n = 1204
(26.3% never smokers,
63.3% former smokers,
10.4% current smokers) Findings (11)
To examine microbiota found in the lower
airway in patients with COPD, smokers without
COPD and non-smokers. n = 37
(18 adults with COPD, 8
smokers with no airways
disease, and 11 healthy
individuals) Culture-independent analysis showed increased Pseudomonas, greatest in the
lower airways of patients with COPD. There was decreased alpha and beta diversity in the COPD group. Bacteroidetes (Prevotella spp) was increased in the non-COPD comparison
groups. Bacteroidetes (Prevotella spp) was increased in the non-COPD comparison
groups. Co-occurrence bacterial taxa and putative core were observed within the lower
airways. Co-occurrence bacterial taxa and putative core were observed within the lower
airways. (12)
To investigate the relation between host
genetics and lifestyles with sputum microbiota
compositions. Lifestyle factors considered
include smoking, alcohol consumption, and
physical activity. n= 257
(13)
To examine the effect of smoking on the
composition of the subgingival microbiome
and associated risk for disease. n = 200
(14)
To investigate the changes in the upper airway
microbiome that result from smoking. n > 4,000 adults. (12)
To investigate the relation between host
genetics and lifestyles with sputum microbiota
compositions. Lifestyle factors considered
include smoking, alcohol consumption, and
physical activity. n= 257
(13)
To examine the effect of smoking on the
composition of the subgingival microbiome
and associated risk for disease. n = 200
(14)
To investigate the changes in the upper airway
microbiome that result from smoking. n > 4,000 adults. Providencia and Bacteroides were influenced by host genetic factors. Smoking had the strongest effect on the overall microbial community structure
compared to other tested lifestyle factors. Veillonella and Megasphaera were increased in current-smokers, and increased
further with the pack-year value and the Fagerstrom Test of Nicotine Dependence
(FTND) score. Haemophilus decreased with the pack-year of smoking and the FTND score. Co-occurrence taxa influenced by host genetics were found together. Subgingival microbial profiles were different at all taxonomic levels in smokers
compared to nonsmokers. Subgingival microbial profiles were different at all taxonomic levels in smokers
compared to nonsmokers. Principle coordinate analysis: microbial community clustering performed based on
smoking status. Smokers were characterized by a highly diverse, pathogen-rich, commensal-poor,
anaerobic microbiome that closely resembles disease-associated communities. Approximately 25,000 sequence reads were generated. Samples clustered in the first principal coordinate by whether they were smokers. (19% of variance). n = 101
(70 CRS patients and 31
control subjects) (10)
To evaluate the relation between smoking
history and sinonasal microbiome alterations in
chronic rhinosinusitis (CRS) and non-CRS
subjects.
n = 101
(70 CRS patients and 31
control subjects) Findings Tobacco smokers had higher relative abundance of Prevotella, lowered
Bacteroides, and lowered Shannon diversity. No significant differences were found in alpha diversity, beta-diversity, or
taxonomic relative abundances between EC users and controls. Bacterial taxa along the Erysipelotrichi-Catenibacterium lineage and
Alphaproteobacteria increased in current smokers. Each taxa exhibited dose-
response associations. Increased abundance of Firmicutes and Actinobacteria in smokers. Decreased
abundance of Bacteroidetes and Proteobacteria on phylum level in smokers. Microbial diversity increased following smoking cessation. Decision tree was successfully able to identify smokers and non-smokers using
operational taxonomic units (OTUs) for analysis. Related OTUs were all found to be uncultured bacteria. Subjects were divided into current smokers and never smokers and were
characterized by alpha and beta diversity of the gut microbiome. Current smokers
had increased alpha (mean 42.3 species) versus never smokers (mean 38.9
species) and exhibited increased beta diversity, Dialister invisus, and
Megasphaera micronuciformis. Decision tree was successfully able to identify smokers and non-smokers using
operational taxonomic units (OTUs) for analysis. Related OTUs were all found to be uncultured bacteria. Subjects were divided into current smokers and never smokers and were
characterized by alpha and beta diversity of the gut microbiome. Current smokers
had increased alpha (mean 42.3 species) versus never smokers (mean 38.9
species) and exhibited increased beta diversity, Dialister invisus, and
Megasphaera micronuciformis. Current smokers had decreased Proteobacteria (4.6%) compared with never
smokers (11.7%) at class, genus and OTU levels
No difference in Proteobacteria was found between former and never smokers. Reduced genera Capnocytophaga, Peptostreptococcus and Leptotrichia in
current smokers compared with never smokers. Functional analysis revealed
these genera were related to carbohydrate, energy, and xenobiotic metabolism
Increased Atopobium and Streptococcus in current smokers compared with
never smokers. Current smokers had decreased Proteobacteria (4.6%) compared with never
smokers (11.7%) at class, genus and OTU levels No difference in Proteobacteria was found between former and never smokers. Reduced genera Capnocytophaga, Peptostreptococcus and Leptotrichia in
current smokers compared with never smokers. Functional analysis revealed
these genera were related to carbohydrate, energy, and xenobiotic metabolism
Increased Atopobium and Streptococcus in current smokers compared with
never smokers. Univariate analysis demonstrated that genus-level compositions of the middle
meatus microbiota are significantly associated with smoking (p= 0.04),
preoperative antibiotics (p= 0.03), and purulence (p= 0.0002). Multivariable model demonstrated that CRS (p= 0.02), polyposis (p= 0.03),
purulence (p= 0.0004), and use of saline rinses (p = 0.5) have significant
interactions with smoking. Nicotine Interaction with Diet in NAFLD
and Obesity y
NAFLD poses a significant health risk, affecting 20 to 40% of
adults in the general American population and over 70% of
individuals with obesity (44). Alongside obesity, nicotine is
acknowledged as a risk factor for NAFLD (162, 163). There are
at least three mechanisms by which smoking and/or nicotine
appear to have adverse effects on the liver: toxic, immunologic,
and oncogenic (164). The toxic effects include oxidative stress,
which results in the activation of stellate cells, leading to fibrosis;
an increase in proinflammatory cytokines (e.g., IL-1, IL-6, IL-8,
TNF alpha) is a direct contributor to liver cell injury. The
immunologic effects of smoking are both cell-mediated (e.g.,
apoptosis of lymphocytes, impaired natural killer cell activity)
and humoral (i.e., suppression of antibody formation). The
oncogenic effects of smoking include carcinogens found in
cigarettes, such as hydrocarbons, nitrosamines, tar, and vinyl
chloride that can lead to NAFLD. Tobacco consumption has also
been implicated in the reduction of p53, a tumor-suppressing
gene, which may be a common pathway of oncogenesis for many
neoplasms (164). Lastly, nicotine may contribute to increased gut permeability
and has been implicated in poor outcomes in IBD patients (174). Miele and colleagues showed that patients with biopsy-proven
NAFLD also experienced significantly greater gut permeability
due to the disruption of intercellular tight junctions in the
intestine compared to healthy volunteers (175). Both increased
gut permeability and the prevalence of small intestinal bacterial
overgrowth (SIBO), which is correlated with the severity of
steatosis in NAFLD patients (175). Since smoking appears to
induce profound changes in the intestinal microbiota (148, 176),
we hypothesize that nicotine with HFD could compound and
lead to increased intestinal permeability, LPS activation of TLRs
and the inflammasome (167), induce changes in SCFAs
metabolism (167), decreased choline availability, and increased
trimethylamine production (167), all of which could contribute In addition to the three mechanisms noted above, nicotine also
appears to exacerbate obesity-induced hepatic steatosis (44) via gut
dysbiosis and its influence on the pathogenesis of NAFLD (44, 165–
167). When nicotine is combined with a HFD in mice, there is a
significant increase in the levels of serum and hepatic triglyceride, as
well as circulating free fatty acids (141, 143, 161). In mice, nicotine
exacerbates hepatic steatosis through increased hepatocellular
apoptosis and oxidative stress, as well as decreased phosphorylation
(i.e., inactivation) of adenosine-5-monophosphate-activated protein
kinase. Findings by whether they were never smokers (17% of variance). Former smokers were
distributed within and between both these clusters. Specific OTUs increased or decreased with respect to each of the two main
clusters. Multivariate analysis revealed increased Bacteroides-Prevotella in smokers (38.4%)
compared with nonsmokers (28.1%). Healthy controls also exhibited increased
Bacteroides-Prevotella (34.8%) compared to nonsmokers (24.1%). Multivariate analysis revealed increased Bacteroides-Prevotella in smokers (38.4%)
compared with nonsmokers (28.1%). Healthy controls also exhibited increased
Bacteroides-Prevotella (34.8%) compared to nonsmokers (24.1%). Pooled multivariate analysis showed patients with CD had higher bifidobacteria,
higher Bacteroides-Prevotella, and lower F. prausnitzii (in comparison to healthy
controls. Pooled multivariate analysis showed patients with CD had higher bifidobacteria,
higher Bacteroides-Prevotella, and lower F. prausnitzii (in comparison to healthy
controls. Decreased gut microbial gene richness (P=0.01), genus diversity (P<0.01), and
species diversity (P=0.01) in smoking patients with CD compared to nonsmoking
patients with CD. Decreased relative abundance of the genera Collinsena (P=0.02), Enterohabdus
(P=0.02), and Gordonibacter (P=0.02) in smoking patients with CD compared to
nonsmoking patients with CD. acetyl-coenzyme A-carboxylase, which yields further hepatic
lipogenesis (44). Nicotine also increases endoplasmic reticulum
(ER) stress (44) that modulates many factors, including nuclear
factor 2 erythroid-related factor 2 (Nrf2), c-Jun N-terminal
kinase (JNK), nuclear factor kB (NF-kB), and c/EBP homologous
protein. These all contribute to the inflammatory process associated
with smoking and are part of the cellular defense against oxidative
stress, often resulting in cell death (44). For instance, Nrf2 serves as a
master regulator of a cellular defense system against oxidative stress
(168, 169) and JNK is activated in several animal models of obesity
and also in patients with NASH. The activation of JNK has been
demonstrated in HFD-induced hepatic steatosis in apoplipoprotein-
E knockout mice (170) and nicotine plus HFD-induced hepatic
steatosis in obese mice (141); the genetic deletion of JNK in animal
models resulted in attenuation of fatty liver (171). NF-kB is an
important transcription factor and primary regulator of
inflammatory pathways. Consistent activation of NF-kB signaling
has been documented in animal models of NAFLD as well as in
patients with NASH (172). Thus, the data suggest that the use of
nicotine-based products results in increased oxidative stress,
upregulated inflammation, perturbed hepatic lipid homeostasis,
apoptosis, and autophagy, which contribute to hepatic steatosis and
progression to NASH (173). tobaccosmokingbutcautionthattheirstudywasonlydoneinasmall
cohort of smokers and e-cigarette users. Findings Stewart and colleagues
proposed a larger, multi-location cohort study with e-cigarettes and
conventional cigarette users to provide insight into their effects on
the microbiome. Findings Similarly, samples clustered in the second principal coordinate June 2021 | Volume 12 | Article 667066 Frontiers in Endocrinology | www.frontiersin.org 7 TABLE 1 | Continued
Study
Study Purpose
Sample Size
Findings
by whether they were never smokers (17% of variance). Former smoke
distributed within and between both these clusters. Specific OTUs increased or decreased with respect to each of the two
clusters. (150)
To assess the relation between smoking and
intestinal microbiota in patients with active
Crohn’s disease (CD). n = 169
(103 subjects with active
CD; 66 healthy controls;
29 smokers with CD; 8
smokers in the control
group)
Multivariate analysis revealed increased Bacteroides-Prevotella in smoke
compared with nonsmokers (28.1%). Healthy controls also exhibited inc
Bacteroides-Prevotella (34.8%) compared to nonsmokers (24.1%). Pooled multivariate analysis showed patients with CD had higher bifidob
higher Bacteroides-Prevotella, and lower F. prausnitzii (in comparison to
controls. (151)
To evaluate changes in gut microbiota
composition associated in smokers versus
nonsmokers with active Crohn’s disease using
a metagenomic approach. n = 42
21 smoking and 21
nonsmoking patients with
CD included
Decreased gut microbial gene richness (P=0.01), genus diversity (P<0.0
species diversity (P=0.01) in smoking patients with CD compared to no
patients with CD. Decreased relative abundance of the genera Collinsena (P=0.02), Entero
(P=0.02), and Gordonibacter (P=0.02) in smoking patients with CD com
nonsmoking patients with CD. Martinez et al. Gut Dysbiosis, Nicotine, and Intestinal M Gut Dysbiosis, Nicotine, and Intestinal Microbiome Martinez et al. Frontiers in Endocrinology | www.frontiersin.org June 2021 | Volume 12 | Article 667066 REFERENCES Chronic Obstructive Pulmonary Disease, Smokers and Healthy non-
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bmj.j5145 17. Nicotine Interaction with Diet in NAFLD
and Obesity This, in turn, results in the up-regulation of sterol response
element-binding protein 1-c, fatty acid synthase, and activation of June 2021 | Volume 12 | Article 667066 8 Gut Dysbiosis, Nicotine, and Intestinal Microbiome Martinez et al. to a nicotine-derived pathway and result in the pathogenesis
of NAFLD. relevant. Effects of nicotine, either alone or in combination
with the WD, on the intestinal microbiome remain to
be elucidated. ACKNOWLEDGMENTS Editorial review was provided by the NIH National Center for
Advancing Translational Science (NCATS) UCLA CTSI Grant
Number UL1TR001881. FUNDING Funded by NIH grants MD012579-01, R25 DA050723, U54
MD007598, DOD CDMRP grant PR190942, VA CDA2
IK2CX001717, and California Tobacco-Related Disease
Research Program (TRDRP) Community Practice-Based
Research Implementation Award 28CP-0040. AUTHOR CONTRIBUTIONS A healthy intestinal microbiome is dependent on a delicate
balance of various microorganisms that is susceptible to
external lifestyle factors, including unhealthy diet, lack of
exercise, smoking and nicotine-exposure. Lifestyle modification
can alter the variable portion of the microbiome. Exercise may
hold numerous potential benefits for the health of the intestinal
microbiome, not only through improved insulin sensitivity,
weight loss, and improved cardiovascular health, but also
through its impacts on the intestinal microbiota composition. Use of nicotine-based products (e-cigarettes and traditional
cigarettes) leads to known health consequences, but also may
be a major contributor to gut dysbiosis and increased gut
permeability. More research is needed to confirm the
importance of avoiding nicotine-based products to optimize
gut health and lessen the risk of gut dysbiosis. Additionally,
the effects of nicotine use on the gut immune system should be
more closely evaluated. Moving forward, the ability of the
microbiome to recover from external factors, such as nicotine
and unhealthy diets, should also be evaluated. With the number
of young adults and teens consuming nicotine via e-cigarettes on
the rise, the long-term effect of nicotine has become more JM and SG performed the literature review. JM wrote the first
draft of the paper. All other authors contributed to the writing
and editing of the paper. All authors contributed to the article
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Jacobs, Sinha-Hikim and Friedman. This is an open-access article distributed under
the terms of the Creative Commons Attribution License (CC BY). The use, distribution
or reproduction in other forums is permitted, provided the original author(s) and the
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Transesophageal echocardiography-guided percutaneous closure of multiple muscular ventricular septal defects with pulmonary hypertension using single device: A case report
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Frontiers in cardiovascular medicine
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Transesophageal
echocardiography-guided
percutaneous closure of multiple
muscular ventricular septal
defects with pulmonary
hypertension using single device:
A case report EDITED BY
Mingxing Xie,
Huazhong University of Science and
Technology, China
REVIEWED BY
Do Tin,
Ho Chi Minh City Medicine and Pharmacy
University, Vietnam
Xiangbin Pan,
Chinese Academy of Medical Sciences and
Peking Union Medical College, China
*CORRESPONDENCE
Sisca Natalia Siagian
sisca.ped.car@gmail.com
SPECIALTY SECTION
This article was submitted to Pediatric
Cardiology, a section of the journal Frontiers in
Cardiovascular Medicine
RECEIVED 09 November 2022
ACCEPTED 03 March 2023
PUBLISHED 23 March 2023 Sisca Natalia Siagian
1*, Radityo Prakoso
1, Brian Mendel
1,2,
Zakky Hazami
3, Valerinna Yogibuana Swastika Putri
1
, Zulfahmi
1,
Damba Dwisepto Aulia Sakti
1 and Ario Soeryo Kuncoro
4 1Department of Cardiology and Vascular Medicine, Division of Pediatric Cardiology and Congenital Heart
Disease, National Cardiovascular Centre Harapan Kita, Universitas Indonesia, Jakarta, Indonesia,
2Department of Cardiology and Vascular Medicine, Sultan Sulaiman Government Hospital, Serdang
Bedagai, Indonesia, 3Department of Cardiology and Vascular Medicine, National Cardiovascular Centre
Harapan Kita, Universitas Indonesia, Jakarta, Indonesia, 4Department of Cardiology and Vascular
Medicine, Division of Non-Invasive Diagnostic and Cardiovacular Imaging, National Cardiovascular Centre
Harapan Kita, Universitas Indonesia, Jakarta, Indonesia Siagian SN, Prakoso R, Mendel B, Hazami Z,
Putri VYS, Zulfahmi, Sakti DDA and Kuncoro AS
(2023) Transesophageal echocardiography-
guided percutaneous closure of multiple
muscular ventricular septal defects with
pulmonary hypertension using single device:
A case report. Background: Surgery is typically used to correct challenging ventricular septal
defects (VSDs), such as VSD with pulmonary hypertension and multiple defects. In this case report, we would like to highlight the feasibility of multiple defects
VSD closure with single device percutaneously using zero-fluoroscopy technique. Case presentation: A 7-year-old child was referred with the main symptom of
shortness
of
breath. She
started
experiencing
repeated
respiratory
tract
infections, feeding issues, and failure to thrive at the age of six months. Her
body weight was only 18 kg. TEE revealed several muscular VSD with 2–3 mm
and 12 mm diameters, 3 mm spacing between VSD, L to R shunt, AR (-), and TR
mild with septal leaflet tricuspid prolapse. Following right heart catheterization
(Qp:Qs 3.5, PVRi 5.23WUmsq, PVR 4.55 WU, PVR/SVR 0.16), we made the
decision to correct the defect using an Amplatzer Septal Occluder (AGA) No. 16
mm
using
transjugular
method. Full
device
deployment
was
successfully
performed with several episodes of PVC storm and severe bradycardia. TYPE Case Report
PUBLISHED 23 March 2023
DOI 10.3389/fcvm.2023.1093563 TYPE Case Report
PUBLISHED 23 March 2023
DOI 10.3389/fcvm.2023.1093563 TYPE Case Report
PUBLISHED 23 March 2023
DOI 10.3389/fcvm.2023.1093563 echocardiography-guided,
muscular
VSD,
pulmonary
hypertension,
single
device,
transjugular Transesophageal
echocardiography-guided
percutaneous closure of multiple
muscular ventricular septal
defects with pulmonary
hypertension using single device:
A case report One and
a half years after the procedure, her TVG dropped to only 18 mmHg, her visible
indicators of PH subsided, and the PA dilator treatment was discontinued. Her
body weight had increased to 28 kg, and she had no complaints. Front. Cardiovasc. Med. 10:1093563. doi: 10.3389/fcvm.2023.1093563 COPYRIGHT
© 2023 Siagian, Prakoso, Mendel, Hazami, Putri,
Zulfahmi, Sakti and Kuncoro. This is an open-
access article distributed under the terms of the
Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other
forums is permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original publication in this
journal is cited, in accordance with accepted
academic practice. No use, distribution or
reproduction is permitted which does not
comply with these terms. 3.1. Management of ventricular septal
defect with pulmonary hypertension The right femoral
artery of the patient was punctured, and the patient then inserted a
4F sheath, heparin 1.000 IU, and MP sidehole 5F catheter. The
right jugular vein was punctured; the MP sidehole was 5F and the
sheath 6F was used. Following right cardiac catheterization (Qp:Qs
3.5, PVRi 5.23WUmsq, PVR 4.55 WU, PVR/SVR 0.16), we made
the decision to correct the defect using an Amplatzer Septal
Occluder (AGA) No. 16 mm using transjugular method. Through
the muscular VSD, a 5F MP sidehole diagnostic catheter was
introduced under the direction of transesophageal echocardiography
(TEE). A 5F MP sidehole diagnostic catheter was introduced under
the direction of transesophageal echocardiography (TEE) from
SVC, RA, RV and LV through muscular VSD (Figures 1B–D). Using 0.035″ Amplatzer stiff wire, we change to 8F delivery sheath The ESC (European Society of Cardiology) 2022 guidelines
recommend shunt closure in individuals with a pulmonary-
systemic flow ratio greater than 1.5:1 based on estimated
pulmonary vascular resistance. When PVR is less than 3 WU
and when PVR is between 3 and 5 WU, VSD closure is advised. After thorough assessment in a specialized facility, shunt closure
may be taken into consideration in patients with PVR > 5 WU
(6). As a result, we chose to treat on this patient’s VSD. 3.1. Management of ventricular septal
defect with pulmonary hypertension A 7-year-old child was referred with the main symptom of
shortness of breath one year prior to admission. She started
experiencing repeated respiratory tract infections, feeding issues,
and failure to thrive at the age of six months, although she was not
bluish. She received a pulmonary TB diagnosis and had 9 months
of therapy. She continued to report having dyspnea, and an
echocardiogram showed that she had multiple muscular VSDs with
a left-to-right shunt and pulmonary hypertension with a total
diameter of 1–1.4 cm. The results of a physical examination
revealed a heart rate of 115 beats per minute, a respiratory rate of
21 breaths per minute, and a room air oxygen saturation of 98%. Her height was 122 cm, and her body weight was 18 kg. Regular
first and second heart sounds were audible during auscultation, as
well as a loud intensity holosystolic murmur grade 3/6 in the lower
left sternal border. The patient had a D-shaped LV and severe PH
with a TVG of 69 mmHg and a transVSD gradient of 15 mmHg. The care of patients with PAH-CHD depends heavily on
behavioral change and identification of relevant risk factors. According to knowledge provided, patients with PAH-CHD who
are functional class III begin treatment with the endothelin
receptor antagonist bosentan (class I, level of evidence B). In
contrast to placebo, bosentan significantly improved exercise
capacity, haemodynamics, and functional class in the BREATHE-
5 (Bosentan Randomised Trial of Endothelin Antagonist-5) trial
and its long-term open label extension research, regardless of the
location of septal defects (4–6). In
patients
with
PAH-CHD,
sildenafil
therapy
has
been
demonstrated to enhance exercise capacity, Borg dyspnoea score,
functional class, quality of life, and hemodynamics. When symptoms
persisted despite taking the maximum amount of medication, as we
did in this patient, right cardiac catheterization should be carried out
to determine whether shunt closure is appropriate (4, 6). We chose to use the jugular vein approach to accomplish
percutaneous transcatheter VSD closure. Preprocedural 98%; patient
underwent general anesthesia. The patient was intubated using ETT
No. 5.5% and 30% FiO2. 100% post-intubation saturation TEE
revealed several muscular VSD with 2–3 mm and 12 mm
diameters, 3 mm spacing between VSD, L to R shunt, AR (-), and
TR mild with septal leaflet tricuspid prolapse. (Figure 1A). The
decision was made to catheterize the right heart. COPYRIGHT © 2023 Siagian, Prakoso, Mendel, Hazami, Putri,
Zulfahmi, Sakti and Kuncoro. This is an open-
access article distributed under the terms of the The use, distribution or reproduction in other
forums is permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original publication in this
journal is cited, in accordance with accepted
academic practice. No use, distribution or
reproduction is permitted which does not
comply with these terms. Conclusions: Our experience demonstrated that percutaneous closure of multiple
VSD with a single device is possible, even with pulmonary hypertension. KEYWORDS echocardiography-guided,
muscular
VSD,
pulmonary
hypertension,
single
device,
transjugular Frontiers in Cardiovascular Medicine 01 frontiersin.org Siagian et al. Siagian et al. 10.3389/fcvm.2023.1093563 10.3389/fcvm.2023.1093563 10.3389/fcvm.2023.1093563 1. Introduction (Figure 1E). Full device deployment was successfully performed
with several episodes of PVC storm and severe bradycardia
(Figures
1F–H). Echocardiography
evaluation
showed
no
complications before, during, and after deployment of the occluder
device (Figure 1H). Her symptoms and appetite steadily improved
a week after the operation. She was able to engage in moderately
intensive activities three months after the treatment without
experiencing any discomfort. Surgery is typically used to correct ventricular septal defects
(VSDs), particularly in challenging cases like those involving
individuals with pulmonary hypertension and multiple defects. In
recent years, muscular VSD found in locations that are challenging
for
surgeons
to
reach
are
often
treated
with
transcatheter
percutaneous closure. However, multiple defects are often repaired
using multiple devices and are typically guided by fluoroscopy (1,
2). Eliminating radiation exposure during procedure is crucial
since fluoroscopy effects are cumulative and raise serious issues,
especially in the younger population (3). In this case report, we
would like to highlight the feasibility of multiple defects VSD
closure with single device using zero-fluoroscopy technique. Her TVG was 60 mmHg and 50 mmHg six months and one
year after the procedure, respectively, with still visible evidence of
PH. However, 1.5 years after the procedure, her TVG dropped to
only 18 mmHg, her visible indicators of PH subsided, and the
PA dilator treatment was discontinued. Her body weight had
increased to 28 kg, and she had no complaints. The patient’s
ECG showed sinus rhythm during our most recent clinic visit. 3. Discussions A 7-year-old child was referred with the main symptom of
shortness of breath one year prior to admission. She started
experiencing repeated respiratory tract infections, feeding issues,
and failure to thrive at the age of six months, although she was not
bluish. She received a pulmonary TB diagnosis and had 9 months
of therapy. She continued to report having dyspnea, and an
echocardiogram showed that she had multiple muscular VSDs with
a left-to-right shunt and pulmonary hypertension with a total
diameter of 1–1.4 cm. The results of a physical examination
revealed a heart rate of 115 beats per minute, a respiratory rate of
21 breaths per minute, and a room air oxygen saturation of 98%. Her height was 122 cm, and her body weight was 18 kg. Regular
first and second heart sounds were audible during auscultation, as
well as a loud intensity holosystolic murmur grade 3/6 in the lower
left sternal border. The patient had a D-shaped LV and severe PH
with a TVG of 69 mmHg and a transVSD gradient of 15 mmHg. We chose to use the jugular vein approach to accomplish
percutaneous transcatheter VSD closure. Preprocedural 98%; patient
underwent general anesthesia. The patient was intubated using ETT
No. 5.5% and 30% FiO2. 100% post-intubation saturation TEE
revealed several muscular VSD with 2–3 mm and 12 mm
diameters, 3 mm spacing between VSD, L to R shunt, AR (-), and
TR mild with septal leaflet tricuspid prolapse. (Figure 1A). The
decision was made to catheterize the right heart. The right femoral
artery of the patient was punctured, and the patient then inserted a
4F sheath, heparin 1.000 IU, and MP sidehole 5F catheter. The
right jugular vein was punctured; the MP sidehole was 5F and the
sheath 6F was used. Following right cardiac catheterization (Qp:Qs
3.5, PVRi 5.23WUmsq, PVR 4.55 WU, PVR/SVR 0.16), we made
the decision to correct the defect using an Amplatzer Septal
Occluder (AGA) No. 16 mm using transjugular method. Through
the muscular VSD, a 5F MP sidehole diagnostic catheter was
introduced under the direction of transesophageal echocardiography
(TEE). A 5F MP sidehole diagnostic catheter was introduced under
the direction of transesophageal echocardiography (TEE) from
SVC, RA, RV and LV through muscular VSD (Figures 1B–D). Using 0.035″ Amplatzer stiff wire, we change to 8F delivery sheath 3.2. Strategy for multiple ventricular septal
defect closure The apical, central, or outflow regions of the interventricular
septum are the most common locations for muscular ventricular
septal defects (VSDs). Up to 20% of VSDs in babies are
muscular VSDs, which can have several occurrences and take on
a “Swiss cheese” appearance (1, 2, 7). The VSD in our instance Frontiers in Cardiovascular Medicine 02 frontiersin.org Siagian et al. 10.3389/fcvm.2023.1093563 FIGURE 1
Antegrade jugular vein approach in multiple-defect muscular VSD closure with pulmonary hypertension. (A) Multiple VSD. (B) 5F sidehole Multipurpose
catheter was directed from SVC towards RA. (C) Catheter was then directed towards RV. (D) The catheter successfully crossed from RV into LV. (E) With
the assistance of 0.035″ Amplatzer stiff wire, the catheter was changed with 8F delivery sheath. (F) The Amplatzer device occluder (AGA) No 16 mm was
delivered and one of the disc was deployed in the LV side, (G) and RV side. (H) Device stowed in place; Red arrowhead showed the position of the
catheter, green arrowhead showed the position of the delivery sheath. Notes: VSD, ventricular septal defect; SVC, superior vena cava; RV, right
ventricle; LV, left ventricle; RA, right atrium; LA, left atrium. FIGURE 1
Antegrade jugular vein approach in multiple-defect muscular VSD closure with pulmonary hypertension. (A) Multiple VSD. (B) 5F sidehole Multipurpose
catheter was directed from SVC towards RA. (C) Catheter was then directed towards RV. (D) The catheter successfully crossed from RV into LV. (E) With
the assistance of 0.035″ Amplatzer stiff wire, the catheter was changed with 8F delivery sheath. (F) The Amplatzer device occluder (AGA) No 16 mm was
delivered and one of the disc was deployed in the LV side, (G) and RV side. (H) Device stowed in place; Red arrowhead showed the position of the
catheter, green arrowhead showed the position of the delivery sheath. Notes: VSD, ventricular septal defect; SVC, superior vena cava; RV, right
ventricle; LV, left ventricle; RA, right atrium; LA, left atrium. was situated mid-apical. In our institution, surgery is typically used
as a method of multiple VSD defect repair. However, it was
determined during the surgical discussion that the defect could
be closed percutaneously. We are only authorized to use one
device per interventional operation at our hospital due to
legislation. There is no agreement on the maximum diameter at
which a single device could close numerous VSD faults at the
moment. Frontiers in Cardiovascular Medicine Ethics statement Ethical review and approval was not required for the study on
human participants in accordance with the local legislation and
institutional
requirements. Written
informed
consent
to
participate in this study was provided by the participants’ legal
guardian/next of kin. Written informed consent was obtained
from the individual(s), and minor(s)’ legal guardian/next of kin,
for the publication of any potentially identifiable images or data
included in this article. Written informed consent was obtained
from the participant/patient(s) for the publication of this case
report. Frontiers in Cardiovascular Medicine 4. Conclusions It is challenging to complete the closure process for a patient who
has a defect adjacent to the apex since the insertion of the occluder
necessitates the catheter bevel. Hemolysis that is brief and self-
contained has been demonstrated by (8) Santos et al. (2018). Both
perimembranous
and
muscular
VSD
that
were
treated
with
Amplatzer Septal Occluder (AGA) had a lower risk of total AV
block (9); however, in our instance, some PVC storms and severe
bradycardia transiently occurred after the defect closure. We
suspected that several PVC storms and bradycardia episodes in our
patient were caused by pulmonary hypertension rather than by the
device. As we know, the most common arrhythmia complications
caused by device closure are AV block and bundle branch block. We also saw no conduction abnormalities until the current follow-
up, so we believe the issues were not caused by the device. Our experience demonstrated that percutaneous closure with a
single device is possible to carry out successfully even in multiple
VSD defects accompanied by pulmonary hypertension. However,
the cardiac team should think about a more direct strategy to
lower the danger and they should pick the patient carefully in
which
patient
with
multiple
defects
should
be
done
percutaneously or surgically. Author contributions SNS and RP: conceived the original idea of the manuscript, and
all authors discussed and agreed with the idea of the paper. BM,
ZH, VYSP and Z: contributed in collecting the patient data and
writing the main text of the paper. The manuscript was
proofread and accepted by all authors. All authors contributed to
the article and approved the submitted version. As far as we know, fluoroscopy may cause certain long-term,
delayed negative effects, particularly in children and newborns. Over the years, there have been more reports of skin injuries, such
as redness, necrosis, and ulceration, which can be painful and
disabling. There has also been an increase in the likelihood of
developing neoplasms, radiation-induced cataracts, and hair loss (3). In this instance, the finding of the VSD from the jugular
approach is simple and is followed by a cross to the LV to close
the defect after the patient has been intubated and is being seen
with TEE. In our scenario, a fluoroscopy-guided operation is also
possible, but since there are two defects, a TEE could provide a As far as we know, fluoroscopy may cause certain long-term,
delayed negative effects, particularly in children and newborns. Over the years, there have been more reports of skin injuries, such
as redness, necrosis, and ulceration, which can be painful and
disabling. There has also been an increase in the likelihood of
developing neoplasms, radiation-induced cataracts, and hair loss (3). Data availability statement The original contributions presented in the study are included
in the article/Supplementary Material, further inquiries can be
directed to the corresponding author/s. Patients with numerous VSDs frequently experience residual
shunt because a proper occluder size is necessary to completely
cover the defect. As a result, the type and placement of the
occlusion should be carefully chosen based on the specific
circumstances. After
the
treatment,
our
patient,
however,
displayed no symptoms of a residual shunt. Our post-procedural
echocardiogram also revealed that the mitral and tricuspid valves
were not damaged, as predicted when we selected and estimated
the distance between the device, and the interventricular septum
that would be covered by the device. 3.3. Feasibility of transesophageal
echocardiography-guided closure Even though ALARA (as low as reasonably achievable)
principle is used, it would be preferable to completely eliminate
radiation exposure risks for both the operators and the patient. In our facility, the technique would be carried out initially
without any fluoroscopy, or if the echocardiography window was
insufficient intraprocedurally, the technique would be converted
to the standardized fluoroscopy process (1, 3, 10, 11). 3.2. Strategy for multiple ventricular septal
defect closure For atrial septal defects, If the distance between defects
is smaller than 0.5 or 0.7, only one device needs to be used (2). also possible to approach the deficiency from the transfemoral
side, although this method carries a higher risk because it is
more complicated, sinuous, and difficult to traverse to the mid-
apical position of the defect. Delivered through the sheath, the multipurpose catheter and
guidewire was then moved to the right ventricle via the tricuspid
valve and readjusted with its top facing the VSD. The guidewire
was gently moved and adjusted to the left ventricle via the VSD
utilizing transesophageal echocardiography (TEE)-guided only. Since the patient was young and just 18 kg in weight, the 5F
sheath expander needed to be removed gradually while the sheath
was inserted into the conduit to prevent heart injury. To prevent
harming nearby intracardiac structures, the sheath shouldn’t be
put straight and should instead be inserted about 3 to 5 cm deep. As the abnormalities in the IVS are more posteriorly
positioned, we attempted to operate through the jugular vein
route to reduce curvature of the delivery sheath and eliminate
resistance
across
the
septum. This
patient’s
jugular
vein
technique also provided a more direct route to the problem. It is 03 frontiersin.org Siagian et al. Siagian et al. 10.3389/fcvm.2023.1093563 10.3389/fcvm.2023.1093563 superior image. With TEE, we could instantly see the position
and visualize it. Therefore, if there were multiple defects, we
would prefer to advise using TEE directed. The shape and
diameter of the VSD may be estimated with precision using TEE. When employing TEE, the danger of oesophageal trauma and
erosion should be taken into account (1, 3, 10, 11). Patients with perimembranous VSD utilize a different type of
closure device than those with muscular VSD. For this patient, we
utilized the Amplatzer Septal Occluder (AGA) No. 16 mm device
because a sufficient size for a muscular VSD device was not
available at the time of the procedure. We also chose Amplatzer
Septal Occluder (AGA) since the retention disc is larger and could
cover both defects with a 3 mm gap. We chose device no. 16 mm
since there were two defects, and we expect that the device will
cover both defects as well as the gap between them. Acknowledgments In this instance, the finding of the VSD from the jugular
approach is simple and is followed by a cross to the LV to close
the defect after the patient has been intubated and is being seen
with TEE. In our scenario, a fluoroscopy-guided operation is also
possible, but since there are two defects, a TEE could provide a We would like to thank those who have supported us in the making
of this study. We are especially grateful to the Department of
Cardiology and Vascular Medicine, Faculty of Medicine Universitas 04 frontiersin.org Siagian et al. 10.3389/fcvm.2023.1093563 10.3389/fcvm.2023.1093563 Indonesia, for their guidance and assistance in teaching the authors
about research methodology and for proof-reading this article. Conflict of interest The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. Publisher’s note All claims expressed in this article are solely those of the
authors and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed
or endorsed by the publisher. 6. Humbert M, Kovacs G, Hoeper MM, Badagliacca R, Berger RM, Brida M, et al.
ESC/ERS guidelines for the diagnosis and treatment of pulmonary hypertension. Eur
Heart J. (2022) 43(38):3618–731. doi: 10.1093/eurheartj/ehac237 7. Wang S, Ouyang W, Liu Y, Zhang F, Guo G, Zhao G, et al. Transcatheter
perimembranous
ventricular
septal
defect
closure
under
transthoracic
echocardiographic
guidance
without
fluoroscopy.
J
Thorac
Dis.
(2018)
10
(9):5222–31. doi: 10.21037/jtd.2018.08.03 8. Santos RAP, Guedes H, Marques L, Lourenco C, Silva JC, Pinot P. Transcatheter
closure of a traumatic VSD with an ASD occluder. Arq Bras Cardiol. (2018) 111(2):223–5. 10. Bu H, Yang Y, Wu Q, Jin W, Zhao T. Echocardiography-guided percutaneous
closure of perimembranous ventricular septal defects without arterial access and
fluoroscopy. BMC Pediatr. (2019) 19:302. doi: 10.1186/s12887-019-1687-0 11. Mendel B, Faris Amin B, Prakoso R. Potency of non-fluoroscopy guided patent
ductus arteriosus closure: a case report. SN Comprehensive Clin Med. (2021) 3
(12):2665–72. doi: 10.1007/s42399-021-01062-4 5. Mendel B, Christianto C, Angellia P, Holiyono I, Prakoso R, Siagian SN. Reversed
potts shunt outcome in suprasystemic pulmonary arterial hypertension: a systematic
review and meta-analysis. Curr Cardiol Rev. (2022) 18(6):95–103. doi: 10.2174/
1573403X18666220509203335 8. Santos RAP, Guedes H, Marques L, Lourenco C, Silva JC, Pinot P. Transcatheter
closure of a traumatic VSD with an ASD occluder. Arq Bras Cardiol. (2018) 111(2):223–5.
9. Carminati M, Butera G, Chessa M, Drago M, Negura D, Piazza L. Transcatheter
closure of congenital ventricular septal defect with amplatzer septal occluders. Am
J Cardiol. (2005) 96(12A):52l–8l. doi: 10.1016/j.amjcard.2005.09.068 9. Carminati M, Butera G, Chessa M, Drago M, Negura D, Piazza L. Transcatheter
closure of congenital ventricular septal defect with amplatzer septal occluders. Am
J Cardiol. (2005) 96(12A):52l–8l. doi: 10.1016/j.amjcard.2005.09.068 6. Humbert M, Kovacs G, Hoeper MM, Badagliacca R, Berger RM, Brida M, et al.
ESC/ERS guidelines for the diagnosis and treatment of pulmonary hypertension. Eur
Heart J. (2022) 43(38):3618–731. doi: 10.1093/eurheartj/ehac237
7. Wang S, Ouyang W, Liu Y, Zhang F, Guo G, Zhao G, et al. Transcatheter
perimembranous
ventricular
septal
defect
closure
under
transthoracic
echocardiographic
guidance
without
fluoroscopy.
J
Thorac
Dis.
(2018)
10
(9):5222–31. doi: 10.21037/jtd.2018.08.03
8. Santos RAP, Guedes H, Marques L, Lourenco C, Silva JC, Pinot P. Transcatheter
closure of a traumatic VSD with an ASD occluder. Arq Bras Cardiol. (2018) 111(2):223–5.
9. Carminati M, Butera G, Chessa M, Drago M, Negura D, Piazza L. Transcatheter
closure of congenital ventricular septal defect with amplatzer septal occluders. Am
J Cardiol. (2005) 96(12A):52l–8l. doi: 10.1016/j.amjcard.2005.09.068
10. Bu H, Yang Y, Wu Q, Jin W, Zhao T. Echocardiography-guided percutaneous
closure of perimembranous ventricular septal defects without arterial access and
fluoroscopy. BMC Pediatr. (2019) 19:302. doi: 10.1186/s12887-019-1687-0
11. Mendel B, Faris Amin B, Prakoso R. Potency of non-fluoroscopy guided patent
ductus arteriosus closure: a case report. SN Comprehensive Clin Med. (2021) 3
(12):2665–72. doi: 10.1007/s42399-021-01062-4 References 1. Mendel B, Laurentius A, Ulfiarakhma D, Prakoso R. Safety and feasibility of
transesophageal
echocardiography
in
comparison
to
transthoracic
echocardiography-guided
ventricular
septal
defect
percutaneous
closure:
an
evidence-based case report. World Heart J. (2020) 12:199–207. 6. Humbert M, Kovacs G, Hoeper MM, Badagliacca R, Berger RM, Brida M, et al. ESC/ERS guidelines for the diagnosis and treatment of pulmonary hypertension. Eur
Heart J. (2022) 43(38):3618–731. doi: 10.1093/eurheartj/ehac237 2. Felice H, Chandran S, Bhamra-Ariza P, Brecker H Transcatheter closure of
multiple defects of the atrial septum: technique and follow-up. Int J Clin Med. (2015) 06(09):591–6. doi: 10.4236/ijcm.2015.69079 3. Siagian SN, Prakoso R, Putra BE, Kurniawati Y, Lelya O, Sembiring AA, et al. Echocardiography-guided percutaneous patent ductus arteriosus closure: 1-year
single center experience in Indonesia. Front Cardiovasc Med. (2022) 9:2–7. doi: 10. 3389/fcvm.2022.885140 4. D’Alto M, Mahadevan VS. Pulmonary arterial hypertension associated with
congenital heart disease,”. Eur Respir Rev. (2012) 21(126):328–37. doi: 10.1183/
09059180.00004712 4. D’Alto M, Mahadevan VS. Pulmonary arterial hypertension associated with
congenital heart disease,”. Eur Respir Rev. (2012) 21(126):328–37. doi: 10.1183/
09059180.00004712 10. Bu H, Yang Y, Wu Q, Jin W, Zhao T. Echocardiography-guided percutaneous
closure of perimembranous ventricular septal defects without arterial access and
fluoroscopy. BMC Pediatr. (2019) 19:302. doi: 10.1186/s12887-019-1687-0 5. Mendel B, Christianto C, Angellia P, Holiyono I, Prakoso R, Siagian SN. Reversed
potts shunt outcome in suprasystemic pulmonary arterial hypertension: a systematic
review and meta-analysis. Curr Cardiol Rev. (2022) 18(6):95–103. doi: 10.2174/
1573403X18666220509203335 11. Mendel B, Faris Amin B, Prakoso R. Potency of non-fluoroscopy guided patent
ductus arteriosus closure: a case report. SN Comprehensive Clin Med. (2021) 3
(12):2665–72. doi: 10.1007/s42399-021-01062-4 11. Mendel B, Faris Amin B, Prakoso R. Potency of non-fluoroscopy guided patent
ductus arteriosus closure: a case report. SN Comprehensive Clin Med. (2021) 3
(12):2665–72. doi: 10.1007/s42399-021-01062-4 05 Frontiers in Cardiovascular Medicine 05 frontiersin.org
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Memory in Elementary School Children Is Improved by an Unrelated Novel Experience
|
PloS one
| 2,013
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cc-by
| 7,630
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Introduction Finding different teaching strategies to improve students’
performance is a subject of great relevance to education. Although
arousing students’ interest in the syllabus might work, this is not
always possible. In particular, some topics are resisted by many
students. Therefore, finding behavioral procedures with enhancing
effects on human memory poses an exciting challenge, with
potential implications for educational strategies. Materials and Methods A prevailing view in neuroscience is that memory formation is a
gradual process. Memories are initially labile but they become
more stable with the passage of time. This memory consolidation
hypothesis was proposed more than 100 years ago [1] and some of
its underlying molecular and cellular mechanisms were well
characterized over the past decades [2,3,4]. Behavioral, hormonal
and neural influences acting during this labile period can regulate
memory consolidation, improving or impairing it. Thus, stress,
arousal, motivation and reward can profoundly affect memory
formation [5,6,7,8,9]. Moreover, there is increasing evidence
supporting the view that the ability to consolidate a memory for a
certain event can be concurrently improved by what has happened
before or after it [10]. In this regard, previous work in rodents has
demonstrated that a novel behavioral experience can promote the
formation of a long-lasting memory for another task when novelty
occurs within a restricted time window around the learning task Abstract This is an open-access article distributed under the terms of the Creative Commons Attribut
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. arini et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
tion, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the Universidad de Buenos Aires and the Agencia Nacional de Promocio´n Cientı´fica y Tecnolo´gica (Argentina). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: hviola@fmed.uba.ar Competing Interests: The authors have declared that no competing interests exist. * E-mail: hviola@fmed.uba.ar [11,12,13,14,15]. This phenomenon is called behavioral tagging and
its underlying mechanisms are currently being studied [16]. In the
present work, we investigated whether a similar approach could be
applied to the school syllabus during students’ regular classes, with
the aim of improving teaching strategies used in the educational
system. Students 1676 Participants (ages 7 to 9 years-old) from 8 different
schools in Buenos Aires, Argentina were tested. Characteristics of
the institutions and the students intervening in the experiments
are shown in table S1. All students were naı¨ve to the procedures. Fabricio Ballarini1, Marı´a Cecilia Martı´nez1, Magdalena Dı´az Perez1, Diego Moncad Fabricio Ballarini1, Marı´a Cecilia Martı´nez1, Magdalena Dı´az Perez1, Diego Moncada1, Hayde´e Viola1,2*
1 Instituto de Biologı´a Celular y Neurociencias Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas de Argentina, Facultad de Medicina, Universidad de Buenos
Aires, Buenos Aires, Argentina, 2 Departamento de Fisiologı´a, Biologı´a Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Fabricio Ballarini , Marıa Cecilia Martınez , Magdalena Dıaz Perez , Diego Moncada , Haydee Viola
1 Instituto de Biologı´a Celular y Neurociencias Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas de Argentina, Facultad de Medicina, Universidad de Buenos
Aires, Buenos Aires, Argentina, 2 Departamento de Fisiologı´a, Biologı´a Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos
Aires Argentina 1 Instituto de Biologı´a Celular y Neurociencias Consejo Nacional de Investigaciones Cientı´ficas y Te´cnicas de Argentina, Facultad de Medicina, Universidad de Buenos
Aires, Buenos Aires, Argentina, 2 Departamento de Fisiologı´a, Biologı´a Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos
Aires, Argentina Abstract Education is the most traditional means with formative effect on the human mind, learning and memory being its
fundamental support. For this reason, it is essential to find different strategies to improve the students performance. Based
on previous work, we hypothesized that a novel experience could exert an enhancing effect on learning and memory within
the school environment. Here we show that novel experience improved the memory of literary or graphical activities when
it is close to these learning sessions. We found memory improvements in groups of students who had experienced a novel
science lesson 1 hour before or after the reading of a story, but not when these events were 4 hours apart. Such promoting
effect on long-term memory (LTM) was also reproduced with another type of novelty (a music lesson) and also after another
type of learning task (a visual memory). Interestingly, when the lesson was familiar, it failed to enhance the memory of the
other task. Our results show that educationally relevant novel events experienced during normal school hours can improve
LTM for tasks/activities learned during regular school lessons. This effect is restricted to a critical time window around
learning and is particularly dependent on the novel nature of the associated experience. These findings provide a tool that
could be easily transferred to the classroom by the incorporation of educationally novel events in the school schedule as an
extrinsic adjuvant of other information acquired some time before or after it. This approach could be a helpful tool for the
consolidation of certain types of topics that generally demand a great effort from the children. Citation: Ballarini F, Martı´nez MC, Dı´az Perez M, Moncada D, Viola H (2013) Memory in Elementary School Children Is Improved by an Unrelated Novel
Experience. PLoS ONE 8(6): e66875. doi:10.1371/journal.pone.0066875
Editor: Thomas Burne, University of Queensland, Australia Citation: Ballarini F, Martı´nez MC, Dı´az Perez M, Moncada D, Viola H (2013) Memory in Elementary School Children Is Improved by an Unrelated Novel
Experience. PLoS ONE 8(6): e66875. doi:10.1371/journal.pone.0066875 rtı´nez MC, Dı´az Perez M, Moncada D, Viola H (2013) Memory in Elementary School Children Is Improved by an Unrelated Nove
6): e66875. doi:10.1371/journal.pone.0066875 Editor: Thomas Burne, University of Queensland, Australia Received November 20, 2012; Accepted May 10, 2013; Published June 19, 2013 Received November 20, 2012; Accepted May 10, 2013; Pu Copyright: 2013 Ballarini et al. Memory in Children Is Improved by a Novel Class Memory in Children Is Improved by a Novel Class Memory in Children Is Improved by a Novel Class Figure 1. Memory of a story is enhanced by a novel science lesson around the time of the reading. A) Novelty was experienced before or
after the reading (24 h, n = 57; 21 h, n = 56; +1 h, n = 119; +4 h, n = 98). Schematic representation of the experimental protocol is presented on the
top left of the panels. Memory index is shown as mean 6 SEM of the ratio between control (dotted line, n = 89) and novelty groups. *** p,0.001 vs. Control, 24 h and +4 h, Newman-Keuls after one-way ANOVA. B) Mean 6 SEM of the percentage of correct answers to the hard level of difficulty
questions. Novel groups (24 h, n = 6; 21 h, n = 9; +1 h, n = 9; +4 h, n = 9, calculated from students analyzed in A) vs. their controls for each time
point. ** p,0.01, ***p,0.001 vs. Control, Student’s t test. C) A story A was read and 3 hours later story B was read. Novelty was experienced one hour
after story B. Schematic representation of the experimental protocol is presented on the top right of the panels. Memory index is shown as mean 6
SEM for the novelty groups at different times. Half of the students were tested on story A (n = 20) and the others on story B (n = 28). *** p,0.001 vs
+4 h, Student’s t test. D) Means 6 SEM of the percentage of correct answers to hard level of difficulty questions are shown corresponding to groups
of students analyzed in C (+4 h, n = 6; +1 h, n = 6). ** p,0.01 +1 h vs. +4 h, Student’s t test. doi:10.1371/journal.pone.0066875.g001 Figure 1. Memory of a story is enhanced by a novel science lesson around the time of the reading. A) Novelty was experienced before or
after the reading (24 h, n = 57; 21 h, n = 56; +1 h, n = 119; +4 h, n = 98). Schematic representation of the experimental protocol is presented on the
top left of the panels. Memory index is shown as mean 6 SEM of the ratio between control (dotted line, n = 89) and novelty groups. *** p,0.001 vs. Control, 24 h and +4 h, Newman-Keuls after one-way ANOVA. Memory in Children Is Improved by a Novel Class B) Mean 6 SEM of the percentage of correct answers to the hard level of difficulty
questions. Novel groups (24 h, n = 6; 21 h, n = 9; +1 h, n = 9; +4 h, n = 9, calculated from students analyzed in A) vs. their controls for each time
point. ** p,0.01, ***p,0.001 vs. Control, Student’s t test. C) A story A was read and 3 hours later story B was read. Novelty was experienced one hour
after story B. Schematic representation of the experimental protocol is presented on the top right of the panels. Memory index is shown as mean 6
SEM for the novelty groups at different times. Half of the students were tested on story A (n = 20) and the others on story B (n = 28). *** p,0.001 vs
+4 h, Student’s t test. D) Means 6 SEM of the percentage of correct answers to hard level of difficulty questions are shown corresponding to groups
of students analyzed in C (+4 h, n = 6; +1 h, n = 6). ** p,0.01 +1 h vs. +4 h, Student’s t test. doi:10.1371/journal.pone.0066875.g001 between different experiment in order to avoid any bias (e.g. if
there were 4 different X groups, 2 of them started hearing to story
A and the other one to story B as control situation). Ethics statement This
study
was
performed
under
the
approval
of
the
Ministerio de Education de la Provincia de Buenos Aires,
Argentina. Also, procedures were reviewed and approved by the
Head of each participating educational institution. Students were
able
to
withdraw
from
the
study
at
any
time
without
consequence. No identifying information was collected during
the study. June 2013 | Volume 8 | Issue 6 | e66875 1 PLOS ONE | www.plosone.org General protocol A literary memory study was carried out. Briefly, a teacher read
a short story (A) to her group (X) of students (Control). In parallel,
another group of students (Y) belonging to the same Institution
and of the same grade was read by their own teacher another short
story (B), which was associated to a novel lesson (Novelty). 24 hours after the reading of the story, long-term memory was
evaluated anonymously in both groups. Two weeks later, these two
groups of students were involved in a second experiment, being
group X assigned the story B and group Y the story A. The stories
A and B were randomly associated to control/novelty groups Subjects experienced the novel lesson 1 or 4 h before or after
the reading of the story. This protocol was performed with
children that attend school from 7:30 am to 12:00 pm (morning
shift) or 13:00 pm to17:30 pm (afternoon shift). Long-term memory was evaluated using a written list of 10
questions of different difficulty levels -easy, intermediate and hard-
related to the story read the day before. The answers to the three
easy questions were used to check that the student paid attention June 2013 | Volume 8 | Issue 6 | e66875 PLOS ONE | www.plosone.org 2 June 2013 | Volume 8 | Issue 6 | e66875 Memory in Children Is Improved by a Novel Class Figure 2. Memory of the story is not enhanced when the students were pre-informed about the contests of a new science lesson or
when just moved around the classroom. Schematic representation of the experimental protocol is presented on the top panels. A) Memory
index for the story is shown as mean 6 SEM for the Control group (n = 25) and Pre-informed group (n = 22). A short story was read to a group of
students that had been previously informed that they would be given a science class outside their habitual classroom one hour after the reading. LTM of the story was evaluated 24 hours after. p.0.05 Students t test. B) Memory index for the story is shown as mean 6 SEM for the Control group
(n = 25) and Moving group (n = 30). A short story was read and one hour after students moved from the classroom to the place where the novel class
would take place and back, without experiencing any class. Specific time-task protocol The other parameter analyzed in this study is the percentage
of correct answers classified by levels of difficulty. This was
calculated as follows: first, it was we obtained the proportion of
correct answers for each separate category –easy, intermediate and
hard- for each group of students. Afterwards, a mean of the
proportions from all the courses was obtained for each category. These means were used to compare the scores between Control
and Novelty groups (i.e. to analyze the effect of novelty in each
category) in the intermediate and hard questions. Easy questions
were taken as a positive control of the students’ attendance to the
lesson and their comprehension of the story. To illustrate the
procedure, here we include an example: to calculate the number of
correct answers from a group of 30 students in control or novelty
conditions, there were 90 possible answers (three questions per
each student, corresponding to one level of difficulty) in each
condition. If control group correctly answered 18 questions out of
90, then their score is 20%. If the novelty group correctly
answered 54 out of those 90 questions, their score is 60%. Using a similar approach as described before, students were
read two stories (A and B) on the same day, with an interval of
three hours between sessions. An hour after the second short story
had been read, a novel science class was presented. 24 h later,
LTM for one of the stories was evaluated on one half of the
students while the other story was evaluated on the second half. As
a control parallel groups of students were read two different stories
(randomized alternate) separated by a 3 hours interval. 24 h later,
LTM was tested for both stories as described above. General protocol LTM of the story was evaluated 24 hours after p.0.05 Students t test. doi:10.1371/journal.pone.0066875.g002 Figure 2. Memory of the story is not enhanced when the students were pre-informed about the contests of a new science lesson or
when just moved around the classroom. Schematic representation of the experimental protocol is presented on the top panels. A) Memory
index for the story is shown as mean 6 SEM for the Control group (n = 25) and Pre-informed group (n = 22). A short story was read to a group of
students that had been previously informed that they would be given a science class outside their habitual classroom one hour after the reading. LTM of the story was evaluated 24 hours after. p.0.05 Students t test. B) Memory index for the story is shown as mean 6 SEM for the Control group
(n = 25) and Moving group (n = 30). A short story was read and one hour after students moved from the classroom to the place where the novel class
would take place and back, without experiencing any class. LTM of the story was evaluated 24 hours after p.0.05 Students t test. doi:10.1371/journal.pone.0066875.g002 (Figures S1, S2, S3). Intermediate difficulty questions addressed
relevant aspects of the story, although only mentioned once. These
questions were correctly answered by about 40% of the students in
the control groups (Figures S1, S2, S3). Hard difficulty questions
were made on details and events unrelated to the core of the plot. These questions were correctly answered by 20% of the students in
the control groups (Figures S1, S2, S3). and understood the reading. One point per each correct answer
was assigned, and Memory index was calculated as the ratio
between the score obtained for both the hard and the intermediate
questions for students belonging to the Novelty group relative to
the mean obtained for its corresponding Control group. Both short
stories were appropriate for the instruction level of the students
and had a comparable complexity (this assumption was based on
the number of correct answers from Control groups). Memory in Children Is Improved by a Novel Class Memory in Children Is Improved by a Novel Class Memory in Children Is Improved by a Novel Class 3 Th
l
i
b
kill d
h
k
h
extremely infrequent to experience this kind of empirical science
lessons
In the class
students were introduced to some basic
Figure 3. Memory of the story is enhanced by a novel, but not a
familiar, music lesson. Schematic representation of the experimental
protocol is presented on the top of the panels. A) Memory index for the
story is shown as mean 6 SEM of the ratio between data from groups
of students that experienced novel (n = 33) or familiar music lesson
(n = 40) and control students (dotted line, n = 52). A short story was read
and a novel music lesson or a familiar music lesson was given 1 hour
later. LTM of the story was evaluated 24 hours after. ** p,0.01 vs
Control and Familiar group, Newman-Keuls analysis after one-way
ANOVA. B) Correct answer percentages corresponding to hard level of
difficulty questions calculated from students analyzed in A (n = 6 for all
groups). ** p,0.01 vs Control, Student’s t test. doi:10.1371/journal.pone.0066875.g003
Figure 4. A graphical memory can also be enhanced by the
experience of a novel science lesson. Schematic representation of
the experimental protocol is presented on the top of the panels. A) LTM
for the complex figure is shown as mean 6 SEM of memory percentage
between the copy and the recall of the figure at the test. Comparisons
were made between control (n = 32) and novel groups (n = 43). ***
p,0.001 vs Control, Student’s t test. B) Mean 6 SEM of the correct
performance percentages corresponding to configural elements and
details of the complex figure are shown for control and novel groups
analyzed in A. ** p,0.01 vs Control, Student’s t test. doi:10.1371/journal.pone.0066875.g004 Figure 3. Memory of the story is enhanced by a novel, but not a
familiar, music lesson. Schematic representation of the experimental
protocol is presented on the top of the panels. A) Memory index for the
story is shown as mean 6 SEM of the ratio between data from groups
of students that experienced novel (n = 33) or familiar music lesson
(n = 40) and control students (dotted line, n = 52). Novel lessons In this work we considered it to be a novel activity if the class/
lesson (science or music) complied with these requirements: 1. The whole group of students was unexpectedly taken from its
classroom and led to a different place to attend a lesson that
was not previously informed about until it started. 2. This novel lesson was given in a place inside the school but not
usually frequented by the students for their lessons, e.g.,
common room, laboratory, patio, hall. Easy questions were based on major aspects of the story such as
characters name and core events in the plot. These questions were
correctly answered by 80% of the students in the control groups June 2013 | Volume 8 | Issue 6 | e66875 June 2013 | Volume 8 | Issue 6 | e66875 PLOS ONE | www.plosone.org 3 Memory in Children Is Improved by a Novel Class A short story was read
and a novel music lesson or a familiar music lesson was given 1 hour
later. LTM of the story was evaluated 24 hours after. ** p,0.01 vs
Control and Familiar group, Newman-Keuls analysis after one-way
ANOVA. B) Correct answer percentages corresponding to hard level of
difficulty questions calculated from students analyzed in A (n = 6 for all
groups). ** p,0.01 vs Control, Student’s t test. d i 10 1371/j
l
0066875 003 Figure 4. A graphical memory can also be enhanced by the
experience of a novel science lesson. Schematic representation of
the experimental protocol is presented on the top of the panels. A) LTM
for the complex figure is shown as mean 6 SEM of memory percentage
between the copy and the recall of the figure at the test. Comparisons
were made between control (n = 32) and novel groups (n = 43). ***
p,0.001 vs Control, Student’s t test. B) Mean 6 SEM of the correct
performance percentages corresponding to configural elements and
details of the complex figure are shown for control and novel groups
analyzed in A. ** p,0.01 vs Control, Student’s t test. doi:10.1371/journal.pone.0066875.g004 extremely infrequent to experience this kind of empirical science
lessons. In the class, students were introduced to some basic
physics and biology principles such as density, gravity, superficial
tension and electrostatics) with playful activities. The novel science
lesson was specially designed and given by the researchers. Besides,
the novel music class was planned and dictated by the music
therapist aiming to resemble those novel features of the science
class. Likewise, students were not informed of this class until its
start and were involved in its activities. Considering that students
habitually have music lessons in their schools (as it is a compulsory
subject), the novel music lesson was based on innovative teaching
methods and it also dealt with topics highly infrequent for the
students. The lesson consisted of games with homemade instru- PLOS ONE | www.plosone.org Complex figure test We designed a task to test graphic memory based on Rey-
Osterrieth’s complex figure test [17]. A complex figure picture
(Figure S4) was shown to the whole group of students and each
student was allowed to copy it on a blank paper for a 2-minute
period. Once finished, drawings were handed in to the teacher. 24 hours later, the memory of this figure was tested by asking
students to draw it again on a blank paper in a 4-minute interval. Memory
percentage
for
each
student
was
assessed
by
comparison of the copy (acquisition session) with the second
drawing (test session). Each of the elements in the figure were
analyzed for location, accuracy and organization by using the Rey-
Osterrieth’s score [18] (Table S2) and an individual score was
obtained. Only those students who had completed more than 75%
of the copy were included in the experiment. We designed a task to test graphic memory based on Rey-
Osterrieth’s complex figure test [17]. A complex figure picture
(Figure S4) was shown to the whole group of students and each
student was allowed to copy it on a blank paper for a 2-minute
period. Once finished, drawings were handed in to the teacher. 24 hours later, the memory of this figure was tested by asking
students to draw it again on a blank paper in a 4-minute interval. Memory
percentage
for
each
student
was
assessed
by
comparison of the copy (acquisition session) with the second
drawing (test session). Each of the elements in the figure were
analyzed for location, accuracy and organization by using the Rey-
Osterrieth’s score [18] (Table S2) and an individual score was
obtained. Only those students who had completed more than 75%
of the copy were included in the experiment. We then wondered to know if another type of novelty, in the
form of a music lesson, given 1 hour after the reading of the story,
could also enhance memory consolidation. As expected, this kind
of novelty also improved the narrative memory (Fig. 3A). Interestingly, the enhancing effect was not observed when the
lesson was familiar (Fig. 3A). For this purpose, the music lesson
had been repeatedly given for two weeks. In the third week the
story was read to the students one hour before the familiar music
lesson. Results Using cognitive memory tests we evaluated the consequences of
undergoing a novel experience in the school environment, before
or after a standard learning session. In our first experiment, this
session consisted of a short story that was read to elementary
school students, between ages 7 and 9 (Table S1). Twenty-four
hours later we evaluated how much they remembered about it
with a questionnaire. The novel experience was science lesson with
contents completely unknown by the students (See Text S1). Memory improvements were observed in those groups of students
who had experienced the novel science lesson 1 hour before or
after the story telling, but not 4 hours before or after it (Fig. 1A). The test list included questions of different levels of difficulty –easy,
intermediate and hard- (See Text S1). The percentage of correct
answers to hard questions was increased in those groups of
students who had a novel science lesson 1 hour before or after the
reading of the story, but not 4 hours before or after it (Fig. 1B). A
similar outcome was observed in the case of intermediate difficulty
answers, without any significant changes in the easy ones (Fig. S1). In addition, to assess the tasks’ time-specificity of this effect,
students were read two different stories separated by a 3-hour
interval, being only one of them followed by a novel lesson 1 hour
later. Memory enhancement was observed for the story in the
adjacent context of novelty but not for the other, in accordance to
the time-dependence effect of novelty observed in Fig. 1A (Fig. 1C). As a control, parallel groups of students were read two different Familiar lesson The same music lesson described above was presented for
2 weeks (one lesson each week) and in the third week this ‘‘familiar
lesson’’ was associated with a short story read 1 hour before the
music lesson. The following day, long-term memory for the story
was tested. Memory in Children Is Improved by a Novel Class stories separated by a 3 hour interval and LTM was tested for
both stories. When the stories were not associated with a novel
lesson, there was no difference between the scores obtained for
each of them. (Memory index for Story A = 1.00160.09470,
n = 39 vs Story B = 1.08060.1050, n = 47; p.0.05 Student’s t
test). Moreover, the percentages of correct answers to the hard
(Fig. 1D) and intermediate difficulty questions (Fig. S2) were
significantly increased for the story read close to novelty in relation
to the story read 4 hours before it. Easy questions were correctly
answered by a similar percentage of students in both groups
(Fig. S2). This suggests the effect of novelty accounts for the
enhancement of memory on the harder tasks. In addition, it is
important to note that the promoting effects of novelty lessons
were not merely due to moving around. The control group of
students that only moved from the classroom to the place where
the novel class took place for other groups showed no enhance-
ment in their Memory index when they experienced this moving
around one hour after the reading (Fig. 2A). Similarly, there was
no memory improvement in the groups of students which had
been previously informed of the contents of the novel class
(Fig. 2B). ments (like rods, x-ray films, buckets and balls) and it was intended
to show that music could even be made with simple elements and
yet they could also form an orchestra with those atypical
instruments. Complex figure test The percentage of correct answers on the hard questions
was significantly increased for the students in the novelty group;
however, if the students were familiarized to the music lesson, such
effect was not observed (Fig. 3B). Intermediate difficulty and easy
questions were answered by a similar proportion of students in
both groups (Fig. S3). These results show that the enhancing effect
is specifically related to the novel nature of the music lesson and
not to the lesson itself. Another parameter analyzed in this experiment is the correct
performance percentage classified by levels of visual com-
plexity. Configural elements are considered to be the most
‘‘global’’ parts of the design, the details as the most ‘‘local’’
elements (Picture S1). An average was obtained from the
individual performances for both configural elements and for
details. Literary and graphical activities were performed by each
group’s own teacher inside the classroom as part of a regular class. For more experimental details, see Text S1. Finally, to analyze the generality of the phenomena, we selected
a different type of learning based on graphical information. Therefore, we implemented an adaptation of the Rey-Osterrieth’s
complex figure task for children [17]. The procedure followed was
the same as before. Students were allowed to copy a figure for
2 minutes and 24 hours later they were asked to draw it again in
4 minutes, based on what they remembered of it. The individual
retention score was calculated for each item in the figure drawn in
the test session by comparing them with the figure drawn the day
before (See Text S1). Analysis of the drawings showed a significant
enhancement in the retention for those students who experienced
a novel science lesson one hour after copying the figure (Fig. 4A). Interestingly, the improvement was observed in the retention of
the Rey-Osterrieth’s complex figure as a whole, since the number
of correct items was increased in both configural objects and in
details as well (Fig. 4B). 3. The lesson was given by a skilled teacher unknown to the
students. 3. The lesson was given by a skilled teacher unknown to the
students. 3. The lesson was given by a skilled teacher unknown to the
students. 3. The lesson was given by a skilled teacher unknown to the
students. 4. It is a short activity (20 min) never experienced before by the
students, with novel contents appropriate for their age. 5. Students are encouraged to actively participate and be attentive
at all times. When the activity finishes, they return to their
habitual classroom. The novel science class was based on simple experiments aiming
to the constant participation of the students and their full
interaction with the elements. In the first years at school it is June 2013 | Volume 8 | Issue 6 | e66875 June 2013 | Volume 8 | Issue 6 | e66875 PLOS ONE | www.plosone.org 4 Supporting Information Though it cannot be demonstrated in this work
that the effect of novelty depends on protein synthesis, we put
forward the hypothesis that the learning experience (story/
drawing) triggers a transient process (which resembles the idea of
a learning tag) that enables the consolidation of those information
by the effects of the novel experience occurring around a critical
time window. Further studies are required on the identity of the
tag and its location. Our results show that novelty has a
symmetrical effect on the promotion of LTM formation, whether
it occurs 1 h before or after the acquisition of the information, but
it does not exert any effect on LTM promotion if it is experienced
at distant time points of the acquisition. Moreover, when the two
stories were read to the same groups of students, novelty specifically
exerted a promoting effect on the memory of the story read 1 h
before and not on the other story that had been read 4 h before. These results suggest that when these events (story and novelty) are
temporally more distant, the processes triggered by both of them
probably do not interact. Besides, the promoting effect of novelty
on memory formation depends on the novel features of the lesson;
when it is familiar, it lacks efficacy. Therefore, taken as a whole,
our findings suggest that a behavioral tagging-like process could be Figure S2
The percentage of correct answers is in-
creased when students experience a novel science lesson
in a specific time lapse around the task. Schematic
representation of the experimental protocol is presented on the
top of the panels. Figures depict the mean 6 SEM of the
percentage of correct answers to easy (+4 h, n = 8; +1 h, n = 8) and
intermediate (+4 h, n = 6; +1 h, n = 6) levels difficulty questions
from the same groups of students analyzed in Fig. 1C and D. Students experienced a novel science lesson in different times
(+1 h and +4 h) from a particular story (A or B) telling,. Half of the
students were tested on the story A and the rest on the story B. *
p,0.05 vs +4 h. Figure S3
Percentage of correct answer is not enhanced
when
the
concurrent
lesson
is
familiar. Schematic
representation of the experimental protocol is presented on the
top of the panels. Supporting Information Figure S1
The percentage of intermediate difficulty
questions correctly answered is increased when stu-
dents experience a novel science lesson around the time
of the reading. Schematic representation of the experimental
protocol is presented on the top panels. A novel Science lesson was
given at different times before (24 h, 21 h) or after (+1 h, +4 h)
the reading of a short story. 24 hours later it was evaluated how
much the students remembered on the story by means of a written
test. Means 6 SEM of the percentage of correct answers to easy
(24 h, n = 8; 21 h, n = 12; +1 h, n = 12; +4 h, n = 12) and
intermediate (24 h, n = 6; 21 h, n = 9; +1 h, n = 9; +4 h, n = 9)
levels of difficulty questions are shown corresponding to the same
groups of students analyzed in Figures 1A and B. Comparisons
were made between control and novel groups for each of the time
points. * p,0.05, ** p,0.01 vs Control, Student’s t test. (TIF) There is a well accepted idea establishing that the entrance of
new information into LTM depends on neural signals triggered by
the novel or rewarding aspects of the stimulus to be encoded
[25,26]. Our findings are in consistent with these observations and
show that the role of novelty is broader than it was previously
thought. In all, we demonstrate that novel lessons exert a
penumbra-like effect on the surrounding events that can improve
the LTM of students for information acquired in temporal
proximity of novelty. Based on ‘‘synaptic tagging and capture’’
hypothesis [27,28,29,30,31], we have shown in rodents that novel
events late-associated to a weak learning synthesized proteins
which could be captured by the learning tags set by the weak
training, resulting in the promotion of LTM formation for this
event [12,14,16]. This phenomenon was named ‘‘behavioral
tagging’’ and it was demonstrated the existence of a window of
sensitivity to novelty which could be explained in terms of the
transient nature of the learning tag induced by the weak training
and also by the kinetics of production and degradation of
plasticity-related protein induced by novelty. Here, we show that
novel lessons can exert late-associative effects on LTM formation
for the story telling or figure drawing, leading to the consolidation
of their memories. Memory in Children Is Improved by a Novel Class operating in humans. Alternatively, any other brain mechanisms
based on factors triggered by a novel lesson acting on an
anterograde and retrograde critical time window close to the
learning session could explain the promoting effects on its LTM
formation. cognitive functions (increased working memory, increased fluid
intelligence, etc), encouraging them to undergo intensive training
[24]. Even in the case of positive outcomes, there is not such a
project of these characteristics in the educative field. Here we show
a consistent enhancement of memory in students of different ages
using a quick non-expensive methodology (it only requires a novel
session of 20 minutes) that can be easily implemented by teachers
in the school setting. Finding different teaching strategies to improve students’
performance is a subject of great relevance to education. Our
findings provide a tool that could be easily transferred to the
classroom by the incorporation of educationally novel events in the
school schedule as an extrinsic adjuvant of other information
acquired some time before or after it. This approach could be a
helpful tool for the consolidation of certain types of topics that
generally demand a great effort from the children. Our results show that educationally relevant novel events
experienced during normal school hours can improve LTM for
tasks learned during regular school lessons. This effect is restricted
to a critical time window around learning and is particularly
dependent on the novel nature of the associated experience. In
addition, we show that this memory enhancement procedure is
applicable to at least two different types of memory, one narrative
(verbal) and the other graphical (visual), by different kinds of novel
experiences. Furthermore, these findings suggest that memory
improvement induced by novelty is not derived from an increase
in the arousal state or from lowering the threshold to learn,
because both types of novel experiences can exert their positive
effect on memory even when they occur after the acquisition of the
information to be remembered. Discussion Different strategies have been used in an attempt to improve
cognitive functions, (e.g. executive functions, memory, attention,
processing
speed)
through
transference,
generalization
and
retrieval [19,20,21,22,23]. In that sense, numerous games (e.g. Brain Age, Big Brain Academy, and Brain Challenge), web pages
and computer programs (e.g. Luminosity, Happy neuron, Who
has the biggest brain?, Brain training, etc) attract thousands of
users under the promise of an accelerated enhancement in several In addition, to assess the tasks’ time-specificity of this effect,
students were read two different stories separated by a 3-hour
interval, being only one of them followed by a novel lesson 1 hour
later. Memory enhancement was observed for the story in the
adjacent context of novelty but not for the other, in accordance to
the time-dependence effect of novelty observed in Fig. 1A (Fig. 1C). As a control, parallel groups of students were read two different PLOS ONE | www.plosone.org June 2013 | Volume 8 | Issue 6 | e66875 June 2013 | Volume 8 | Issue 6 | e66875 5 References 16. Moncada D, Ballarini F, Martinez MC, Frey JU, Viola H (2011) Identification
of transmitter systems and learning tag molecules involved in behavioral tagging
during memory formation. Proc. Natl. Acad. Sci. 108, 12931–6. 1. Muller G, Pilzecker A (1900) Experimentelle Beitrage zur jehere von Gedachtiss. Psychol. 1, 1–288. 2. McGaugh JL (1966) Time-dependent processes in memory storage. Science 153,
1351–8. 17. Rey A (1959) Test de copie et de reproduction de memoire de figures
geometriques complexes [A test of copy and recall of a complex geometric
figure]. Editions du centre de Psycholegie Appliquee. 3. McGaugh JL (2000) Memory – a century of consolidation. Science 287, 248–51. 4. Dudai Y, Eisenberg M (2004) Rites of passage of the engram: reconsolidation
and the lingering consolidation hypothesis. Neuron 44, 93–100. 18. Shin MS, Park SY, Park SR, Seol SH, Kwon JS (2006) Clinical and empirical
applications of the Rey-Osterrieth Complex Figure Test. Nature Protocols 1,
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Reward-motivated learning: mesolimbic activation precedes memory formation. Neuron 50, 507–17. 19. Jaeggi SM, Buschkuehl M, Jonides J, Perrig WJ (2008) Improving fluid
intelligence with training on working memory. Science 105, 6829–6833. 6. McGaugh JL (2004) The amygdala modulates the consolidation of memories of
emotionally arousing experiences. Annu. Rev. of Neurosci. 27, 1–28. 20. Owen A, Hampshire A, Grahn JA, Stentor R, Dajani S, et at. (2010) Putting
brain training to the test. Nature 465, 775–8. emotionally arousing experiences. Annu. Rev. of Neurosci. 27, 1 7. Roozendaal B, McGaugh JL (2011) Memory modulation. Behav.Neurosci., 125,
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Affective Domain: Increasing Cognitive and Affective Executive Control
through Emotional Working Memory Training. PloS ONE 6, doi:10.1371/
journal.pone.0024372. 8. Schwabe L, Bohringer A, Chatterjee M, Schachinger H (2008) Effects of pre-
learning stress on memory for neutral, positive and negative words: Different
roles of cortisol and autonomic arousal. Neurobiol. Learn. Mem. 90, 44–53. 22. Karpicke JD, Roediger HL (2008) The critical importance of retrieval for
learning. Science 319, 966–968. 9. Wittmann BC, Schott BH, Guderian S, Frey JU, Heinze HJ, et al. (2005)
Reward-related FMRI activation of dopaminergic midbrain is associated with
enhanced hippocampus-dependent long-term memory formation. Neuron 45,
459–67. 23. Roediger HL, Butler AC (2011) The critical role of retrieval practice in long-
term retention. TiNS 15(1):20–7. 24. Supporting Information Memory for the story is shown as mean 6 SEM
of the ratio between data from groups of students that experienced
novel or familiar music lesson and control students. A short story
was read and a novel music lesson or familiar music lesson was
given 1 hour later. 24 hours later it was evaluated how much the
students remembered about the short story. Correct answer
percentages corresponding to easy (n = 8 for all groups) and
intermediate (n = 6 for all groups) levels of difficulty questions are
shown for the same groups of students analyzed in Fig. 2
compared against its control group. p.0.05 vs. Control, Student’s
t test. (TIF) Picture S1
Rey- Osterrieth’s complex figure for chil-
dren. Students were given this image to copy during acquisition
session and 24 hours later their memory for the figure was tested June 2013 | Volume 8 | Issue 6 | e66875 PLOS ONE | www.plosone.org 6 Memory in Children Is Improved by a Novel Class by asking them to draw what they recalled of it (test session). In this
figure we considered 4 Configural elements, here pictured in black
(circle, triangle, rectangle and square), and the 7 detail elements,
here pictured in green (two dots, a cross, semicircle, 4 lines inside
the semicircle, diagonal inside the square, black dot inside the
square and equal symbol). (DOC) drawing the ROCF(2). If the student completes all the figures and
places them in the right position, the corresponding score will be of
22 points. (DOC) drawing the ROCF(2). If the student completes all the figures and
places them in the right position, the corresponding score will be of
22 points. (DOC) Text S1
Supplementary methods. (DOC) Table S1
Characteristics of the institutions and the
students intervening in the experiments. Detailed infor-
mation on the participating school shifts, age and number of the
students. All the students attend to only one shift in these schools. The schools that offer both morning and afternoon shifts, have
separate groups of students in the respective morning and
afternoon shifts. Table S1
Characteristics of the institutions and the
students intervening in the experiments. Detailed infor-
mation on the participating school shifts, age and number of the
students. All the students attend to only one shift in these schools. Author Contributions Conceived and designed the experiments: FB HV. Performed the
experiments: FB. Analyzed the data: FB HV. Contributed reagents/
materials/analysis tools: FB MCM MDP DM HV. Wrote the paper: FB
MCM HV. Acknowledgments We thank J. H. Medina, T. Bekinschtein, P. Bekinschtein and L. Muller
Igaz for their helpful comments and discussion of the manuscript. We thank J. H. Medina, T. Bekinschtein, P. Bekinschtein and L. Muller
Igaz for their helpful comments and discussion of the manuscript. Supporting Information The schools that offer both morning and afternoon shifts, have
separate groups of students in the respective morning and
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Training Game Improves Executive Functions and Processing Speed in the
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and capture hypothesis. Nat. Rev. Neurosci. 12, 17–30. 11. Almaguer-Melian W, Bergado-Rosado J, Pavo´n-Fuentes N, Alberti-Amador E,
Mercero´n-Martı´nez D, et al. (2012) Novelty exposure overcomes foot shock-
induced spatial-memory impairment by processes of synaptic-tagging in rats. Proc. Natl. Acad. Sci. 109, 953–958. p
25. Lisman J, Grace AA (2005) The hippocampal-VTA loop: controlling the entry of
information into long-term memory. Neuron 46, 703–713. 26. Lisman J, Grace AA, Duzel E (2011) A neoHebbian framework for episodic
memory: role of dopamine-dependent late LTP. TiNS, 34, 536–47. 12. Ballarini F, Moncada D, Martinez MC, Alen N, Viola H (2009) Behavioral
tagging is a general mechanism of long-term memory formation. Proc. Natl. Acad. Sci. 106, 14599–14604. 27. Barco A, Lopez de Armentia M, Alarcon JM (2008) Synapse-specific
stabilization of plasticity processes: the synaptic tagging and capture hypothesis
revisited 10 years later. Neurosci. Biobehav. Rev. 32, 831–51. ,
13. Lu Y, Ji Y, Ganesan S, Schloesser R, Martinowich K, et al. (2011) TrkB as a
Potential Synaptic and Behavioral Tag. J. Neurosci. 31, 11762–11771. revisited 10 years later. Neurosci. Biobehav. Rev. 32, 831–51. . Lu Y, Ji Y, Ganesan S, Schloesser R, Martinowich K, et al. (2011 28. Frey U, Morris RG (1997) Synaptic tagging and long-term potentiation. Nature
385, 533–536. 14. Moncada D, Viola H (2007) Induction of long-term memory by exposure to
novelty requires protein synthesis: evidence for a behavioral tagging. J. Neurosci. 27, 7476–7481. 29. Frey U, Morris RG (1998a) Synaptic tagging: implications for late maintenance
of hippocampal long-term potentiation. TiNS 21, 181–8. 30. Frey U, Morris RG (1998b) Weak before strong: dissociating synaptic tagging
and plasticity-factor accounts of late-LTP. Neuropharmacol. 37, 545–52. 15. Wang SH, Redondo RL, Morris RGM (2010) Relevance of synaptic tagging and
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813–20. June 2013 | Volume 8 | Issue 6 | e66875 PLOS ONE | www.plosone.org 7
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English
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Expressions of Carbohydrate Response Element Binding Protein and Glucose Transporters in Liver Cancer and Clinical Significance
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Pathology and oncology research/Pathology oncology research
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cc-by
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Abstract Carbohydrate response element binding protein (ChREBP) is a glucose-sensing transcription factor that mediates the induction of
glycolytic and lipogenic genes in response to glucose. We investigated the expression patterns of ChREBP and glucose transporters
(GLUTs) in human hepatocellular carcinoma (HCC) and their association with HCC progression. ChREBP, GLUT2 and GLUT1
immunohistochemistry were performed on liver tissue array containing normal liver tissue, HCC adjacent tissue and cancer tissue of
different HCC stages. The effect of HCC malignancy on protein expression was analyzed with one-way ANOVA. The correlations
between protein expressions were analyzed with Pearson Correlation test. We found that ChREBP protein expression tended to be
positively correlated to liver malignancy. GLUT2 protein expression was significantly reduced in human HCC as compared to normal
liver tissue and its expression in HCC was inversely associated to malignancy (p < 0.001). In contrast, GLUT1 was significantly
increased in cancer cells and its expression was positively correlated to malignancy (p < 0.001). Furthermore, GLUT1 expression was
positively associated to ChREBP expression (r = 0.481, p < 0.0001, n = 70) but negatively correlated to GLUT2 expression (r =
−0.320, p = 0.007, n = 70). Notably, ChREBP-expressing hepatocytes did not express GLUT2 but GLUT1. This is the first report
unveiling expressions of ChREBP and GLUT2/GLUT1 and their relations in HCC. The expression patterns are related to malignancy
and this information would facilitate evaluation of clinical behavior and treatment of HCC. Keywords ChREBP . Glucose transporters . Hepatocellular carcinoma . Diagnostic marker . Glycolysis Pathology & Oncology Research
https://doi.org/10.1007/s12253-019-00708-y
(2020) 26:1331–1340 Pathology & Oncology Research
https://doi.org/10.1007/s12253-019-00708-y
(2020) 26:1331–1340 ORIGINAL ARTICLE Expressions of Carbohydrate Response Element Binding Protein
and Glucose Transporters in Liver Cancer and Clinical Significance Yu Lei1,2,3 & Qiaoling Hu1 & Jiang Gu1,3,4 Received: 10 June 2019 /Accepted: 1 August 2019
# The Author(s) 2019
/Published online: 12 August 2019 4
Department of Pathology, Beijing University Health Science Center,
Beijing 100083, China Introduction promotes lipogenesis and nucleotide biosynthesis, and en-
hances tumor cell growth and proliferation by providing es-
sential synthetic and bioenergetic molecules [9, 10]. hepatic ChREBP expression has not been examined in human
HCC and the relationship between ChREBP expression and
the degree of liver tumor malignancy has yet to be
investigated. Cancer cells acquire energy from glucose to satisfy their
high metabolic demands. Glucose and related hexoses are
transported into cells via glucose transporter (GLUT) family
proteins (Solute carrier, SLC2A family) [11]. To date, 14
GLUT family members have been identified, each of them
with different affinities for glucose and their expression pat-
terns are tissue dependent [12, 13]. Many studies have dem-
onstrated that the expression of glucose transporters, especial-
ly GLUT1, is increased in a variety of tumors, including pan-
creatic, breast, esophageal, and brain cancers [14, 15]. GLUT1
overexpression is associated with tumor progression and poor
overall survival in various malignant tumors [16, 17]. GLUT2
is expressed in liver, pancreatic islet cells and retina and it is
the most abundant glucose transporter in liver [18]. GLUT2
mRNA expression was shown to be increased in gastric tu-
mors [19] but decreased in insulinomas when compared to
normal tissues [20]. However, data on these glucose transport-
er expression in different stages of HCC have been lacking. In this study, we analyzed GLUT family members GLUT2
and GLUT1, and ChREBP protein expression levels in human
liver tissue array composed of normal, HCC and adjacent liver
tissue. We found that the expression of ChREBP showed a
tendency to increase with liver malignancy, but unexpectedly,
that GLUT2 protein expression was decreased in cancer cells
compared to normal hepatocytes and its expression was neg-
atively associated with advanced stages of HCC. GLUT1 was
increased in cancer tissues and its expression was significantly
correlated with clinical stage. Moreover, ChREBP and
GLUT1 expressions were positively correlated to each other
but GLUT1 and GLUT2 expressions were negatively corre-
lated. Therefore, a combined evaluation of ChREBP, GLUT1
and GLUT2 protein expression profile may provide a new
diagnostic and prognostic marker for HCC, that might be use-
ful in improving patient treatment and survival. One of the master regulators of intracellular glucose me-
tabolism is carbohydrate responsive element binding protein
(ChREBP), a basic helix–loop–helix leucine zipper (bHLH-
LZ) transcription factor expressed in liver, white and brown
adipose tissues, intestine, muscle, and pancreatic β-cells [21,
22]. Introduction have progressed to advanced stages at time of diagnosis,
resulting in poor prognoses and high mortality. Despite vari-
ous treatment options, survival of HCC is poor due to late
diagnosis and resistance to chemotherapy. The current gold
standard and most commonly used diagnostic marker for
HCC is plasmic alpha-fetoprotein (AFP) along with ultra-
sound every 6 to 12 months, but this is far from satisfactory. Only serum AFP levels of more than 400 ng/mL are consid-
ered truly diagnostic, but such high values are observed only
in a small percentage of patients with HCC at advanced stages
[1]. Although many molecules have been considered to asso-
ciate with diagnosis and prognosis, their presumed merits are
controversial [2, 3]. Accordingly, new diagnostic and prog-
nostic factors are needed to improve HCC diagnosis and
treatment. Hepatocellular carcinoma (HCC) is the fifth most common
cancer in men and the seventh in women. Many HCC patients Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s12253-019-00708-y) contains supplementary
material, which is available to authorized users. * Jiang Gu
2523381625@qq.com 1
Department of Pathology and Pathophysiology, Provincial Key
Laboratory of Infectious Diseases and Immunopathology,
Collaborative and Creative Center, Shantou University Medical
College, Shantou 515041, Guangdong, China
2
Department of Pediatrics, University of Groningen, University
Medical Center Groningen, 9713, GZ Groningen, The Netherlands
3
Jinxin Research Institute for Reproductive Medicine and Genetics,
Chengdu Jinjiang Hospital for Maternal and Child Health Care, 66
Jingxiu Road, Chengdu 610066, China
4
Department of Pathology, Beijing University Health Science Center,
Beijing 100083, China 1
Department of Pathology and Pathophysiology, Provincial Key
Laboratory of Infectious Diseases and Immunopathology,
Collaborative and Creative Center, Shantou University Medical
College, Shantou 515041, Guangdong, China Evidence is accumulating that perturbed cellular metabo-
lism predisposes human to tumor development. Metabolic
diseases such as obesity and diabetes are associated with in-
creased risk to develop various cancers [4–6]. In addition,
many human tumors display a high rate of aerobic glycolysis,
de novo fatty acid synthesis and nucleotide biosynthesis [7, 8]. It has been proposed that increased glucose metabolism 2
Department of Pediatrics, University of Groningen, University
Medical Center Groningen, 9713, GZ Groningen, The Netherlands 3
Jinxin Research Institute for Reproductive Medicine and Genetics,
Chengdu Jinjiang Hospital for Maternal and Child Health Care, 66
Jingxiu Road, Chengdu 610066, China 4
Department of Pathology, Beijing University Health Science Center,
Beijing 100083, China 1332 Y. Lei et al. Materials and Methods Human hepatic carcinoma and normal tissue microarray was
purchased from AURAGENE (TC0145, Changsha, China). The tissue array contains 40 cases of hepatocellular carcino-
ma, 17 cases of normal tissue and 13 cases of adjacent normal
tissue. In addition, 5 pairs of cancer and adjacent normal tis-
sues were collected from the Second Affiliated Hospital of
Shantou University Medical College, Shantou, China. This
study has been approved by Shantou University medical col-
lege Ethic Committee. All methods were performed in accor-
dance with the relevant guidelines and regulations. Informed
consent was obtained from all participants and/or their legal
guardians. Introduction Elevated insulin, in response to increased glucose levels,
promotes ChREBP dephosphorylation and its translocation
from the cytoplasm to the nucleus where, in association with
its binding partner MLX (Max-like interacting protein), it
binds to carbohydrate response elements, present in the pro-
moters of target genes. Glucose metabolites F6P and acetyl-
CoA can increase ChREBP activity via O-linked glycosyla-
tion and acetylation, respectively [23–25]. The ChREBP/Mlx
heterodimer controls glucose and lipid metabolism through
regulating glycolytic (Pklr, Fk, Glut2, Glut4), gluconeogenic
(G6pc), and lipogenic (Fasn, Acc1, Scd1, Elovl6) gene expres-
sions [22, 26, 27], suggesting that ChREBP may have an
important role in the pathogenesis of metabolic diseases and
cancer. So far, most of the work on ChREBP has focused on
its function as a hepatic transcription factor, its activation by
glucose metabolites and its role in the regulation of lipogene-
sis. Little is known about the role of ChREBP in cancer cells. Genomic analysis of ChREBP target gene expression in hu-
man hepatocellular carcinoma cell line HEPG2 by ChIP-
sequencing showed that ChREBP regulates genes associated
with a tumor metabolic phenotype and malignant progression,
such as tumor differentiation and motility [28]. Suppression of
ChREBP in hepatocellular carcinoma and colorectal cancer
cells led to reduced lipogenesis and nucleotide synthesis and
decreased proliferative and tumorigenic potential in mice [29]. In breast cancer, the level of ChREBP protein expression is
positively correlated with tumor progression [30]. However, ChREBP Protein Expression Tended to Increase
with Malignancy The stain-decolorize-stain method was performed to show
whether GLUT2 and ChREBP are expressed in the same
hepatocytes/pattern according to an established procedure
[32]. Briefly, immunohistochemistry of ChREBP was first
performed on the liver microarray. After visualization and
taking photos, the slides were decolorized with 80% alcohol
for half an hour at room temperature and then heated in a
microwave oven for 10 min to remove the bound antibodies. The microarray and slides were then incubated with GLUT2
antibody at 4 °C overnight. After visualization with the AEC
kit, photos were taken at the same fields as for the first immu-
nostaining. Appropriate controls were carried out and the
specificity of this technique has been reported previously [32]. Immunohistochemistry demonstrated that 4 out of 5 pairs of
liver sample clearly showed stronger positive intensity of
ChREBP protein in malignant tissues than the adjacent normal
tissues (Fig. 1a). Only 1 pair of tissue showed similar intensity
of ChREBP expression in malignant and adjacent normal tis-
sue. The scatter positive signals in non-hepatocytes were T
lymphocytes. All the CD3, T lymphocyte marker, −positive
cells showed positive signals for ChREBP (Fig. S1A-B). Most
of the CD68, macrophage marker, −positive cells did not
show clear ChREBP signals, but a few cells showed mild
positive signals (Fig. S1C-D). ChREBP immunohistochemistry was also performed in
tissues of different HCC clinical stages to determine whether
there is an association between ChREBP expression and ma-
lignancy. The staining results showed a trend of increasing
intensity of ChREBP with HCC malignant progression as de-
fined by histopathological diagnosis (Fig. 1b). The staining
positivity was quantified with a score defined by staining in-
tensity and size of positive area (Table 2). In normal and HCC
adjacent tissues, about 40% of the sections showed no
ChREBP positivity and about 37% showed mild ChREBP
positivity, but no sections showed strong or very strong
stainings. However, in stage II and stage III HCC tissues,
about 30% of the sections showed no ChREBP expression, Patient Characteristics A total of 70 liver samples were analyzed, of which 40 were
defined as HCC. Mean age of the 40 cancer patients was
49 years and 82% were males. Stages I, II and III accounted
for 6%, 19% and 33% of total liver samples, respectively. There
were 14 normal liver tissues and 13 cancer adjacent tissues, and
they accounted for 20% and 19% of total, respectively (Table 1). Immunohistochemistry Immunohistochemistry was carried out following an
established protocol [31]. Briefly, liver tissue microarray or
paraffin tissue sections were dewaxed, rehydrated through
graded ethanol and incubated with 3% hydrogen peroxide
for 30 min. Antigen retrieval was performed by heating the
sections to 95 °C in 0.01 mol/l citrate buffer (pH 6.0) for
15 min. Slides were then washed in PBS for 15 min and
treated with 10% normal horse serum for 30 min and incubat-
ed with primary antibody at 4 °C overnight. The reaction
products were detected with 3-amino-9-ethylcarbozole
(AEC) substrate-chromogen kit after incubating with the sec-
ondary antibody of Dako REAL EnVision Detection Kit
(Dako, Carpinteria, CA) for 30 min and washing in 0.1 M
PBS at room temperature. Staining with AEC resulted in red Expressions of Carbohydrate Response Element Binding Protein and Glucose Transporters in Liver Cancer and... 1333 Results signals. The primary antibody for GLUT2 (Novus
Biologicals, NBP2-22218SS, USA), GLUT1 (Abcam,
ab115730, USA), were used at a 1:500 dilution and anti-
ChREBP (Novus Biologicals, NB400–135, USA) was used
at a 1:200 dilution. The antibodies against GLUT2 and
ChREBP were rabbit-derived polyclonal and the antibody
against GLUT1 was rabbit-derived monoclonal. For negative
controls, the primary antibodies were replaced with PBS. signals. The primary antibody for GLUT2 (Novus
Biologicals, NBP2-22218SS, USA), GLUT1 (Abcam,
ab115730, USA), were used at a 1:500 dilution and anti-
ChREBP (Novus Biologicals, NB400–135, USA) was used
at a 1:200 dilution. The antibodies against GLUT2 and
ChREBP were rabbit-derived polyclonal and the antibody
against GLUT1 was rabbit-derived monoclonal. For negative
controls, the primary antibodies were replaced with PBS. Semiquantition of Stainings of ChREBP and Glucose
Transporters Semiquantitative scoring was carried out as described in a
previous study [33]. Briefly, tissue microarray sample ‘spots’
were viewed at 400× magnification and an overall score was
assigned according to intensity and area of positive immuno-
staining. Sample scorings are as follows: 0, no red staining at
all (negative); 1, pink staining in the minority of tissue (mild);
2, pink staining in the majority of the tissue (middle); 3, red
staining in the majority of the tissue (strong) and 4, dark-red
staining in all the tissue (very strong) following a well
established protocol [33]. To reduce the variation of scoring,
all slides, including microarray, were scored by two observers
independently (Y. L. and Q. H.). Table 1
Patient information
Total samples
70
Average age of cancer patient (mean ± SD)
49 ± 10
Number
Percentage (%)
Male with cancer
33
82%
Female with cancer
7
18%
Normal tissue
14
20%
Adjacent normal tissue
13
19%
Stage I
4
6%
Stage II
13
19%
Stage III
23
33%
Fatty degeneration
3
4% Statistical Analysis The effect of HCC malignancy on protein expression was
analyzed with one-way ANOVA. The correlation between 2
protein expression levels were analyzed with Pearson
Correlation test. If protein expression levels were normally
distributed and had equal variance, Student’s t test was used. If the scores were not normally distributed, we used the Mann-
Whitney U test. p < 0.05 was considered statistically signifi-
cant. All figures and statistics were made with Graphpad
Prism 7 (CA, USA). 1334 Y. Lei et al. patient. Scale bar = 30 μm. B ChREBP staining in liver cancer tissues
with different malignant progression. a, normal liver tissue; b, stage I liver
cancer; c, stage II liver cancer; d, stage III liver cancer. Scale bar = 30 μm Fig. 1 ChREBP immunohistochemistry in HCC and normal liver
tissue. A The strong ChREBP positive staining in malignant liver (a)
compared with mild staining in adjacent normal tissue (b) in a same
patient. Scale bar = 30 μm. B ChREBP staining in liver cancer tissues
with different malignant progression. a, normal liver tissue; b, stage I liver
cancer; c, stage II liver cancer; d, stage III liver cancer. Scale bar = 30 μm Fig. 1 ChREBP immunohistochemistry in HCC and normal liver
tissue. A The strong ChREBP positive staining in malignant liver (a)
compared with mild staining in adjacent normal tissue (b) in a same
patient. Scale bar = 30 μm. B ChREBP staining in liver cancer tis
with different malignant progression. a, normal liver tissue; b, stage I
cancer; c, stage II liver cancer; d, stage III liver cancer. Scale bar = 30 patient. Scale bar = 30 μm. B ChREBP staining in liver cancer tissues
with different malignant progression. a, normal liver tissue; b, stage I liver
cancer; c, stage II liver cancer; d, stage III liver cancer. Scale bar = 30 μm Fig. 1 ChREBP immunohistochemistry in HCC and normal liver
tissue. A The strong ChREBP positive staining in malignant liver (a)
compared with mild staining in adjacent normal tissue (b) in a same 25% showed mild staining, 9% showed strong expression,
and 16% showed very strong positivity (Table 2). The strong
and very strong stainings were only seen in highly malignant
(Stages II and III) HCC tissues (Fig. 4a and Table 2). However, the result of one-way ANOVA of ChREBP expres-
sion and clinical stage found no significant difference (p =
0.172). GLUT2 Protein Expression Negatively Correlates
to Malignancy Since it has been reported that glycolysis is increased in various
cancer cells [34, 35] and GLUT2 is the main glucose transporter
in liver [18], GLUT2 protein expression was examined (Fig. 2). In HCC, no GLUT2 protein expression was detected (Fig. 2A,
a), while GLUT2 was highly expressed in the adjacent normal
liver tissue, mainly at the hepatocyte membrane (Fig. 2A, b). In
addition, GLUT2 expression was decreased with malignant pro-
gression (Fig. 2B and Fig. 4b). In normal hepatocytes, GLUT2
protein was clearly detectable at the hepatocyte membrane (Fig. 2B, a). In stage I liver cancer tissues, no positive signal could be Statistical Analysis Therefore, there was only a tendency of increased
ChREBP expression with malignancy. detected (Fig. 2B, b). In stage II, most of the samples showed
mild positivity (Fig. 2B, c), but in stage III most tissues showed
no signal (Fig. 2B, d). Quantification of stain positivity was
carried out based on the positivity scores that were defined by
staining intensity and size of positive area (Table 2 and Fig. 4b). It showed that in normal tissue about 36% showed medium
GLUT2 positivity, 40% showed strong or very strong staining. Only a few normal liver tissue sections showed negative or mild
staining. In stage I HCC, 100% of the sections showed no stain-
ing. This may be attributed to limited number of samples in
Stage I. In stage II HCC, about 77% of the sections showed
negative or mild GLUT2 staining. In stage III HCC, about 90%
of the sections showed negative or mild GLUT2 staining (Fig. 4b). The result of one-way ANOVA of GLUT2 protein expres-
sion and clinical stage revealed that GLUT2 protein expression
negatively correlates to liver malignancy (P < 0.001) (Fig. 4b). GLUT1 Protein Expression Positively Correlates
to Liver Malignancy Since it has been reported that GLUT1 expression might in-
crease to contribute to increased glycolysis in some cancers Expressions of Carbohydrate Response Element Binding Protein and Glucose Transporters in Liver Cancer and... 1335 Expressions of Carbohydrate Response Element Binding Protein and Glucose Transporters in Liver Cancer and... Table 2 Expression of ChREBP,
GLUT2 and GLUT1 in different
clinical stages
ChREBP expression in different clinical
stages
Negative
(0)
Mild
(1)
Middle
(2)
Strong
(3)
Very strong
(4)
Normal
36%
36%
28%
0%
0%
Adjacent
46%
39%
15%
0%
0%
I
50%
25%
25%
0%
0%
II
31%
23%
23%
8%
15%
III
30%
26%
17%
9%
18%
GLUT2 expression in different clinical stages
Normal
7%
14%
36%
21%
22%
Adjacent
15%
15%
39%
31%
0%
I
100%
0%
0%
0%
0%
II
54%
23%
15%
8%
0%
III
61%
26%
9%
4%
0%
GLUT1 expression in different clinical stages
Normal
64%
36%
0%
0%
0%
Adjacent
69%
23%
8%
0%
0%
I
25%
50%
25%
0%
0%
II
8%
31%
23%
38%
0%
III
9%
26%
35%
26%
4%
Expressions of Carbohydrate Response Element Binding Protein and Glucose Transporters in Liver Cancer and... 1335 Table 2 Expression of ChREBP,
GLUT2 and GLUT1 in different
clinical stages ChREBP expression in different clinical
stages
Negative
(0)
Mild
(1)
Middle
(2)
Strong
(3)
Very strong
(4)
Normal
36%
36%
28%
0%
0%
Adjacent
46%
39%
15%
0%
0%
I
50%
25%
25%
0%
0%
II
31%
23%
23%
8%
15%
III
30%
26%
17%
9%
18%
GLUT2 expression in different clinical stages
Normal
7%
14%
36%
21%
22%
Adjacent
15%
15%
39%
31%
0%
I
100%
0%
0%
0%
0%
II
54%
23%
15%
8%
0%
III
61%
26%
9%
4%
0%
GLUT1 expression in different clinical stages
Normal
64%
36%
0%
0%
0%
Adjacent
69%
23%
8%
0%
0%
I
25%
50%
25%
0%
0%
II
8%
31%
23%
38%
0%
III
9%
26%
35%
26%
4% Fig. 2 GLUT2 immunohistochemistry in HCC and normal liver
tissue. A The GLUT2 staining was negative in liver cancer (a) but
positive on the membrane of the adjacent normal hepatocytes (b). B
GLUT2 staining in liver cancer tissues with different malign
progression. a, normal liver tissue; b, stage I liver cancer; c, stage II li
cancer; d, stage III liver cancer. GLUT1 Protein Expression Positively Correlates
to Liver Malignancy Scale bar = 30 μm GLUT2 staining in liver cancer tissues with different malignant
progression. a, normal liver tissue; b, stage I liver cancer; c, stage II liver
cancer; d, stage III liver cancer. Scale bar = 30 μm GLUT2 staining in liver cancer tissues with different malignant
progression. a, normal liver tissue; b, stage I liver cancer; c, stage II liver
cancer; d, stage III liver cancer. Scale bar = 30 μm Fig. 2 GLUT2 immunohistochemistry in HCC and normal liver
tissue. A The GLUT2 staining was negative in liver cancer (a) but
positive on the membrane of the adjacent normal hepatocytes (b). B g. 2 GLUT2 immunohistochemistry in HCC and normal liver
sue. A The GLUT2 staining was negative in liver cancer (a) but
sitive on the membrane of the adjacent normal hepatocytes (b) B 2 GLUT2 immunohistochemistry in HCC and normal liver GLUT2 staining in liver cancer tissues with different m GLUT2 staining in liver cancer tissues with different malignant
progression. a, normal liver tissue; b, stage I liver cancer; c, stage II liver
cancer; d, stage III liver cancer. Scale bar = 30 μm Fig. 2 GLUT2 immunohistochemistry in HCC and normal liver
tissue. A The GLUT2 staining was negative in liver cancer (a) but
positive on the membrane of the adjacent normal hepatocytes (b). B Y. Lei et al. 1336 Fig. 3 GLUT1 immunohistochemistry in HCC and normal liver
tissue. A The GLUT1 staining was positive on the membrane and in
the cytoplasm in malignant hepatocytes (a) but negative in the adjacent
normal hepatocytes (b). B GLUT1 staining in liver cancer tissues with
different malignant progression. a, normal liver tissue; b, stage I liver
cancer; c, stage II liver cancer; d, stage III liver cancer. Scale bar = 30 μm normal hepatocytes (b). B GLUT1 staining in liver cancer tissues with
different malignant progression. a, normal liver tissue; b, stage I liver
cancer; c, stage II liver cancer; d, stage III liver cancer. Scale bar = 30 μm normal hepatocytes (b). B GLUT1 staining in liver cancer tissues with
different malignant progression. a, normal liver tissue; b, stage I liver
cancer; c, stage II liver cancer; d, stage III liver cancer. Scale bar = 30 μm Fig. 3 GLUT1 immunohistochemistry in HCC and normal liver
tissue. GLUT1 Protein Expression Positively Correlates
to Liver Malignancy A The GLUT1 staining was positive on the membrane and in
the cytoplasm in malignant hepatocytes (a) but negative in the adjacent [36], we examined GLUT1 expressions on the liver tissues. We found that GLUT1 expression in HCC was significantly
higher than that of the adjacent normal tissue (Fig. 3a). The
non-hepatocyte positive cells were red blood cells because of
the lacking of nuclei (Fig. S2). Besides, GLUT1 expression
was increased with malignant progression (Fig. 3b and Fig. 4c). In normal hepatocytes, no GLUT1 positive signal
could be detected (Fig. 3B, a). In stage I liver cancer
tissues, only weak positive signals were detected (Fig. 3B, b). In stage II, most of the samples showed mild
positivity (Fig. 3B, c), and in stage III, most hepatocytes
showed strong positive signals on cell membrane and in [36], we examined GLUT1 expressions on the liver tissues. We found that GLUT1 expression in HCC was significantly
higher than that of the adjacent normal tissue (Fig. 3a). The
non-hepatocyte positive cells were red blood cells because of
the lacking of nuclei (Fig. S2). Besides, GLUT1 expression
was increased with malignant progression (Fig. 3b and Fig. Fig. 4 Histograms of protein expression in different liver tissues. Histograms of scores of ChREBP (a), GLUT2 (b), GLUT1(c)
expression levels in different stages of liver cancer. 0, absent of positive
signal; 1, mild staining; 2, middle staining; 3, strong staining; 4, very Fig. 4 Histograms of protein expression in different liver tissues. Histograms of scores of ChREBP (a), GLUT2 (b), GLUT1(c)
expression levels in different stages of liver cancer. 0, absent of positive
signal; 1, mild staining; 2, middle staining; 3, strong staining; 4, very
strong staining. One-way ANOVA was used to analyze the correlation
between protein expression and liver malignancy. Student-T test or
Mann-Whitney U test was used to analyze the protein expression differ-
ences between 2 different clinical stages. **, P < 0.01; ***, P < 0.001 Fig. 4 Histograms of protein expression in different liver tissues. Histograms of scores of ChREBP (a), GLUT2 (b), GLUT1(c)
expression levels in different stages of liver cancer. 0, absent of positive
signal; 1, mild staining; 2, middle staining; 3, strong staining; 4, very strong staining. One-way ANOVA was used to analyze the correlation
between protein expression and liver malignancy. Student-T test or
Mann-Whitney U test was used to analyze the protein expression differ-
ences between 2 different clinical stages. Correlations between Different Protein Expression
Levels cytoplasm (Fig. 3B, d). Quantification of the staining in-
tensity (Table 2) showed that in normal tissues, only 36%
showed mild positivity and majority (64%) of the sections
showed no GLUT1 staining. In HCC adjacent tissues,
similar expression pattern was observed to that in the
normal tissues. In stage I HCC, 25% of the sections
showed negative staining, while mild positivity represent-
ed 50% and medium positivity 25%. No strong or very
strong positivity was observed in stage I HCC. In stage
II HCC, however, 8% of the sections showed GLUT1
negative expression, 31% showed mild positivity, 23%
showed medium positivity and 38% showed strong pos-
itivity. In stage III HCC, 9% of the sections showed
GLUT1 negative expression, 26% showed mild positiv-
ity, 35% showed medium positivity and 30% showed
strong and very strong positivity (26% strong, 4% very
strong) (Fig. 4c). The result of one-way ANOVA of
GLUT1 expression and clinical stage showed that GLUT1
protein expression was increased significantly (P < 0.001) fol-
lowing malignant progression of HCC (Fig.4c). cytoplasm (Fig. 3B, d). Quantification of the staining in-
tensity (Table 2) showed that in normal tissues, only 36%
showed mild positivity and majority (64%) of the sections
showed no GLUT1 staining. In HCC adjacent tissues,
similar expression pattern was observed to that in the
normal tissues. In stage I HCC, 25% of the sections
showed negative staining, while mild positivity represent-
ed 50% and medium positivity 25%. No strong or very
strong positivity was observed in stage I HCC. In stage
II HCC, however, 8% of the sections showed GLUT1
negative expression, 31% showed mild positivity, 23%
showed medium positivity and 38% showed strong pos-
itivity. In stage III HCC, 9% of the sections showed
GLUT1 negative expression, 26% showed mild positiv-
ity, 35% showed medium positivity and 30% showed
strong and very strong positivity (26% strong, 4% very
strong) (Fig. 4c). The result of one-way ANOVA of
GLUT1 expression and clinical stage showed that GLUT1
protein expression was increased significantly (P < 0.001) fol-
lowing malignant progression of HCC (Fig.4c). To study the relationships among expressions of ChREBP,
GLUT1 and GLUT2, the Pearson Correlation test was per-
formed between any two different proteins. Although
ChREBP had the trend to increase and GLUT2 expression
decreased with HCC malignant progression, there was no cor-
relation between these 2 proteins (r = −0.031, p = 0.796, n =
70). GLUT1 Protein Expression Positively Correlates
to Liver Malignancy **, P < 0.01; ***, P < 0.001 1337 Expressions of Carbohydrate Response Element Binding Protein and Glucose Transporters in Liver Cancer and... Correlations between Different Protein Expression
Levels However, GLUT1 expression was significantly positively
correlated with ChREBP expression, and both proteins in-
creased with HCC malignancy (r = 0.481, p < 0.0001, n =
70). GLUT1 expression was inversely associated with
GLUT2 expression (r = −0.320, p = 0.007, n = 70). Discussion It has been proposed that the glucose-sensitive transcription
factor ChREBP plays a role in cancer pathology. It has been
reported that suppression of ChREBP mRNA inhibited cell
proliferation in vitro and reduced tumor growth in vivo [29]. ChREBP protein expression was positively correlated to
breast cancer malignancy, i.e., more malignant cancers ex-
press more ChREBP protein [30]. However, in hepatocellular
carcinoma, the relationship between ChREBP expression and
cancer malignancy has not been established. In this study, we
found that ChREBP protein expression tended to increase
with cancer progression although, with the limited number
of samples, could not reach statistical significance in compas-
sion to normal liver tissue. In normal liver tissue, there was no
or weak ChREBP protein expression, but in liver cancers the
expression was abundant. In stage III HCC patients, 32% of
the samples showed strong staining of ChREBP. The in-
creased ChREBP expression may serve to support increased
glycolysis and lipogenesis in cancerous hepatocytes. Although ChREBP protein levels can be used as a novel di-
agnostic and prognostic marker in breast cancer [30], variation
in HCC appears to be too large for this purpose. Nevertheless,
ChREBP may contribute to diagnosis of cancer grading in
combination with other markers. Taken together, this is to our best knowledge the first to
report of ChREBP expression in HCC and its relationship
with glucose transporters GLUT1 and GLUT2. Analysis of
combined profile of ChREBP, GLUT1 and GLUT2 expres-
sion should be helpful for HCC diagnosis and shed light on
improvement of HCC treatment and patient survival. Most cancer cells have increased aerobic glycolysis under
both anaerobic and aerobic conditions [37]. This increased gly-
colysis, accompanied by accelerated glucose uptake, is known
as the Warburg effect, after the German biochemist Otto
Warburg who first described the phenomenon in 1920s [38]. Increased glucose uptake is contributed by transmembrane
transport mediated by specific glucose transporters [39, 40]
and increased activity of hexokinases [41, 42]. Many studies
have demonstrated that the expression of glucose transporters,
especially GLUT1, was increased in a variety of malignancies
and GLUT1 overexpression was associated with invasiveness
and poor overall survival of various malignant tumors [43–47]. GLUT2 mRNA expression was inversely associated with over-
all survival in HCC [18] and invasiveness in insulinomas [20]. Thus our data confirm that GLUT2 can be potentially used for
HCC diagnosis and prognostic prediction [18]. GLUT1 but Not GLUT2 Protein Co-Expressed
with ChREBP in HCC To further investigate ChREBP, GLUT2 and GLUT1 protein
expression patterns, stain-decolorize-stain method was Immunohistochemistry of ChREBP-negative malignant hepatocytes (b)
showing GLUT2 positive on the membrane (d) but GLUT1 negative
staining (f). Arrow indicating the same cell. Scale bar = 30 μm Fig. 5 GLUT1 but not GLUT2 protein co-expressed in ChREBP-
positive hepatocytes in HCC. Immunohistochemistry of ChREBP-
positive malignant hepatocytes (a) showing GLUT2 negative (c) but
GLUT1 positive (e) staining. Arrow heads indicating the same cell. Immunohistochemistry of ChREBP-negative malignant hepatocytes (b)
showing GLUT2 positive on the membrane (d) but GLUT1 negative
staining (f). Arrow indicating the same cell. Scale bar = 30 μm Fig. 5 GLUT1 but not GLUT2 protein co-expressed in ChREBP-
positive hepatocytes in HCC. Immunohistochemistry of ChREBP-
positive malignant hepatocytes (a) showing GLUT2 negative (c) but
GLUT1 positive (e) staining. Arrow heads indicating the same cell. Immunohistochemistry of ChREBP-negative malignant hepatocytes
showing GLUT2 positive on the membrane (d) but GLUT1 negati
staining (f). Arrow indicating the same cell. Scale bar = 30 μm Immunohistochemistry of ChREBP-negative malignant hepatocytes (b)
showing GLUT2 positive on the membrane (d) but GLUT1 negative
staining (f). Arrow indicating the same cell. Scale bar = 30 μm Fig. 5 GLUT1 but not GLUT2 protein co-expressed in ChREBP-
positive hepatocytes in HCC. Immunohistochemistry of ChREBP-
positive malignant hepatocytes (a) showing GLUT2 negative (c) but
GLUT1 positive (e) staining. Arrow heads indicating the same cell. 1338 Y. Lei et al. performed. The results showed that GLUT1 but not GLUT2
positive staining could be detected in ChREBP-positive hepa-
tocytes (Fig. 5a c and e). However strong GLUT2 but not
GLUT1 positive staining was detected in ChREBP-negative
hepatocytes (Fig. 5b, d and f). All slides showed that ChREBP
and GLUT1 but not GLUT2 co-expressed in the same cells. performed. The results showed that GLUT1 but not GLUT2
positive staining could be detected in ChREBP-positive hepa-
tocytes (Fig. 5a c and e). However strong GLUT2 but not
GLUT1 positive staining was detected in ChREBP-negative
hepatocytes (Fig. 5b, d and f). All slides showed that ChREBP
and GLUT1 but not GLUT2 co-expressed in the same cells. influences on GLUT2 expression. This notion, however, re-
quires further investigation. Discussion It has been re-
ported that GLUT2 is a target of ChREBP [22], but its expres-
sion had an opposite pattern compared to ChREBP in our study. The possible reason could be that GLUT2 is not only regulated
by ChREBP but also that other regulators could have stronger Acknowledgements We would like to thank Prof. Folkert Kuipers and
Dr. Maaike H. Oosterveer for suggestions and revision for this
manuscript. Author’s Contributions Y. L. conceived the experiments, Y.L. and Q.H. conducted the experiments, Y. L. and J.G. analysed the results. Y.L. wrote
the manuscript. J.G. revised the manuscript. All authors reviewed the
manuscript. Funding This study was funded by National Natural Science Foundation
of China (no. 81872334) and Li Kashing Foundation. GLUT1 but Not GLUT2 Protein Co-Expressed
with ChREBP in HCC It has been reported that the increased glucose transport
was due, at least in part, to increased expression of GLUT1
in cancers compared to normal tissues [36] and that GLUT2
was replaced by GLUT1 in human hepatocellular carcinoma
cell line HepG2 [48]. These results suggest that the increased
expression of GLUT1 gene may closely relate to cellular
transformation. In our study, we found that GLUT1 was sig-
nificantly increased in HCC compared to adjacent normal he-
patocytes and its expression was positively associated with
HCC progression. Therefore, GLUT1 might serve as a diag-
nostic marker for HCC. Moreover, we found that ChREBP-
expressing hepatocytes do not express GLUT2 protein but
express GLUT1, and that ChREBP-negative hepatocytes ex-
press GLUT2 but not GLUT1. This indicates that ChREBP
and GLUT1 have a similar expression profile. Upon statistical
analysis, we found that ChREBP and GLUT1 protein levels
were significantly positively correlated to each other and
GLUT1 and GLUT2 levels were significantly reversely asso-
ciated. Different glucose transporters have different binding
affinities for glucose. GLUT1/HKIV has high affinity for glu-
cose [46] but GLUT2/GCK has relatively low affinity for
glucose, mannose and galactose [47]. Therefore, a transition
from GLUT2/GCK to GLUT1/HKIV mediated glucose me-
tabolism could contribute to cancer differentiation because of
losing sensitivity of the liver to circulating glucose levels. Compliance with Ethical Standards Competing Interests
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Pronuclear formation by ICSI using chemically activated ovine oocytes and zona pellucida bound sperm
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Journal of Animal Science and Biotechnology/Journal of animal science and biotechnology
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* Correspondence: bet@xanum.uam.mx
2División de Ciencias Biológicas y de la Salud, Departamento de Ciencias de
la Salud, Universidad Autónoma Metropolitana-Iztapalapa, San Rafael Atlixco
186, CP 09340 Ciudad de México, Mexico
Full list of author information is available at the end of the article RESEARCH Open Access © The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Pronuclear formation by ICSI using
chemically activated ovine oocytes
and zona pellucida bound sperm J. E. Hernández-Pichardo1,6, Y. Ducolomb2, S. Romo3, M. E. Kjelland4, R. Fierro2, F. Casillas5 and M. Betancourt2* Hernández-Pichardo et al. Journal of Animal Science and Biotechnology (2016) 7:65
DOI 10.1186/s40104-016-0124-6 Hernández-Pichardo et al. Journal of Animal Science and Biotechnology (2016) 7:65
DOI 10.1186/s40104-016-0124-6 * Correspondence: bet@xanum.uam.mx Background because mechanical activation alone, by means of sperm
injection, is sometimes not sufficient for inducing oocyte
activation [16, 17]. To improve results, appropriate sperm
treatment and oocyte activation is necessary to achieve
fertilization [17]. In recent decades, assisted reproductive techniques (ARTs)
in mammals, have been developed for its use in super-
ovulation, embryo transfer (ET), in vitro fertilization
(IVF), intracytoplasmic sperm injection (ICSI) and
cloning by somatic cell nuclear transfer. Despite the
great advances in the protocols of these ARTs, their ef-
ficiency still remains low [1]. Oocyte activation has not been shown to be crucial in
several species such as mice, hamsters and rabbits; how-
ever, it is essential in pigs, cattle and sheep [18]. The
chemical activation of ovine oocytes after ICSI has been
performed using: ionomycin (ION) [17, 19, 20], ION+ 6
dimethylaminopurine (6-DMAP) [4, 9, 17, 21, 22], etha-
nol (ETL) [4, 21], DTT [9] and calcium ionophore (CAI)
[16]. However, even with such chemical treatments, the
percent of sheep blastocysts produced after ICSI and
cultured in vitro was around 20 %. Moreover most in-
vestigations have evaluated ED but not fertilization,
determined by PN formation, to assure that the zygotes
are diploid. This condition is essential for the normal de-
velopment of the embryos [23]. When oocytes are artifi-
cially activated, a high probability that the resultant
blastocysts will be parthenogenic exists [17, 24, 25]. There-
fore, the objective of the present study was to determine
the efficiency of fertilization using ICSI with chemically
activated ovine oocytes with sperm selected by swim-up
(SU) or SU plus binding to ZP (SU + ZP). Among ARTs, ICSI is the current procedure for avoid-
ing polyspermy and increasing the number of produced
zygotes, as well as allowing for the study of several as-
pects of fertilization such as gamete interaction, sperm
decondensation, oocyte activation [2] and gene transfer
[3, 4]. In human, this technique has been developed with
great success [5]; however, its application in livestock is
still not well established, mainly due to the different re-
productive physiology among species of economically
important domestic animals such as cattle, pigs, horses
and sheep. In general, embryo development (ED) using
the ICSI procedure is lower than that of IVF [2, 6]. Background Although ovine oocyte fertilization by means of the in-
jection of immobilized sperm has been done before, and
normal lambs have been born [7, 8], ICSI success is
markedly restricted due to sperm head descondensation
failure and low male pronuclei (PN) formation [9]. In
turn, several studies have tried to solve this issue in
sheep using sperm pretreatments before performing
ICSI. In order to induce sperm head decondensation
and male PN formation, sperm have been pre-treated
with lysophosphatidylcholine (LPC) [10], triton X-100
[11], dithiothreitol (DTT), sodium dodecyl sulphate
(SDS) or by freezing and thawing without cryoprotec-
tants [9]. These treatments are directed at removing the
sperm membranes and dissolving the nuclear proteins
allowing the head decondensation. Abstract Journal of Animal Science and Biotechnology (2016) 7:65 Hernández-Pichardo et al. Journal of Animal Science and Biotechnology (2016) 7:65 Page 2 of 8 Page 2 of 8 Methods The ovaries were collected from a slaughterhouse, “El
Rojo”. The aforementioned facility has the animal health
federal law authorization under the number 150810056630. Except for the maturation and development media, which
were prepared in a commercial laboratory (In Vitro S.A.,
México), all chemicals were purchased from Sigma Chem-
ical Company (ST Louis, MO USA). All incubation condi-
tions were performed at 38.5 °C in an atmosphere with 5 %
CO2, 95 % air, and humidity at saturation (NUAIRE, USA). In vitro maturation (IVM) and ICSI procedures were per-
formed under mineral oil (Fisher Scientific, USA). All
media were stored at 4 °C for not more than three wk and
supplemented 24 to 48 h before use. In human, physiological strategies are being explored
to investigate the selection of sperm that are more
capable of achieving successful ICSI, compared to the
subjective way that the embryologists currently use
[12], increasing fertilization. A strategy for improving
ICSI is using sperm attached to the zona pellucida
(ZP) [13, 14]. Studies [12, 14, 15] have concluded that
the ZP has the ability to select functionally normal
sperm and even those with a superior quality. Further,
the attachment to the ZP during fertilization is neces-
sary for inducing the acrosome reaction (AR), which is
an important factor for ICSI success. Abstract Background: In order to improve ICSI, appropiate sperm selection and oocyte activation is necessary. The objective
of the present study was to determine the efficiency of fertilization using ICSI with chemically activated ovine
oocytes and sperm selected by swim up (SU) or swim up + zona pellucida (SU + ZP) binding. Results: Experiment 1, 4–20 replicates with total 821 in vitro matured oocytes were chemically activated with
ethanol, calcium ionophore or ionomycin, to determine oocyte activation (precense of one PN). Treatments showed
similar results (54, 47, 42 %, respectively) but statistically differents (P < 0.05) than mechanical activated oocytes in
sham, ICSI and sham injection (13, 25, 32 %, respectively) (10–17 replicates; n = 429). j
p
y
p
Experiment 2: Twelve ejaculates and 28 straws of semen were used (11–19 replicates). Sperm were selected by SU
in BSA-TCM 199-H medium. A total of 2,294 fresh sperm and 2,760 from frozen-thawed semen were analyzed after
SU or SU + ZP binding. Fresh sperm selected by SU showed acrosome reaction (AR) of 59 %, the sperm selected by
SU + ZP binding increased AR to 91 %. In comparison, the AR of frozen-thawed sperm using SU or SU + ZP binding
was 77 and 86 %, respectively (P < 0.05). y
Experiment 3: fertilization in 200 mechanical activativated oocytes (17 replicates) was 4 %, but fertilization increased
in ethanol activated oocytes after ICSI (12-28 %) (5–6 replicates). When fresh sperm only selected by SU were injected
to 123 oocytes, a fertilization rate (28 %) was achieved; in sperm selected by SU + ZP was 25 % (73 oocytes). In
comparison, in frozen-thawed sperm selected by SU, fertilization was 13 % (70 oocytes), whereas sperm from
SU + ZP binding displayed 12 % (51 oocytes) (P > 0.05). Conclusions: Chemical activation induces higher ovine oocyte activation than mechanical activation. Ethanol
slightly displays higher oocyte activation than calcium ionophore and ionomicine. Sperm selection with SU + ZP
increased AR/A and AR/D rates in comparison with SU in fresh and frozen-thawed sperm. According to this, in
terms of fertilization rates, chemical activation after ICSI increased oocyte PN formation compared to mechanical
activation. Also, fresh sperm treated with SU and SU + ZP were significantly different than frozen-thawed sperm,
but between sperm treatments no significant differences were obtained. Keywords: Ethanol, ICSI, Oocyte activation, Ovine, Pronucleus, Zona pellucida Hernández-Pichardo et al. Oocyte activation For experiment 1, mature denuded oocytes (only with a
PB) were distributed randomly among the following
groups to perform activation; mechanical activation: (1)
Sham: oocytes manipulated as in ICSI without injection,
(2) ICSI, (3) Sham injection: oocyte pierced without
sperm insertion, and for chemical activation without
sperm injection: (4) 7 % ETL for 5 min, (5) 50 μmol/L
CAI for 10 min, and (6) 5 μmol/L ION for 5 min. After Experimental design The experimental design consisted of three separate experi-
ments. Experiment 1 was conducted to identify the percent
activation of oocytes matured in vitro only exposed to
chemical activation with ETL (n = 393), CAI (n = 350), ION
(n = 78) or mechanical activation through ICSI (n = 200),
sham injection (false injection) (n = 78) or sham (only ma-
nipulation of the gametes without injection) (n = 151), and
evaluated by the PN formation. Other important factor is the oocyte activation which
is the resumption of meiosis II during fertilization in-
creasing calcium ion levels in the cytoplasm. Without
this step, the inseminated oocyte prevents the deconden-
sation of the sperm head. Notably, artificial stimuli can
mimic the action of sperm during fertilization. Chemical
activation of oocytes has been necessary with ICSI Experiment 2 was conducted to determine the viability
and sperm acrosome status after selection by SU or SU +
ZP of fresh (n = 1,194, SU; n = 1,100 SU + ZP) and frozen-
thawed sperm (n = 718, SU; n = 922 SU + ZP). Page 3 of 8 Page 3 of 8 Hernández-Pichardo et al. Journal of Animal Science and Biotechnology (2016) 7:65 Experiment 3 was conducted to determine fertilization
evaluated by the formation of two PN and without the
presence of a sperm head when performing ICSI with
fresh or frozen-thawed sperm selected by SU or SU + ZP. After ICSI, oocyte chemical activation with 7 % ETL for
5 min (n = 123 SU; n = 73 SU + ZP and n = 70 SU; n = 51
SU + ZP respectively) and mechanical activation (n = 200)
were performed. activation, oocytes were washed three times in modified
Tris-buffered Medium (mTBM) consisting of 13.1 mmol/L
NaCl, 3 mmol/L KCl, 7.5 mmol/L CaCl2 · 2H2O, 20 mmol/
L Tris, 11 mmol/L glucose, 5 mmol/L sodium pyruvate,
supplemented with 0.4 % bovine serum albumin (BSA)
fraction V, and 2.5 mmol/L caffeine benzoate and placed in
200 mL of medium and incubated during 18 h. Exposure of sperm to mature oocytes ZP y
In each assay, four oocytes in metaphase II were placed
in 400 μL drops of BSA-TCM-H and 1 % gentamycin,
followed by the addition of 100 μL of the sperm suspen-
sion and incubated for 1 h. The oocytes were transferred
to a 300 μL drop of TCM-H medium and washed by re-
peated aspirations with a fine glass pipette to eliminate
the non- bound ZP sperm. Two oocytes with sperm
tightly bound to the ZP were placed in 20 μL drops of
the same medium and covered with mineral oil. The
sperm bound to the ZP were removed by repeated aspir-
ation of the oocytes through a fine glass pipette, for ICSI
[13, 29], while the other two oocytes were placed in
100 μL drops to obtain the sperm for viability and AR
analysis. Oocyte collection and maturation After incubation, oocytes were stained with 10 μg/mL
bisbenzimide (Hoechst 33258) in cold ETL for 15 min,
protected from the light. Further, oocytes were placed
on a slide in a microdrop of PBS-glycerol (1:9) [9], and
examined under an epifluorescence microscope at 400×
(Eclipse E600, Nikon, Japan). Ovaries were collected from adult ewes and transported,
less than three h, to the laboratory in 0.157 mol/L NaCl
solution at 37 °C with antibiotics (10,000 UI/mL ampy-
cillin,
10,000
μg/mL
streptomycin
and
25
μg/mL,
amphotericyn (In Vitro S.A., México). Ovaries were
rinsed three times in the fresh solution described above. Follicular fluid was aspirated from 2 to 6 mm follicles
punctured with an 18-gauge needle fixed to a 10 mL dis-
posable syringe containing 1 mL of modified Tyrode’s
medium supplemented with 10 mmol/L sodium lactate,
10 mmol/L HEPES and 0.1 % polyvinilic alcohol (PVA)
(TL-HEPES-PVA) with a pH of 7.3–7.4, supplemented
with 200 UI/mL heparine. Follicular fluid was pooled in a
50 mL conical tube (PISA, México). Cumulus cell-oocyte
complexes (COCs) with compact cumulus mass and
oocytes with uniform cytoplasm were selected and
washed three times in 500 μL drops of maturation
medium TCM-199 with Earle’s salts, 26.2 mmol/L sodium
bicarbonate and L glutamine (In Vitro S.A., México), sup-
plemented with 0.1 % PVA, 3.05 mmol/L D-glucose,
0.91 mmol/L sodium pyruvate, 0.57 mmol/L cysteine and
10 ng/mL epidermal growth factor [26]. Non-activated oocytes were determined by the identi-
fication of the metaphase II, whereas activated oocytes
presented a PN. Sperm preparation Semen was collected from a creole ram of proven fertil-
ity, located in the animal facilities at the Universidad
Autónoma Metropolitana-Xochimilco, as approved under
the regulations of the Committee for Care and Use of
Animals. One hundred μL of fresh or frozen semen
were added to 2 mL of TCM 199-H supplemented with
3 mg/mL BSA (BSA-TCM 199-H), and incubated for
1 h for SU to help capacitation. Afterwards, 500 μL of
medium were gently removed from the top of the sus-
pension, then 3 mL of BSA-TCM 199-H were added
and centrifuged at 200 ×g for 10 min [9, 27]. The pellet
was suspended to give a final concentration of 1×106
sperm/mL [28]. For oocyte maturation, sterile four well plates were used
(Thermo-Scientific Nunc, Rochester NY). First, 500 μL of
maturation medium with 0.5 μg/mL LH, 0.5 μg/mL FSH
and 10 % (v/v) fetal calf serum (FBS) (In Vitro S.A.,
México) was added to each well. Next, 20–30 COCs were
placed in the well with the maturation medium and incu-
bated for 24 h [19]. After IVM, COCs were placed in
500 μL of TCM 199-HEPES (TCM 199-H) with 300 IU of
hyaluronidase for three min, then washed three times in
TCM 199-H [13]. Maturation was determined by identifi-
cation of the first polar body (PB) using an inverted micro-
scope (Nikon Eclipse TE200, Japan). Nuclear staining Eighteen h after ICSI, oocytes were stained with 10 μg/mL
Hoechst 33258 as mentioned, to determine the following
stages: 1) non-activated: oocytes in metaphase II and
without a decondensed sperm head; 2) activated oo-
cytes: formation of the female PN and without a decon-
densed sperm head; and 3) fertilized oocytes: oocytes
with two PN and without the presence of the sperm
head [9, 19, 33]. Intracytoplasmic sperm injection Intracytoplasmic sperm injection
For ICSI, a 50 μL drop of TCM 199-H, supplemented
with 2 % FBS and 1 % gentamycin containing five in
vitro matured denuded oocytes, and one 10 μL drop of
sperm suspension with 10 % polivinilpirrolidine (PVP)
(1:1 dilution) were added to a 6 cm diameter Petri dish
[8, 16]. Microinjection was performed as previously re-
ported by Shirazi et al. [9], using an inverted microscope
at 200× (Nikon Eclipse TE200, Japan). The microinjec-
ton pipette was immersed in the sperm suspension-PVP
and one sperm was immobilized by scoring the midpiece
and aspirating the tail-first, and then injected into the
ooplasma through the ZP. The first PB was either in the 6
or 12 o’clock position, and the injection pipette was in the
3 o’clock position. During the injection, cytoplasm was as-
pirated to ensure that the oolema was punctured. After
ICSI, oocytes were washed three times in BSA-TCM 199-
H. Mechanical and chemical activated oocytes were incu-
bated in 100 μL of mTBM for 18 h. A total of 517 oocytes
were injected (5–17 replicates). Viability and acrosomal status of sperm after SU and ZP
binding Fresh and frozen-thawed sperm were selected by SU or
SU + ZP binding. To determine their viability and acrosome Hernández-Pichardo et al. Journal of Animal Science and Biotechnology (2016) 7:65 Page 4 of 8 Page 4 of 8 alcohol 9000-02) in TCM 199-H at room temperature
for 5 min [32]. After activation, oocytes were washed
and incubated in mTBM during 18 h. status, propidium iodide (PI) and fluorescein isothiocyanate
with peanut agglutinin (FITC-PNA) double staining was
developed [30, 31]. From each sperm suspension sample,
100 μL were obtained. Next, 5 μL of FITC-PNA and 5 μL
of PI were added, followed by incubation for 5 min. Ten μL
of the sperm suspension were mixed with 10 μL 1.6 % para-
formaldehyde in a slide and analyzed under a fluorescence
microscope (Eclipse E600, Nikon, Japan) at 400×. The func-
tional status of the sperm was classified according to the
following staining patterns: live sperm with intact acro-
some: cells with FITC-PNA stain at the acrosome, without
PI stain at the post-acrosomal region. Live sperm with AR:
cells without FITC-PNA acrosome stain and without PI nu-
clear stain. Dead sperm with intact acrosome: cells with
FITC-PNA stain in the acrosome region, and with PI nu-
clear stain. Dead sperm with AR: without FITC-PNA stain
in the acrosome region, and with PI nuclear stain. Oocyte activation The mechanical activation of oocytes with sham injec-
tion was higher than sham (P < 0.05). However, no differ-
ences were observed between sham injection and ICSI. Chemical activation with ETL, CAI and ION was higher
than the mechanical activation (P < 0.05). When compared
to chemical activation with ETL, the three chemical acti-
vation treatments had a similar proportion of PN with a
slightly increase in the ETL group, shown in Table 1. Based on these results, oocytes subjected to ICSI were
hereafter treated only with 7 % ETL for 5 min. Statistical analysis With regard to the oocyte activation, 4–20 replicates
were performed. For sperm viability and acrosome status,
11–19 replicates were performed for fresh and frozen-
thawed semen. To assess fertilization 5–6 replicates were
performed for each group. ANOVA and Bonferroni mul-
tiple post hoc test were used for comparing the results of
the following variables: oocyte activation, viability and ac-
rosome status of fresh sperm compared to frozen-thawed
sperm, and fertilization with mechanical and chemical
activation, but also between sperm treatments [34],
considering significant differences between the groups
when P < 0.05. a,b,cValues with different letters indicate statistically significant differences (P < 0.05) ICSI The oocytes treated with ETL had an increased fertilization
rate with ICSI (presence of two PN without the sperm
head) compared to mechanical activation. Fertilization
was increased when fresh semen was used compared to
frozen-thawed semen, independent of the sperm selec-
tion procedure (P < 0.05). The comparison of the four
groups of oocytes exposed to chemical activation with
ETL were significant different than mechanical activa-
tion (P < 0.05), shown in Table 3. Also, a significant dif-
ference in fertilization was observed between the fresh
and frozen-thawed sperm but between selection (SU
and SU + ZP) no significant differences were obtained
(Table 3). The percentage of activated oocytes was greater
than percentage of fertilized oocytes, indicating that the
proportion of sperm decondensation is still lower when
the oocytes are activated. Also, in the present study, the use of CAI resulted in
an oocyte activation of 47 %. However, little is known
about sheep CAI oocyte activation. Nevertheless, studies
in cattle reported 22 % [39]. Oocyte activation using
ION resulted in 42 % PN formation, which is similar to
that reported by Shirazi et al. [36], who evaluated activa-
tion through ED. These results support the use of these
chemical activators with sheep oocytes, which are essen-
tial in ICSI. Oocyte chemical activation after ICSI For experiment 3, 10–20 ICSI inseminated oocytes were
activated in 100 μL drops of 7 % ETL (Baker Absolute Table 1 Activation procedures in sheep oocytes matured in vitro
Mechanical activation
Chemical activation
Sham
ICSI
Sham Injection
ETL
CAI
ION
N
13
17
10
20
19
4
n
151
200
78
393
350
78
Activated oocytes,%
2,0
13 ± 1.6a
4,9
25 ± 0.5ab
25
32 ± 3.4b
21,1
54 ± 1.3c
16,4
47 ± 1.3c
3,3
42 ± 5.0c
Sham: oocytes manipulated as in ICSI but no injection; Sham injection: oocyte pierced but no sperm insertion; ETL: 7 % Ethanol 5 min; CAI: 50 μmol/L Calcium
Ionophore 10 min; ION: 5 μmol/L Ionomicine 5 min. Mechanically activated oocytes: Oocytes in metaphase II. Activated oocytes: Presence of one pronucleus
N number of replicates; n number of oocytes examined
Percentage data are presented as mean ± SD
a,b,cValues with different letters indicate statistically significant differences (P < 0.05) Table 1 Activation procedures in sheep oocytes matured in vitro
Mechanical activation Table 1 Activation procedures in sheep oocytes matured in vitro Sham: oocytes manipulated as in ICSI but no injection; Sham injection: oocyte pierced but no sperm insertion; ETL: 7 % Ethanol 5 min; CAI: 50 μmol/L Calcium
Ionophore 10 min; ION: 5 μmol/L Ionomicine 5 min. Mechanically activated oocytes: Oocytes in metaphase II. Activated oocytes: Presence of one pronucleus
N number of replicates; n number of oocytes examined
Percentage data are presented as mean ± SD
a,b,cValues with different letters indicate statistically significant differences (P < 0.05) Page 5 of 8 Page 5 of 8 Hernández-Pichardo et al. Journal of Animal Science and Biotechnology (2016) 7:65 Hernández-Pichardo et al. Journal of Animal Science and Biotechnology (2016) 7:65 Viability and acrosome reaction Other chemical activators include CAI and ION, which
induce the liberation of intracellular calcium stored in
the endoplasmic reticulum, evident by the activation of
several proteolytic pathways calcium dependent that
drive the destruction of cyclin B, reducing the activity of
MPF [25, 36, 38]. Independently of whether the sperm were alive or dead,
the proportion with AR was greater in those selected
with SU + ZP than SU only (control) (P < 0.05), either in
fresh or frozen-thawed semen. These results indicate
that if ICSI is performed using sperm selection through
ZP binding, the probability of injecting sperm with AR is
very high, independently of the viability and fresh or
frozen-thawed condition, shown in Table 2. In regard to experiment 1 the present study demon-
strated that chemical activation induced a higher female
PN formation than that produced by mechanical activa-
tion. The results obtained with ETL oocyte activation
were similar to those reported by Shirazi et al. (50 %)
[36] who studied the effect of sheep oocyte maturation
on parthenogenetic development. In other mammalian
domestic species, oocyte activation with ETL generally
leads to a lower incidence of pronuclear formation. Des-
pite this, Wang et al. [39] activated cow oocytes and re-
ported 42 % activation. Additionally, Yi and Park [40]
observed an increase in the activation of pig oocytes
with ETL exposure (34 %). thin rows, among the variables corresponding to fresh and frozen-thawed semen counterparts, significant difference was observed (P g
p
alues in the same column with different letters are significantly different (P < 0.05) nted as mean ± SD
mn with different letters are significantly different (P < 0.05) g
p
a,bValues in the same column with different letters are significantly different (P < 0.05) Discussion Journal of Animal Science and Biotechnology (2016) 7:65 Hernández-Pichardo et al. Journal of Animal Science and Biotechnology (2016) 7:65
Page 6 of 8 Page 6 of 8 Table 3 Effect of oocyte activation, semen type and sperm selection technique for ICSI on fertilization percentage
Semen type
Frozen-thawed semen
Fresh
Frozen-thawed
Oocyte treatment
Mechanical activation
Activated with 7 % Ethanol
Sperm Treatment
“Swim-up”
“Swim-up”
“Swim-up” + Zona pellucida
“Swim-up”
“Swim-up” + Zona pellucida
N
17
5
6
5
5
n
200
123
73
70
51
Activated ooctyes, %
4,9
25 ± 2.7
7,5
61 ± 1.3*
3,6
49 ± 1.6*
4,0
57 ± 3.2*
3,1
61 ± 6.3*
Fertilized ooyctes, %
8
4 ± 0.2
3,5
28 ± 1.7*
18
25 ± 1.7*
9
13 ± 1.2*▲
6
12 ± 0.9▲
Activated oocytes: Formation of female pronucleus and no decondensed sperm head; Fertilized oocytes: Oocytes with two pronuclei without sperm head
N number of replicates, n Number of oocytes examined
Percentage data are presented as mean ± SD
*Indicates significant differences with respect to mechanical activated oocytes (P < 0.05)
▲Indicates significant differences with respect to SU fresh (P < 0.05) Activated oocytes: Formation of female pronucleus and no decondensed sperm head; Fertilized oocytes: Oocytes with two pronuclei without sperm head
N number of replicates, n Number of oocytes examined
Percentage data are presented as mean ± SD
*Indicates significant differences with respect to mechanical activated oocytes (P < 0.05)
▲Indicates significant differences with respect to SU fresh (P < 0.05) by Shirazi et al. [17], besides, they reported that mechanical
stimulation (ICSI or sham injection) is not sufficient to ac-
tivate ovine oocytes for PN formation, indicating that
chemical activation is essential for ICSI in sheep. during the removal of cumulus cells, since this enzyme
is capable of producing oocyte activation in mice, cattle
and human itself [32]. With regard to the activation
using sham injection or ICSI, our results indicate that
the injection by itself induces sheep oocyte activation
and not necessarily the presence of a sperm. Oocyte treatment with ETL after ICSI increased the
oocyte activation percentage from 49 to 61 %, and 12 to
28 % for fertilization, compared to mechanical activated
(25 and 4 %, respectively). Discussion The fertilization rate with
fresh sperm was only 50 % with respect to the propor-
tion of the activated oocytes; while that in frozen-thawed
sperm was not higher than 25 %. If the evaluation of
these oocytes was through the ED, more than 50 %
could be parthenotes. Similar results have been reported
in cattle oocytes treated with ETL after ICSI with
frozen-thawed sperm heads, with 52 % of activation, but
only 27 % of fertilization, being 56 % parthenote em-
bryos [46]. In equine oocytes with the same treatment,
56 % activation, 16 % fertilization, and 40 % parthenotes
have been obtained; indicating that even when oocytes
are activated, the sperm decondensation may not occur,
but artificial activation can induce parthenogenetic de-
velopment in oocytes with ICSI [47]. Several factors influence oocyte sensitivity to artificial
activation and this can explain the differences between
the results obtained in the present study and those re-
ported previously. Factors influencing oocyte activation
include: differences among species [37], in vivo or in vitro
maturation, presence or absence of cumulus cells [32, 36]
and type of stimuli [32]. For improving ICSI results, new methods of sperm se-
lection are being developed in human, as injecting sperm
previously bound to the ZP of oocytes. To our knowledge,
this is the first study that evaluates the acrosome status
and viability of sperm from fresh and frozen-thawed ovine
sperm after being in contact with the in vitro matured
oocyte ZP of the same species. It is important to con-
sider that the use of ZP does not just select sperm, but
also induces AR, which has been reported to be essen-
tial for decondensation of sperm chomatin in ooplasm
[9]. In experiment 2 the percentage of sperm with AR
after the ZP binding was 91 and 86 % for fresh and
frozen-thawed sperm, respectively, which are similar to
those reported with fresh goats semen, [42] pigs [43]
and human [44]. Notably, a significant difference was
observed between the SU + ZP and SU selection proce-
dures. Discussion In the present study, mechanical activation by ICSI
displayed 25 % PN formation, similar to that reported by
Gómez et al. [8, 33] with 31 % (determined through ED)
and 38 % (resumption of second meiotic division), while
Shirazi et al. [17] observed a PN formation of 5 % only. The effect of the sham injection resulted in 32 % of the
oocytes undergoing PN formation. The results of this
study are superior to those reported by Gómez et al. [10], with 22 %. The chemical activation of the sheep oocytes during
ICSI is necessary due to the low percentages of sperm
decondensation and PN formation. Oocyte activation
implies an increase of intracellular free calcium, causing
the degradation of the maturation promoting factor
(MPF), which is essential for the resumption of the second
meiotic division and the formation of both PN [25, 35]. ETL is among the most utilized chemical activators
through the formation of inositol 1,4,5-triphosphate and
its union to the membrane receptors in the endoplasmic
reticulum releasing of intracellular calcium [25, 36, 37]. The sham group showed that 13 % of the oocytes were
activated, which is similar to those by Catt and Rhodes
[41]. This could be due to the use of hyluronidase Table 2 Viability and acrosome status of sperm selected by swim-up or swim-up and oocyte ZP binding Table 2 Viability and acrosome status of sperm selected by swim-up or swim-up and oocyte ZP binding
Fresh Semen, %
Frozen-thawed semen, %
AR/A
AR/D
NAR/A
NAR/D
AR/A
AR/D
NAR/A
NAR/D
SU
N = 12
N = 14
n = 1,194
52,8
44 ± 1.8a
17,9
15 ± 0.8a
34,0
28 ± 8.3a
14,7
12 ± 0.6a
n = 1410
71,8
51 ± 0.9a*
37,3
26 ± 0.9a*
17,5
12 ± 0.3a*
14,4
10 ± 0.5a
SU + ZP
N = 11
N = 19
n = 1,100
64,8
59 ± 0.9b
34,7
32 ± 1.1b
40
4 ± 0.3b
65
6 ± 0.2b
n = 1350
92,2
68 ± 1.8b*
23,7
18 ± 0.9b*
14,3
11 ± 0.5a*
48
4 ± 0.2b*
SU swim-up, ZP Zona pellucida, AR/A acrosome reaction/alive, AR/D acrosome reaction/dead, NAR/A no acrosome reaction/alive, NAR/D no acrosome reaction/dead,
N number of replicates, n number of sperm examined
Percentage data are presented as mean ± SD osome status of sperm selected by swim-up or swim-up and oocyte ZP binding Hernández-Pichardo et al. Conclusions Chemical activation induces higher ovine oocyte activation
than mechanical activation. ETL slightly displays higher
oocyte activation than CAI and ION. Sperm selection with
SU + ZP increased AR/A and AR/D rates in comparison
with SU in fresh and frozen-thawed sperm. According to
this, in terms of fertilization rates, chemical activation
after ICSI increased oocyte PN formation compared to
mechanical activation. Fresh sperm selected by SU and
SU + ZP were significantly different than frozen-thawed
sperm, but between both selection procedures no differ-
ences were obtained. Therefore, this study demonstrates
that chemical activation is recquired to activate oocytes
after injection and acrosome reaction is not essential for
fertilization. In turn, fresh sperm was more efficient in
terms of fertilization capacity when performing ICSI. 4. Pereyra-Bonnet F, Gibbons A, Cueto M, Sipowicz P, Fernández-Martín R,
Salamone D. Efficiency of sperm-mediated gene transfer in the ovine by
laparoscopic insemination, in vitro fertilization and ICSI. J Reprod Dev. 2011;57:188–96. 4. Pereyra-Bonnet F, Gibbons A, Cueto M, Sipowicz P, Fernández-Martín R,
Salamone D. Efficiency of sperm-mediated gene transfer in the ovine by
laparoscopic insemination, in vitro fertilization and ICSI. J Reprod Dev. 2011;57:188–96. 5. Gerris JMR. Single embryo transfer and IVF/ICSI outcome: a balanced
appraisal. Hum Reprod Update. 2005;11:105–21. 5. Gerris JMR. Single embryo transfer and IVF/ICSI outcome: a balanced
appraisal. Hum Reprod Update. 2005;11:105–21. 6. Casillas F, Ducolomb Y, Lemus AE, Cuello C, Betancourt M. Porcine embryo
production following in vitro fertilization and intracytoplasmic sperm injection
from vitrified immature oocytes matured with a granulosa cell co −culture
system. Cryobiology. 2015;71:299–305. 6. Casillas F, Ducolomb Y, Lemus AE, Cuello C, Betancourt M. Porcine embryo
production following in vitro fertilization and intracytoplasmic sperm injection
from vitrified immature oocytes matured with a granulosa cell co −culture
system. Cryobiology. 2015;71:299–305. 7. Catt JW. Intracytoplasmic sperm injection (ICSI) and related technology. Anim Reprod Sci. 1996;42:239–50. 7. Catt JW. Intracytoplasmic sperm injection (ICSI) and related technology. Anim Reprod Sci. 1996;42:239–50. 8. Gómez MC, Catt JW, Evans G, Maxwell WMC. Cleavage, development and
competence of sheep embryos fertilized by intracytoplasmic sperm
injection and in vitro fertilization. Theriogenology. 1998;49:1143–54. 8. Gómez MC, Catt JW, Evans G, Maxwell WMC. Cleavage, development and
competence of sheep embryos fertilized by intracytoplasmic sperm
injection and in vitro fertilization. Theriogenology. 1998;49:1143–54. 9. Shirazi A, Derakhshan-Horeh M, Pilvarian AA, Ahmadi E, Nazari H, Heidari B. Funding 12. De-Yi L. Could using the zona pellucida bound sperm for intracytoplasmic
sperm injection (ICSI) enhance the outcome of ICSI? Asian J Androl. 2011;13:197–8. This study was not financially supported. References 1. Franco MM, Prickett AR, Oakey RJ. Minireview. The role of CCCTC-Binding
Factor (CTCF) in genomic imprinting, development, and reproduction. Biol
Reprod. 2014;91:1–9. 2. García-Roselló E, García-Mengual E, Coy P, Alfonso J, Silvestre MA. Intracytoplasmic sperm injection in livestock species: an update. Reprod
Domest Anim. 2009;44:143–51. 3. Bevacqua RJ, Pereyra-Bonnet F, Fernandez-Martin R, Salamone DF. High
rates of bovine blastocyst development after ICSI-mediated gene transfer
assisted by chemical activation. Theriogenology. 2010;74:922–31. Conclusions Effect of pre-treatment of ovine sperm on male pronuclear formation and
subsequent embryo development following intracytoplasmic sperm
injection. Reprod Domest Anim. 2011;46:87–94. 10. Gómez MC, Catt JW, Evans G, Maxwell WMC. Effect of culture, incubation
and acrosome reaction of fresh and frozen-thawed ram spermatozoa for in
vitro fertilization and intracytoplasmic sperm injection. Reprod Fertil Dev. 1997;9:665–73. Availability of data and materials Availability of data and materials 13. Black M, de Liu Y, Bourne H, Baker HW. Comparison of outcomes of
conventional intracytoplasmic sperm injection and intracytoplasmic sperm
injection using sperm bound to the zona pellucida of immature oocytes. Fertil Steril. 2010;93:672–4. 13. Black M, de Liu Y, Bourne H, Baker HW. Comparison of outcomes of
conventional intracytoplasmic sperm injection and intracytoplasmic sperm
injection using sperm bound to the zona pellucida of immature oocytes. Fertil Steril. 2010;93:672–4. The authors declare that they do not apply new software and databases. Discussion Metropolitana-Iztapalapa, San Rafael Atlixco 186, CP 09340 Ciudad de México,
Mexico. 3Departamento de Ciencias Pecuarias, Facultad de Estudios
Superiores Cuautitlán, UNAM, Ciudad de México, Estado de México, Mexico. 4Conservation, Genetics & Biotech, LLC, Valley City, ND, USA. 5División de
Ciencias Biológicas y de la Salud, Departamento de Biología de la
Reproducción, Universidad Autónoma Metropolitana-Iztapalapa, Ciudad de
México, Mexico. 6Doctorado en Biología Experimental, Universidad
Autónoma Metropolitana-Iztapalapa, Ciudad de México, Mexico. Received: 12 May 2016 Accepted: 14 October 2016 Received: 12 May 2016 Accepted: 14 October 2016 Consent for publication
Not applicable. 17. Shirazi A, Ostad-Hosseini S, Ahmadi E, Heidari B, Shams-Esfandabadi N. In
vitro developmental competence of ICSI-derived activated ovine embryos. Theriogenology. 2009;71:342–8. Authors’ contributions Direction of the study: MB, YD, RF and SR. Established the experimental
design: JEHP and MB. Managed the animals and performed the experiments:
JEHP. Analyzed the results and wrote the manuscript: JEHP, MB, MK, YD, SR
and FC. All authors verify reading and approving the final manuscript. 14. Henkel R. Sperm preparation: state-of-the art-physiological aspect and
application of advanced sperm preparation methods. Asian J Androl. 2012;14:260–9. 14. Henkel R. Sperm preparation: state-of-the art-physiological aspect and
application of advanced sperm preparation methods. Asian J Androl. 2012;14:260–9. 15. Parmegiani L, Cognigni GE, Filicori M. New Advances in Intracytoplasmic
Sperm Injection (ICSI), Advances in Embryo Transfer. In: Bin Wu, editor . InTech. [On line]. 2015. http://cdn.intechopen.com/pdfs-wm/32057.pdf. Acknowledgments The authors thank the slaughterhouse “El Rojo”, Estado de México for the
donation of ovaries. This publication represents partial fulfillment of the
requirements for the degree of Doctor in Biología Experimental for José
Ernesto Hernández-Pichardo. 11. Pereyra-Bonnet F, Gibbons A, Cueto M, Naim P, Fernández-Martín F,
Salamone D. Transgénesis mediada por espermatozoides en ovejas
prepuberes. VII Simp Int Reprod Anim. IRAC. 2007;213. Funding
This study was not financially supported. Competing interests The authors declare that they have no competing interests. 16. Gómez MC, Catt JW, Evans G, Maxwell WMC. Sheep oocyte activation after
intracytoplasmic sperm injection (ICSI). Reprod Fertil Dev. 1998;10:197–205. Discussion Among the factors that affect the trigger of the AR
through ZP contact, is the state of oocyte maturation; im-
mature porcine oocytes in contact with fresh semen for
10 min had an AR of 34 %, while mature oocytes 42 %
underwent AR, similar tendency was observed when
frozen-thawed sperm was used with immature (37 %) and
mature oocytes (45 %) [45]. In the present study, an increase in fertilization was
not observed using sperm previously bound to oocyte
ZP and with an AR higher than 85 %. This finding is in
agreement with that reported by Gómez et al. [10], in
which an increase in fertilization was not observed
(22 %) after ICSI with AR sperm (86 %) compared to
that obtained with untreated sperm (33 %). These results
indicate that there is not a correlation between the per-
centage of sperm with AR and the number of oocytes
fertilized, suggesting that it is not necessary to induce AR
before ICSI in sheep to obtain fertilization. In human, Black
et al. [13] did not observe a significant difference in
fertilization between ICSI performed with sperm bound to
the ZP and conventional ICSI; however, higher quality em-
bryos and a greater number of pregnancies were produced. In experiment 3, a significant increase in the percentage
of activated and fertilized oocytes was observed when stim-
ulated with ETL after ICSI, similar results were obtained A significant difference in the fertilization rate was ob-
served between oocytes fertilized with frozen-thawed Hernández-Pichardo et al. Journal of Animal Science and Biotechnology (2016) 7:65 Hernández-Pichardo et al. Journal of Animal Science and Biotechnology (2016) 7:65 Page 7 of 8 Page 7 of 8 Page 7 of 8 and fresh sperm. This is consistent with Gómez et al. [10], reporting higher fertilization rates after ICSI with
fresh sperm than frozen-thawed sperm. Also Gómez et
al. [10] results could be due to acrosome-membrane
damage which may occur during the freeze-thaw process;
thereby predisposing sperm to further membrane changes
or some other deleterious effect on sperm DNA. In
addition, in human sperm, it has been reported that the
freeze-thaw process produces an oxidative stress due to
the generation of oxygen free radicals and an alteration of
the sperm antioxidant system [48]. The aforementioned
oxidative stress can cause the peroxidation of membrane
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activation and parthenogenetic development of bovine oocytes following
intracytoplasmic sperm injection. Zygote. 1999;7:233–7. 23. Nicoli A, Capodanno F, Rondini I, Valli B, Villani MT, Morini D, et al. Pronuclear
morphology evaluation in vitro fertilization (IVF) intracytoplasmic sperm
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for in vitro embryo production: A review. Adv Biosci Biotechnol. 2013;4:170–82. 48. Gadea J, Molla M, Selles E, Marco MA, Garcia‐Vazquez FA, Gardon JC. Reduced glutathione content in human sperm is decreased after
cryopreservation: Effect of the addition of reduced glutathione to the
freezing and thawing extenders. Cryobiology. 2011;62:40–6. 25. Kharche SD, Birade HS. Parthenogenesis and activation of mammalian oocytes
for in vitro embryo production: A review. Adv Biosci Biotechnol. 2013;4:170–82. 26. Fernández RF, Ducolomb Y, Romo S, Casas E, Salazar Z, Betancourt M. Viability, maturation and embryo development in vitro of immature porcine
and ovine oocytes vitrified in different devices. Cryobiology. 2012;64:261–6. 49. Thomson L, Fleming S, Aitken R, De Iuliis G, Zieschang JA, Clark A. Cryopreservation‐induced human sperm DNA damage is predominantly
mediated by oxidative stress rather than apoptosis. Hum Reprod. 2009;24:2061–70. 27. Paramio MT, Izquierdo D. Current status of in vitro embryo production in
sheep and goats. Reprod Domest Anim. 2014;49:37–8. 28. McPartlin LA, Suarez SS, Czaya CA, Hinrichs K, Bedford-Guaus SJ. Hyperactivation of stallion sperm is required for successful in vitro
fertilization of equine oocytes. Biol Reprod. 2009;81:199–206. 29. Liu F, Qiu Y, Zou Y, Deng ZH, Yang H, Liu YD. Use of zona pellucida-bound
sperm for intracytoplasmic sperm injection produces higher embryo quality
and implantation than conventional intracytoplasmic sperm injection. Fertil
Steril. 2011;95:815–8. 30. Ortgies F, Klewitz J, Görgens A, Martinsson G, Sieme H. Effect of procaine,
pentoxifylline and trolox on capacitation and hyperactivation of stallion
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1 18. Yong HY, Pyo BS, Hong JY, Kang SK, Lee BC, Lee ES, et al. A modified
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Agrícola y Animal, Universidad Autónoma Metropolitana-Xochimilco, Ciudad
de México, Mexico. 2División de Ciencias Biológicas y de la Salud,
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and we will help you at every step: Submit your next manuscript to BioMed Central
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on preimplantation development, apoptosis and ploidy of bovine
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A Benthic Terrain Classification Scheme for American Samoa
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Marine geodesy
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Marine Geodesy, 29: 89–111, 2006
Copyright © Taylor & Francis Group, LLC
ISSN: 0149-0419 print / 1521-060X online
DOI: 10.1080/01490410600738021 EMILY R. LUNDBLAD,1,2 DAWN J. WRIGHT,1
JOYCE MILLER,3 EMILY M. LARKIN,4,5
RONALD RINEHART,4 DAVID F. NAAR,6
BRIAN T. DONAHUE,6 S. MILES ANDERSON,7 AND
TIM BATTISTA8 for American Samoa that extends and improves the NOAA Biogeography scheme, which,
although developed for Pacific island nations and territories, is only applicable to a
maximum depth of 30 m, due to the limitations of satellite imagery. The scheme may be
suitable for developing habitat maps pinpointing high biodiversity around coral reefs
throughout the western Pacific. Keywords terrain analysis, bathymetry, benthic habitat, marine GIS, American Samoa,
corals The high productivity of coral reef ecosystems demands a quantifiable analysis of the
complexity and diversity present there. Many people depend on the resources and services
that coral reef ecosystems provide, and the direct connection with adjacent coastal
ecosystems is important to increasing coastal populations (Culliton 1998). Natural and
anthropogenic processes threaten natural and cultural resources in these areas in the form of
storms, global warming, sea water level rise, disease, over-fishing, ship grounding, sediment
runoff, trade in coral and live reef species, marine debris, invasive species, security training
activities, offshore oil and gas exploration, and coral bleaching (Miller and Crosby 1998;
Evans et al. 2002). Although corals can recover from natural disasters, their ecosystems
may not bounce back in the face of destructive anthropogenic threats (Miller and Crosby
1998; Weier 2001; Green et al. 1999). Downloaded by [Professor Dawn J. Wright] at 14:01 03 November 2014 The U.S. Coral Reef Task Force (CRTF) was established by NOAA in June 1998 as an
overseer of coral reef protection. The CRTF Mapping and Information Working Group has
a goal to characterize and map all priority shallow water (<30 m) coral reefs and deep water
(>30 m) coral reef systems in the U.S. and Trust Territories by 2009 (Evans et al. 2002). Until 2001, U.S. coral reefs were not mapped at a resolution useful enough for assessing
and managing resources (i.e., multibeam bathymetry at 5-m horizontal resolution or lower
and extensive underwater video and photography along transects). The work described here
is a first step in meeting that objective for coral reef systems around the island of Tutuila,
American Samoa. As growing populations flock to coastal homes, residents, resource managers,
researchers, scientists and other professionals alike are becoming more aware of the
importance of marine environments and their resources. To protect such environments
there is a need for documenting baseline information about them on an ecosystem level, for
long-term monitoring, and for estimating the geographic extent of critical habitats. EMILY R. LUNDBLAD,1,2 DAWN J. WRIGHT,1
JOYCE MILLER,3 EMILY M. LARKIN,4,5
RONALD RINEHART,4 DAVID F. NAAR,6
BRIAN T. DONAHUE,6 S. MILES ANDERSON,7 AND
TIM BATTISTA8 1Department of Geosciences, Oregon State University, Corvallis, Oregon, USA
2Now at Joint Institute for Marine and Atmospheric Research, University of
Hawaii—Manoa, Contractor to NOAA Pacific Islands Fisheries Science Center,
Honolulu, Hawaii, USA 3Joint Institute for Marine and Atmospheric Research, University of
Hawaii—Manoa, Contractor to NOAA Pacific Islands Fisheries Science Center,
Honolulu, Hawaii, USA
4D
t
t f G
i
O
St t U i
it
C
lli
O
USA 3Joint Institute for Marine and Atmospheric Research, University of
Hawaii—Manoa, Contractor to NOAA Pacific Islands Fisheries Science Center,
Honolulu, Hawaii, USA 4Department of Geosciences, Oregon State University, Corvallis, Oregon, USA
5Now at NOAA National Ocean Service, Silver Spring, Maryland, USA
6University of South Florida, St. Petersburg, Florida, USA
7Analytical Laboratories of Hawaii, LLC, Honolulu, Hawaii, USA
8NOAA National Centers for Coastal Ocean Science, Center for Coastal
Monitoring and Assessment, Biogeography Program, Silver Spring,
Maryland, USA Coral reef ecosystems, the most varied on earth, continually face destruction from
anthropogenic and natural threats. The U.S. Coral Reef Task Force seeks to characterize
and map priority coral reef ecosystems in the U.S./Trust Territories by 2009. Building
upon NOAA Biogeography shallow-water classifications based on Ikonos imagery,
presented here are new methods, based on acoustic data, for classifying benthic terrain
below 30 m, around Tutuila, American Samoa. The result is a new classification scheme Received 23 February 2005; accepted 26 February 2006. This research was supported by NOAA Coastal Services Center (CSC) Special Projects for the
Pacific Islands Initiative, Grant #NA03NOS4730033 to D. Wright and additional NOAA support
to D. Naar. We acknowledge the logistical and financial support from Marine Sanctuary Director,
Nancy Daschbach, as well as the warm hospitality provided by the local villages adjacent to Fagatele
Bay and Vatia Bay. Others instrumental in this project were Will White (formerly of Department
of Marine and Wildlife Resources), John Rooney (CRED), Peter Craig (National Park of American
Samoa (NPAS)), Allison Graves (formerly of NPAS), Curt Whitmire (NOAA Northwest Fisheries
Science Center), Anita Grunder (Oregon State University), Joshua Murphy and Lori Cary-Kothera
(CSC), Jeff Jenness (Jenness Enterprises), Pat Iampietro (California State University, Monterey Bay),
and Will Smith (Hawaii Institute of Marine Biology). The comments of two anonymous reviewers
significantly improved the manuscript. Address correspondence to Emily R. Lundblad, Joint Institute for Marine and Atmospheric
Research, University of Hawaii—Manoa, Manoa, Hawaii, USA. E-mail: emily.lundblad@noaa.gov 89 90 E. R. Lundblad et al. EMILY R. LUNDBLAD,1,2 DAWN J. WRIGHT,1
JOYCE MILLER,3 EMILY M. LARKIN,4,5
RONALD RINEHART,4 DAVID F. NAAR,6
BRIAN T. DONAHUE,6 S. MILES ANDERSON,7 AND
TIM BATTISTA8 With this
in mind, the National Oceanic and Atmospheric Administration’s (NOAA) Biogeography
Program turned to the use of satellite imagery to delineate potential benthic habitats within
coastal regions down to about 30 m (e.g., Monaco et al. 2005) using a habitat classification
scheme that is coral centric. By using heads-up digitizing to analyze Ikonos imagery,
airborne color aerial photography, and airborne hyperspectral imagery, researchers have
successfully classified habitats in much of the shallow water regions around the coastal
U.S. and Territories (i.e., Puerto Rico and the U.S. Virgin Islands, Northwest Hawaiian
Islands, Monterey Bay, American Samoa, the Commonwealth of the Northern Mariana
Islands, Guam) (Buja et al. 2002; Coyne et al. 2001, 2002a, 2002b; NCCOS 2005). Scientists and researchers are extending their understanding to regions deeper than
30 m in order to protect and monitor coral reef ecosystems, some of the most varied on
earth. To locate and study resources associated with particular terrains, it is necessary
to map benthic terrain at a fine-scale. New data collection and mapping techniques are
required to extend the shallow water classifications for potential habitats in deeper water,
at this fine-scale. The methods and results presented here explain a way to classify benthic
terrains in 30 to 150 m depths with a 1 to 2 m resolution. These terrains provide information, Benthic Terrain Classification for American Samoa 91 based on an integration of marine data into a geographic information system (GIS), which
scientists may use to identify areas of high biodiversity and species’ potential habitats. Use and furtherance of the GIS and the resulting benthic terrain classification may lead to
more efficient management of marine protected areas, simpler decision making with the
use of more detailed, extensive, and accurate science, advancements in marine and coastal
research, and improvements on georeferenced marine mapping (e.g., Wright and Bartlett
2000; Valavanis 2002). In this case, layers (i.e., multibeam bathymetry, slope, bathymetric
position index (at multiple scales), and rugosity) were combined in a GIS and assessed with
unique algorithms to produce classification maps. A significant ongoing goal in seafloor exploration is to define a common classification
scheme that all characterization studies can use effectively and efficiently. The development
of a common classification scheme would make sharing results and data easier. EMILY R. LUNDBLAD,1,2 DAWN J. WRIGHT,1
JOYCE MILLER,3 EMILY M. LARKIN,4,5
RONALD RINEHART,4 DAVID F. NAAR,6
BRIAN T. DONAHUE,6 S. MILES ANDERSON,7 AND
TIM BATTISTA8 However,
in this effort there is an understanding among researchers, scientists, and managers that the
use of a single, common classification scheme applicable to all environments is not a reality
yet. The seafloor mapping community is striving for such a scheme at regional and local
levels (http://www.ngu.no/geohab, Marine Geological and Biological Habitat Mapping
Network). The contribution of this study is that it presents such a scheme specifically
for American Samoa, at depths of 30–150 m. This extends and improves the NOAA
Biogeography scheme, which, although developed for Pacific island nations and territories,
is only applicable to a maximum depth of 30 m, due to the limitations of satellite imagery. Review of Seafloor Classification Approaches: Multibeam and Visual Data They followed Weiss’s (2001) methods to develop
TPI grids that, at a fine-scale, can describe micro- and macroscale habitats while, at a
broad-scale, can describe meso- and megascale habitats. Another derivative of bathymetry
that they applied was rugosity. Rugosity is a measure of roughness or bumpiness (classified
as high, medium, and low) that is quantified with a ratio of surface area to planar area. g ,
,
)
q
p
Coops et al. (1998) further developed and tested procedures to predict topographic
position from digital elevation models for species mapping. In this study, topographic
position is a “loosely defined variable” that attempts to describe topography with spatial
relationships. Quantitative assessments of such terrains are rarely reported. Topographic
position can help researchers understand how patterns, processes, and species are spatially
related. While qualitative analyses can describe processes on slopes at different scales,
a quantitative assessment determines primary units within the context of a process. Depending on the scale of the landscape in interest, more or fewer divisions of topographic
position may be quantified. This overview describes a landscape classification scheme by
Speight (1990). Speight (1990) defined morphology types with eleven different classes:
crests, depressions (open and closed), flats, slopes (upper, mid, lower and simple), ridges,
and hillocks. The topographic position analysis in this study incorporates local relief,
elevation percentile, plan and profile curvature, slope, and variance threshold. After
defining crests, depressions, flats and slopes, the study goes further to define the more
detailed classes. Unfortunately, the study does not attempt to subdivide the depressions into
open and closed though the classification scheme recognizes the need for the distinction. Also, because of their fine scale complexity, hillocks and ridges were not quantified
(Coops et al. 1998). Downloaded by [Professor Dawn J. Wright] at 14:01 03 November 2014 Some historical studies have taken approaches to relate topographic features with
populations of particular species. Schmal et al. (2003) used multibeam bathymetric maps
to guide submersibles that allowed the researchers to identify detailed biotopes and species
within geomorphic zones (e.g., coral species zones within midshelf banks <36 m depth, or
within banks <50 m depth, or on the soft bottom). The bathymetry served as an effective
base layer in a GIS to use for their investigation. Review of Seafloor Classification Approaches: Multibeam and Visual Data Most benthic terrain classification studies have relied primarily on analysis and interpreta-
tion of multibeam bathymetry. They also had access to some kind of visually-observed
survey data (e.g., transect video, still photos, grab samples, etc.) to make qualitative
and/or quantitative inferences. Multibeam backscatter (i.e., the intensity of the acoustic
returns) has been used as well but is a separate issue, not discussed here, due to the
greater complexities in processing acoustic strength versus travel time, and of interpreting
seafloor sediment types and inhomogeneities in subbottom layers (as explained in Zhou and
Chen 2005). Greene et al. (1999) developed a successful classification scheme for fish habitats
offshore of Central California. It was recently updated in Greene et al. 2005 and describes
broad classes such as megahabitats (based on depth and general physiographic boundaries),
meso/macrohabitats (based on scale), seafloor slope, seafloor complexity, and geologic
units. The scheme continues with more detailed habitat characteristics interpreted from
video, still photos or direct observation via SCUBA. They are macro/microhabitats (based
on observed small-scale seafloor features), seafloor slope (estimated from in situ surveys),
and seafloor complexity (estimated rugosity). Weiss (2001) made a unique classification scheme for understanding watershed metrics
by using a topographic position and landform analysis. To form a topographic position index
(TPI), he used algorithms that perform an analysis on each grid cell in an elevation model. Each grid cell is assigned a TPI value that indicates its position (higher than, lower than, or
the same elevation) in the overall landscape. By combining TPI with slope position, Weiss
(2001) found methods to apply a landform classification scheme to watersheds around Mt. Hood, Oregon, USA, and the west slope of the Oregon Cascades. The scheme includes 10
landform classes: canyons, deeply incised streams; midslope drainages, shallow valleys;
upland drainages, headwaters; U-shape valleys; plains; open slopes; upper slopes, mesas;
local ridges/hills in valleys, midslope ridges, small hills in plains; mountain tops, high 92 E. R. Lundblad et al. ridges. Weiss (2001) considered two scales of landforms in order to incorporate structures
found within broad landscapes. His techniques are well suited to benthic classifications that
serve as a predictor for habitat suitability and biodiversity (Guisan et al. 1999). Iampietro and Kvitek (2002) derived descriptive grids from multibeam bathymetry to
quantify seafloor habitats for the nearshore environment of the entire Monterey peninsula
in central California, USA, with GIS. Existing NOAA Biogeography Approach: Satellite Imagery NOAA’s Biogeography Program developed a classification scheme for benthic habitats
throughout the Pacific Islands (Coyne et al. 2002b), based on high-resolution Ikonos satellite
imagery, in order to meet the needs of resource managers and scientists. This extends the
approaches described in the previous section because with the synoptic coverage of satellite
imagery, it allows greater areas of the seafloor to be classified. The drawback here, of course,
is that it is only good to 30 m in clear waters. A hierarchical classification was chosen to
define and delineate habitats and was influenced by management requests, the existing
classification schemes, past knowledge of mapping coral reefs, the minimum mapping unit,
quantitative data, and limitations of the imagery (Coyne et al. 2002b). Their hierarchical
scheme uses two categories of classes (zones and habitats) thereby allowing the user to
expand and collapse the scheme. Zones describe a benthic community’s location. Habitats,
which occur within zones, are based on geomorphologic structure and biological cover
type. The structure and cover component are then further divided into major and detailed
levels resulting in a GIS polygon habitat map product with each polygon populated with
one zone and four habitat attributes. The structural component of the maps is divided into
four major and seventeen detailed designations. The biological cover component is divided
into nine major designations with each subdivided into four density classes. Classes that
were determined to be undetectable from the imagery were not included in the scheme. This approach was first developed by the Caribbean Fishery Management Council (Coyne
et al. 2002b; Christensen et al. 2003) and subsequently refined for use in Hawaii and the
U.S. Pacific Territories. Downloaded by [Professor Dawn J. Wright] at 14:01 03 November 2014 In these maps, polygon boundaries are visually interpreted and manually delineated
on computer screen based of the color, texture, and relative location of the feature in the
remotely sensed imagery. Extensive field observations are conducted to determine habitat
types in areas where uncertainty existed in the visual interpretation of the imagery, where
gradients exist through habitat types or where habitat diversity is highly heterogeneous. Field surveys were also conducted to acquire ground truth needed to establish a
statistically robust assessment of the thematic accuracy of these products (Congalton 1991;
Rosenfield et al. 1982; Cohen 1960; Ma and Redmond 1995; Hudson and Ramm 1987). Review of Seafloor Classification Approaches: Multibeam and Visual Data Another study that effectively related
species to their habitat locations used side scan sonar mosaics to find the relationship
between population abundance and the benthoscape (undersea landscapes; Zajac et al. 2003). With the use of backscatter imagery, they classified large scale benthoscapes such
as muddy sands, fine sands and muds, boulder, cobble and outcrop, sand wave fields,
and mixed. They paid close attention to transitions between benthoscapes where infaunal
populations were readily identified at a finer scale. The approach taken in the current study takes into consideration the many applications
of many types of data that are used for benthic habitat mapping (e.g., Hall et al. 1999). Most
often there is a need for a baseline of information. Usually, the baseline, or framework, used
for a habitat study is a basic data set that describes the surficial characteristics of the seafloor
in some useful fashion (Dartnell and Gardner 2004). Then, based on what the seafloor looks
like, a biologist, geologist, ecologist, geophysicist, or other interested party will supplement
that framework with specific data sets. A biologist may add a layer of information about
amount of relief or the thickness of sediments. A geologist may add data revealing sediment
size or rock type. Depending on the interest of the research, different layers of information
are needed. Therefore, a method has been developed here that results in separate data Benthic Terrain Classification for American Samoa 93 sets that may be combined at different scales and in different combinations to serve as
a baseline of information for researchers, scientists, and managers. But as explained in
the next section, we combine this with the satellite based approach NOAA Biogeography,
considering and extending their classifications into a new classification scheme for Pacific
island deepwater habitats. Existing NOAA Biogeography Approach: Satellite Imagery This statistical treatment generates overall accuracy, Kappa and Tau statistics, as well as
user and producer accuracy of the thematic content of the map products at both the major
and detailed level of the classification scheme. Accuracy of the zone attribute was not
tested. Ikonos satellite imagery was used to generate all of these map products for the coral
reefs of American Samoa. The overall thematic accuracy was greater the 85% (Kappa and
Tau >0.85) at the major level of the classification scheme and greater than 75% (Kappa
and Tau >0.75) for the detailed level of the classification scheme, but again, only for a
maximum depth of 30 m. The classification scheme introduced in this article bridges the multibeam approaches
of the previous section with the satellite-based approach of NOAA Biogeography. Using
those earlier approaches, we effectively take the NOAA scheme into deeper water. A
primary objective of the current study was to extend this existing classification below 30
m, the reach of what is viewable and classifiable in Ikonos imagery. 94 E. R. Lundblad et al. Deep Water (30 m–200 m) Data Collection for This Study Extensive data have been collected in American Samoa since 2001, including multibeam
bathymetry, towed diver videos, accuracy assessment photography, field notes, and
information from a rebreather dive (Wright 2002; Wright et al. 2002). Multibeam mapping
systems allow surveyors to collect bathymetry by ensonifying massive areas of the seafloor
with high accuracy (e.g., Blondel and Murton 1997; Mayer et al. 2000). Scores of acoustic
beams form a swath that fans out up to several times the water depth. Multibeam mapping
systems are set up on research vessels that navigate across the study area making real-time
adjustments for sound velocity, heave, roll, pitch, and speed (3–12 knots) (e.g., Blondel
and Murton 1997). Most modern multibeam mapping systems also collect backscatter data
which are often useful for classifying seafloor bottom characteristics (e.g., sediments versus
lava flows). Downloaded by [Professor Dawn J. Wright] at 14:01 03 November 2014 The first scientific surveys of depths beyond 30 m in coral reef ecosystems around
American Samoa collected bathymetric data from 3 to 160 m depth in 2001 and 2002
with the University of South Florida’s Kongsberg Simrad EM3000, 300 kHz, multibeam
mapping system (Wright et al. 2002; Wright 2002). The 2001 survey collected bathymetry
and backscatter for Fagatele Bay National Marine Sanctuary (FBNMS), part of the National
Park, Pago Pago Harbor, the western portion of Taema Bank, and Faga’itua Bay (Figure 2). Sites surveyed in November 2002 are eastern Taema Bank, Coconut Point, Fagatele Bay,
and Vatia Bay (Figure 2). The NOAA Coral Reef Ecosystem Division (CRED), part of the Pacific Island
Fisheries Science Center (PIFSC), conducted surveys in February/March 2002 and
February/March 2004. CRED towed diver video (0–25 m), deeper (20–100 m) towed
photographic and video data, and single beam/bottom classification data resulting from
the 2002 surveys were used for this analysis. Extensive multibeam (bathymetry and Figure 2. Location of high-resolution multibeam bathymetry surveys around Tutuila, American
Samoa (1-m horizontal spatial resolution and ±1-m vertical accuracy for all areas except for the
National Park where data were collected at 2-m resolution with a vertical accuracy of ∼±5 m). Projection: Geographic, WGS84. Bathymetry was collected in April and May of 2001 and November
of 2002 with the Kongsberg Simrad EM3000 (http://dusk.geo.orst.edu/djl/samoa). Figure 2. Study Site and its Threats American Samoa, a small, remote territory in the heart of the South Pacific, is the only
U.S. territory south of the equator and consists of about 197 km2 of land cover. It lies about
14o south of the equator, about 4,700 km southwest of Honolulu, Hawaii (Figure 1). It
neighbors the independent nation of (western) Samoa as the eastern portion of the Samoan
archipelago. American Samoa’s five volcanic islands (Tutuila, Aunu’u, Ofu, Olosega, and
Ta’u) and two coral atolls (Rose and Swains) are surrounded by true tropical reefs, which
are extremely rare in U.S. waters. The coral reef ecosystems around American Samoa are being threatened by natural
and adverse anthropogenic patterns and processes (Evans et al. 2002). For example, coral
bleaching events related to sea temperature rise have increased in the region, including a
particularly destructive event in 1994 (FBNMS 2004). An infestation of crown-of-thorns
starfish killed vast amounts of coral in the late 1970s owing to their habits of eating live coral
(Craig 2002). In addition, coral around the South Pacific islands are threatened annually
by tropical cyclones. American Samoa suffered from the effects of hurricane Ofa in 1990,
hurricane Val in 1991, and most recently hurricane Heta in January 2004 (Craig 2002;
FBNMS 2004; FEMA 2004). Anthropogenic threats such as gill netting, spear fishing,
poison and dynamite fishing, nonpoint pollution and cumulative impacts challenge and
stunt coral reef recovery from natural disasters (ASG: DOC 2004). wnloaded by [Professor Dawn J. Wright] at 14:01 03 November 2014 Downloaded by [Professor Dawn J. Wright] at 14:01 03 November 2014 Figure 1. Location Map of American Samoa, a U.S. Territory that is home to priority coral reefs
that are part of the National Action Plan to Conserve Coral Reefs. American Samoa is part of the
Samoan archipelago and is comprised of five volcanic islands and two coral atolls. Figure 1. Location Map of American Samoa, a U.S. Territory that is home to priority coral reefs
that are part of the National Action Plan to Conserve Coral Reefs. American Samoa is part of the
Samoan archipelago and is comprised of five volcanic islands and two coral atolls. Benthic Terrain Classification for American Samoa 95 Deep Water (30 m–200 m) Data Collection for This Study Location of high-resolution multibeam bathymetry surveys around Tutuila, American
Samoa (1-m horizontal spatial resolution and ±1-m vertical accuracy for all areas except for the
National Park where data were collected at 2-m resolution with a vertical accuracy of ∼±5 m). Projection: Geographic, WGS84. Bathymetry was collected in April and May of 2001 and November
of 2002 with the Kongsberg Simrad EM3000 (http://dusk.geo.orst.edu/djl/samoa). 96 E. R. Lundblad et al. backscatter) and video data were also collected in early 2004, but it was not possible
to incorporate these data here. These multibeam bathymetric data are now available online
at http://www.pifsc.noaa.gov/cred/hmapping. backscatter) and video data were also collected in early 2004, but it was not possible
to incorporate these data here. These multibeam bathymetric data are now available online
at http://www.pifsc.noaa.gov/cred/hmapping. Bathymetry The 300 kHz multibeam bathymetry data from the 2001 and November 2002 surveys
were used for analysis, after postprocessing, as a 3-column XYZ ASCII file with positive
depth values based on a mean low low water datum at full resolution of the Kongsberg
Simrad EM3000 system. For Fagatele Bay, Coconut Point and Taema (Eastern), the XYZ
bathymetry was gridded at 1m spacing in MB-System (Caress et al. 1996). MB-System
outputs grids in the format of Generic Mapping Tools (GMT) for a UNIX environment. GMT is a public suite of tools used to manipulate tabular, time-series, and gridded data sets,
and to display these data in appropriate formats for data analysis (Wessel and Smith 1991). Then the GMT grids were converted to a format compatible with Arc/INFOő using a suite
of tools called ArcGMT (Wright et al. 1998). For Taema Bank (Western), the XYZ data
were gridded with Fledermaus and exported as an ArcView ASCII file, then converted to
a grid with ArcToolbox. After importing the grids into the Arc/INFOő raster grid format,
algorithms were run in ArcGISTM to calculate derivatives. Bathymetric Derivatives: Bathymetric Position Index, Slope First, positive depth values were converted to negative. Slope, or the measure of steepness
first-order derivative, was simply derived using the ArcGISTM spatial analyst extension’s
surface analysis. Output slope values (raster grids) are derived for each cell as the maximum
rate of change from the cell to its neighbor. Bathymetric Position Index (BPI) is a second-order derivative (as it is derived from
the first derivative, slope) of bathymetry as modified from topographic position index as
defined in Weiss (2001) and Iampietro and Kvitek (2002). BPI was derived as a measure of
where a georeferenced location, with a defined elevation, is relative to the overall landscape. The derivation involves evaluating elevation differences between a focal point and the mean
elevation of the surrounding cells within a user defined rectangle, annulus, or circle. For example, where a user has an elevation grid that has 1 m resolution, he/she may
choose to analyze the grid with an annulus. The annulus, having an inner radius of 2
units and an outer radius of 4 units, would be used to analyze spatially each grid cell in
comparison to its neighboring cells that fall within that annulus (Figure 3). BPI was calculated in the ArcGISTM raster calculator using the focal mean calculation
described above; the resulting grid values are converted to integers to minimize the storage
size of the grid and to simplify symbolization (Algorithm 1). Algorithm 1 creates a BPI grid using bathymetry and user defined radii: scalefactor = outer radius in map units multiplied by bathymetric data resolution, irad = inner radius of annulus in cells, orad = outer radius of annulus in cells, bathy = bathymetric grid, and rad = radius (if using circle instead of annulus). Benthic Terrain Classification for American Samoa 97 Figure 3. Example of the variables used to derive bathymetric position index (BPI) from bathymetry. The grid cells here (1 m resolution) represent bathymetry as negative values. The annulus has an
outer radius of 4 and an inner radius of 2. Therefore the BPI scalefactor is 4 (outer radius multiplied
by bathymetry resolution). Figure 3. Example of the variables used to derive bathymetric position index (BPI) from bathymetry. The grid cells here (1 m resolution) represent bathymetry as negative values. The annulus has an
outer radius of 4 and an inner radius of 2. Therefore the BPI scalefactor is 4 (outer radius multiplied
by bathymetry resolution). BPI⟨scalefactor⟩= int((bathy – focalmean(bathy, annulus, irad, orad)) + 0.5)
or
BPI⟨scalefactor⟩= int((bathy—focalmean(bathy, circle, rad)) + 0.5) BPI⟨scalefactor⟩= int((bathy – focalmean(bathy, annulus, irad, orad)) + 0.5)
or
BPI⟨scalefactor⟩= int((bathy—focalmean(bathy, circle, rad)) + 0.5) The cells in the output grid are assigned values within a range of positive and negative
numbers (Figure 4). The 0.5 is added before the integer conversion and is meant to force
floating point values, regardless of the sign of the value, to round up if the value has a
decimal of greater than.5 and to round down if the value has a decimal of less than.5. The
variable is not necessary if a user chooses to allow the floating point values to be rounded
downward consistently for positive and negative values. A negative value represents a cell that is lower than its neighboring cells (valleys). A positive value represents a cell that is higher than its neighboring cells (ridges). Larger
numbers represent benthic features that differ greatly from surrounding areas (such as sharp
peaks, pits or valleys). Flat areas or areas with a constant slope produce near-zero values. In this example, the cells with a value of 1 are higher than those with the value of 0,
and the values of 2 are higher than the others (Figure 5). The diagonally linear pattern of
cells with the value of 1 starting from the top, left corner of the grid may represent a crest
in the benthoscape. Furthermore, the grid cells with values of 2 along that pattern may be
narrow crests on top of the larger crest. Also, notice that the other groups of BPI values of
1 may represent small mounts within the benthoscape. The values of 0 are all flat areas or
constant slopes. Whether they are flats or slopes would be determined in another algorithm
that considers slope along with BPI. This derivation is discussed in the development of
classification scheme for American Samoa section. This example does not include negative 98 E. R. Lundblad et al. Figure 4. A description of the resulting bathymetric position index (BPI) values that are derived
from bathymetry. These are based on a topographic position index by Weiss (2001). Top describes
fine scale BPI values. Bottom describes broad scale BPI values. (Courtesy of Weiss 2001.) Figure 4. A description of the resulting bathymetric position index (BPI) values that are derived
from bathymetry. These are based on a topographic position index by Weiss (2001). Top describes
fine scale BPI values. BPI⟨scalefactor⟩= int((bathy – focalmean(bathy, annulus, irad, orad)) + 0.5)
or
BPI⟨scalefactor⟩= int((bathy—focalmean(bathy, circle, rad)) + 0.5) Bottom describes broad scale BPI values. (Courtesy of Weiss 2001.) BPI values. If negative values were present in this grid sample, they would represent patterns
of depressions. The scalefactor of the resulting grid is 4, where scalefactor is the resolution
multiplied by the outer radius. The results of BPI are scale dependent; different scales identify fine or broad benthic
features. To achieve the best BPI zone and structure classifications several large and
small-scale grids were created for each study site. The fine scale grids were created with
scalefactors of 10, 20, and 30, and the broad scale grids were created with scalefactors
of 50, 70, 125, and 250. BPI⟨20⟩and BPI⟨250⟩were used to classify Fagatele Bay and
Taema Bank. These scalefactors were chosen because, at these sites, the small seascape
features (distance between relatively small ridges) are, on average, about 20 m across;
the large seascape features (e.g., the distance across the deep channel on the west end of
Taema Bank and the length of the peninsula in Fagatele Bay) are about 250 m across. This
is based on close examination of the bathymetry prior to the BPI calculation, especially
in the Fledermaus 3-D visualization system. For Coconut Point, features of interest were Benthic Terrain Classification for American Samoa 99 Figure 5. Example of the variables used to derive bathymetric position index (BPI) from bathymetry. The grid cells represent a derived BPI grid. Negative values are lower than their neighbors. Positive
values are higher than their neighbors. Values of zero are flat areas or areas with constant slope. Figure 5. Example of the variables used to derive bathymetric position index (BPI) from bathymetry. The grid cells represent a derived BPI grid. Negative values are lower than their neighbors. Positive
values are higher than their neighbors. Values of zero are flat areas or areas with constant slope. identified from about 10 m to 70 m across, so BPI⟨10⟩and BPI⟨70⟩were used. See Tables
1 and 2 for the values used to derive BPI for each study site. Prior to the classification of the final zones and structures, BPI was standardized. Conclusions about the structure of the overall seascape can be made with spatial analysis
by applying an algorithm that combines standardized BPI grids of different scales with
slope and bathymetry. BPI⟨scalefactor⟩= int((bathy – focalmean(bathy, annulus, irad, orad)) + 0.5)
or
BPI⟨scalefactor⟩= int((bathy—focalmean(bathy, circle, rad)) + 0.5) In Arc/INFOő GRID, the final algorithms for classifying BPI zones
and structures are based on combined broad scale and fine scale standardized BPI grids,
slope, and depth. Table 2 Rugosity can be associated with
attributes recorded during dives and with comments and attributes recorded in accuracy
assessment surveys conducted in 2001 (Figure 6). From qualitative analysis, the derived
rugosity grid and the towed-diver surveys are not directly related. However, from this type
of comparison, divers may possibly standardize their observations among different divers
in order to collect less subjective information about the environment. Table 2 Table 2
Parameters used for calculating broad scale bathymetric position index grids Parameters used for calculating broad scale bathymetric position index grids g
y
p
g
Study site
Resolution
(meters)
circle, annulus,
or rectangle
Irad
Orad
Broad
scalefactor
Fagatele Bay
3
Annulus
16
83
250
Coconut Point
1
Circle
70
70
Taema Bank 2002 (Eastern)
3
Annulus
16
83
250
Taema Bank 2001 (Western)
3
Annulus
16
83
250 area ratio. Rugosity was derived with the ArcViewő Surface Area from Elevation Grids
extension (Jenness 2003) using a 3×3 neighborhood analysis to calculate surface area
based on a 3-D interpretation of cells’ elevations. Rugosity values near one indicate flat,
smooth locations; higher values indicate areas of high-relief. Rugosity calculated using this
technique is highly correlated with slope. The highest rugosity values show a relationship
with the high slope and lower rugosity with low slope. Rugosity classifications extend
the classes used by CRED for habitat complexity in their 2002 towed-diver surveys. The
classes were assigned with the following standard deviation divisions in ArcViewő 3.3:
Very High (>3 std. dev.), High (2–3 std. dev.), Medium High (1–2 std. dev.), Medium (0–1
std. dev.), Medium Low (Mean), Low (−1–0 std. dev.). Rugosity can be associated with
attributes recorded during dives and with comments and attributes recorded in accuracy
assessment surveys conducted in 2001 (Figure 6). From qualitative analysis, the derived
rugosity grid and the towed-diver surveys are not directly related. However, from this type
of comparison, divers may possibly standardize their observations among different divers
in order to collect less subjective information about the environment. area ratio. Rugosity was derived with the ArcViewő Surface Area from Elevation Grids
extension (Jenness 2003) using a 3×3 neighborhood analysis to calculate surface area
based on a 3-D interpretation of cells’ elevations. Rugosity values near one indicate flat,
smooth locations; higher values indicate areas of high-relief. Rugosity calculated using this
technique is highly correlated with slope. The highest rugosity values show a relationship
with the high slope and lower rugosity with low slope. Rugosity classifications extend
the classes used by CRED for habitat complexity in their 2002 towed-diver surveys. The
classes were assigned with the following standard deviation divisions in ArcViewő 3.3:
Very High (>3 std. dev.), High (2–3 std. dev.), Medium High (1–2 std. dev.), Medium (0–1
std. dev.), Medium Low (Mean), Low (−1–0 std. dev.). Rugosity Analysis The rugosity analysis resulted in descriptive maps that help identify areas with potentially
high biodiversity. Rugosity describes topographic roughness with a surface area to planar Parameters used for calculating fine scale bathymetric position index grids
Study site
Resolution
(meters)
circle, annulus,
or rectangle
Radius
Fine
scalefactor
Fagatele Bay
1
Circle
20
20
Coconut Point
1
Circle
10
10
Taema Bank 2002 (Eastern)
1
Circle
20
20
Taema Bank 2001 (Western)
1
Circle
20
20 Parameters used for calculating fine scale bathymetric position index grids 100 E. R. Lundblad et al. Development of Classification Scheme for American Samoa Since postprocessing of the 2001 and 2002 data was completed during the initial phase
of this study, only these multibeam bathymetry data were used to classify the seafloor on
the basis of bathymetric position index (BPI) and rugosity. The methods developed were
based on the topographic position index algorithms of Guisan et al. (1999), Weiss (2001)
and Iampietro and Kvitek (2002) and the rugosity algorithm of Jenness (2003) as applied
in Iampietro and Kvitek (2002). Their application to American Samoa bathymetry is the
first extension of existing shallow water benthic classification to depths beyond 30 m. (Figure 7). Spatial analysis was used to derive, from the original bathymetry, indices of slope and
multiple scales of BPI (i.e., BPI zones and structures). The resulting derivative grids were
combined with a new algorithm to develop final products: BPI zones, BPI structures and
rugosity classification maps for the study sites. The maps introduce the first deepwater
benthic classification scheme for American Samoa that may also be extended to other coral
reef systems. The mapping steps for the classifications, including the classification scheme,
are summarized in the flowchart in Figure 8. The process identifies four BPI zones and 13
structure classes. The algorithm that combines these data sets uses standard deviation units where 1
standard deviation is 100 grid value units; slope and depth values are defined by the user. Benthic Terrain Classification for American Samoa 101 Figure 6. Rugosity derived in ArcViewő 3.3 and towed-diver video transects symbolized by habitat
complexity observations. Transects overlaid on rugosity grid shows the relationship between the two
data sets. Figure 6. Rugosity derived in ArcViewő 3.3 and towed-diver video transects symbolized by habitat
complexity observations. Transects overlaid on rugosity grid shows the relationship between the two
data sets. The algorithms for BPI zones and structures use different combinations of the grids. The
following is an example of how BPI zones were derived (Algorithm 2). Algorithm 2 creates an output grid classified by BPI zones by combining the attributes
of BPI and slope: B-BPI = broad scale BPI grid, B-BPI = broad scale BPI grid, B-BPI = broad scale BPI grid, out zones = name of the output grid, out zones = name of the output grid, slope = the slope grid derived from bathymetry, and p
p g
y
y
gentle = the user defined slope value indicating a gentle slope. gentle = the user defined slope value indicating a gentle slope. If (B-BPI> = 100) out zones = 1,
else if (B-BPI< = −100) out zones = 2,
else if (B-BPI> −100 and B-BPI < 100 and slope < = gentle) out zones = 3,
else if (B-BPI> −100 and B-BPI < 100 and slope > gentle) out zones = 4. If (B-BPI> = 100) out zones = 1,
else if (B-BPI< = −100) out zones = 2,
else if (B-BPI> −100 and B-BPI < 100 and slope < = gentle) out zones = 3,
else if (B-BPI> −100 and B-BPI < 100 and slope > gentle) out zones = 4. The unique numbers assigned to classes in algorithm 2 are the following, as defined in th
classification scheme for BPI zones: (1) Crests, (2) Depressions, (3) Flats, and (4) Slopes Structures were derived with a similar algorithm as that used for BPI zones, however
both scales of BPI were considered in order to pinpoint finer features. Also, the variable of
depth was added to identify different flat structures that may represent different habitats. The decision tree below shows the path of decisions that the algorithm uses to derive 102 E. R. Lundblad et al. Figure 7. The amount of overlap that exists between NOAA Biogeography habitat classifications
that were made using Ikonos imagery and the coverage of multibeam bathymetry as of 2002. wnloaded by [Professor Dawn J. Wright] at 14:01 03 November 2014 Downloaded by [Professor Dawn J. Wright] at 14:01 03 November 2014 Figure 7. The amount of overlap that exists between NOAA Biogeography habitat classifications
that were made using Ikonos imagery and the coverage of multibeam bathymetry as of 2002. Figure 8. A flowchart showing the data sets used to derive BPI zones and structures. Benthic Terrain Classification for American Samoa 103 Figure 9. A flowchart of the decisions made by the algorithms that derive zone and structure classes
from broad scale bathymetric position index (B-BPI), fine scale BPI (F-BPI), slope and depth. Downloaded by [Professor Dawn J. Wright] at 14:01 03 November 2014 Figure 9. B-BPI = broad scale BPI grid, BPI Zones for Fagatele Bay National Marine Sanctuary. Program’s scheme for shallow water classifications (NWHI 2003). This biogeography
scheme is being extended into deeper water by scientists at CRED are working primarily
with multibeam and underwater video data (Rooney and Miller pers. comm. 2004) in
20–200 m water depths. The NOAA/NOS classification schemes, the Weiss (2001) landform
scheme, and the Speight (1990) scheme, were closely analyzed to develop agreeable terms
for the BPI zones and structures that extend below 30 m depth around American Samoa. Program’s scheme for shallow water classifications (NWHI 2003). This biogeography
scheme is being extended into deeper water by scientists at CRED are working primarily
with multibeam and underwater video data (Rooney and Miller pers. comm. 2004) in
20–200 m water depths. The NOAA/NOS classification schemes, the Weiss (2001) landform
scheme, and the Speight (1990) scheme, were closely analyzed to develop agreeable terms
for the BPI zones and structures that extend below 30 m depth around American Samoa. In this scheme, broad refers to seafloor characteristics defined by broad scale BPI
grids and fine refers to seafloor characteristics defined by fine scale BPI grids. BPI has been
described in more detail in the data analysis section. B-BPI = broad scale BPI grid, A flowchart of the decisions made by the algorithms that derive zone and structure classes
from broad scale bathymetric position index (B-BPI), fine scale BPI (F-BPI), slope and depth. structure classes (Figure 9). The algorithm assigns a unique number to each of the 13
structures. The unique numbers assigned to classes are the following, as defined in the
classification scheme for structures: (1) Narrow depression, (2) Local depression on flat,
(3) Lateral midslope depression, (4) Depression on crest, (5) Broad depression with an
open bottom, (6) Broad flat, (7) Shelf, (8) Open slopes, (9) Local crest in depression,
(10) Local crest on flat, (11) Lateral midslope crest, (12) Narrow crest, and (13) Steep
slope. Specific values for slope and depth are sensitive to interpretation at specific study sites. Each study site has a unique composition of depth and slope ranges. The methods are best
applied where slope and depth values are considered on the condition of transition zone
locations and the presence of two or more significant depth ranges within the study site. In
order to develop a uniform classification for all the American Samoa study sites, common
values that are suitable for sites around Tutuila were used in the classification algorithms. Gentle slopes were defined 5◦and steep slopes were defined as 70◦. A depth of −22 m
was used to define the difference between shelves and broad flats. These slopes and depths
were determined using 3-D visualization in Interactive Visualization System’s Fledermaus
software. Multiple schemes were reviewed to aid in the development of classifications for the
reefs of American Samoa (Coops et al. 1998; Dartnell and Gardner 2004; Greene et al. 1999; Iampietro and Kvitek 2002; Schmal et al. 2003; Speight 1990; Zajac et al. 2003;
White et al. 2003; Greene et al. 2005). Weiss’s (2001) landform scheme is also valuable as it
classifies slope position and landform types as predictors of habitat suitability, community
composition, and species distribution. In this study, similar landform classes are interpreted
only to describe the seafloor and as a baseline for future habitat studies, given the availability
of future data on species counts and distributions. The terminology used in the classification
scheme (for zones) presented here is well-matched with the NOAA/NOS Biogeography 104 E. R. Lundblad et al. Figure 10. BPI Zones for Fagatele Bay National Marine Sanctuary. Downloaded by [Professor Dawn J. Wright] at 14:01 03 November 2014 Figure 10. Classification Scheme for BPI Zones A surficial characteristic of the seafloor based on a BPI value range at a broad scale and on
slope values. 1. Crests—High points in the terrain where there are positive bathymetric position
index values greater than one standard deviation from the mean in the positive
direction 2. Depressions—Low points in the terrain where there are negative bathymetric
position index values greater than one standard deviation from the mean in the
negative direction 2. Depressions—Low points in the terrain where there are negative bathymetric
position index values greater than one standard deviation from the mean in the
negative direction Benthic Terrain Classification for American Samoa 105 Figure 11. Structures for Fagatele Bay National Marine Fisheries. Figure 11. Structures for Fagatele Bay National Marine Fisheries. 3. Flats—Flat points in the terrain where there are near zero bathymetric position
index values that are within one standard deviation of the mean. Flats have a slope
that is < = 5◦. 3. Flats—Flat points in the terrain where there are near zero bathymetric position
index values that are within one standard deviation of the mean. Flats have a slope
that is < = 5◦. 4. Slopes—Sloping points in the terrain where there are near zero bathymetric position
index values that are within one standard deviation of the mean. Slopes have a
slope that is >5◦. Slopes are otherwise called escarpments in the NOAA/NOS
classification scheme. Discussion The classification developed in this study is the first for deepwater environments in Ameri-
can Samoa and should make a critical contribution to benthic habitat mapping in the Pacific. It gives a unique picture of coral reef environments on two different scales. The descriptive
name of each class in the scheme provides users a way to recognize patterns of terrain based
on zones and structures. The classification uses general terms that can apply to coral reef
environments as seen by scientists with several different interests (e.g., biological habitats,
coral disturbance after natural disasters, algae growth, and marine mammal distribution). By using general descriptions of the zones and structures based on the analytical procedures
described in this article, researchers and scientists may view coral reef environments with
the focus of most specialties. The classifications advantageously use several data sets
to describe basic features of the coral reef environment. The basic descriptions, using
standard language among other classifications schemes, allow the classifications to be
used for qualitative analyses or for quantitative analyses. The quantitative nature would be
accomplished from raster calculations to determine spatial statistics within and among the
classes. One disadvantage to the classification is that the scheme does not incorporate shape
of features. For instance, if a crest is linear it should be recognizable by sight as a linear
ridge. However, the classification scheme will not indicate the difference between a group
of crests that form a linear ridge and a group that forms a mound. The same challenge exists
for classifying patterns of depressions (e.g., channels vs. holes). The classification developed in this study is the first for deepwater environments in Ameri-
can Samoa and should make a critical contribution to benthic habitat mapping in the Pacific. It gives a unique picture of coral reef environments on two different scales. The descriptive The classification does not provide a complete description of benthic habitats, but adds
to the resources that may be used in an integrated GIS. By using multibeam bathymetry and
derivative data sets to describe the structures of the seafloor, researchers have a better idea of
how to combine the data in a fashion that will answer important scientific questions. While
the resulting data sets (BPI zones, structures, and rugosity) each provide a unique picture
of the benthic environment, a combined analysis may reveal more, important information. Classification Scheme for Structures A surficial characteristic of the seafloor based on a BPI value range at a combined fine scale
and broad scale, on slope values and on depth. 1. Narrow depression—A depression where both fine and broad features within the
terrain are lower than their surroundings. g
2. Local depression on flat—A fine scale depression within a broader flat terrain. 3. Lateral midslope depression—fine scale depression that laterally incises a slope. 4. Depression on crest—A fine scale depression within a crested terrain. 5. Broad depression with an open bottom—A broad scale depression with a U-shape
where any nested, fine scale features are flat or have constant slope. 6. Broad flat—A broad flat area where the terrain contains few, nested, fine scale
features. 106 E. R. Lundblad et al. 7. Shelf—A broad flat area where the terrain contains few, nested, fine scale features. A shelf is shallower than 22 m depth. (This depth value was decided on based on
3-D visualization and the NOAA/NOS classification scheme (NWHI 2003)). The
NOAA/NOS scheme defines a shelf as ending between 20 and 30 m depth. 8. Open slopes—A constant slope where the slope values are between 5◦and 70◦and
there are few, nested, fine scale features within the broader terrain. 8. Open slopes—A constant slope where the slope values are between 5◦and 70◦and
there are few, nested, fine scale features within the broader terrain. 9. Local crest in depression—A fine scale crest within a broader depressed terrain. 10. Local crest on flat—A fine scale crest within a broader flat terrain. 11. Lateral midslope crest—A fine scale crest that laterally divides a slope. This often
looks like a ledge in the middle of a slope. 11. Lateral midslope crest—A fine scale crest that laterally divides a slope. This often
looks like a ledge in the middle of a slope. g
p
12. Narrow crest—A crest where both fine and broad features within the terrain are
higher than their surroundings. 12. Narrow crest—A crest where both fine and broad features within the terrain are
higher than their surroundings. 13. Steep slope—An open slope with a slope value greater than 70◦. Discussion Researchers at CRED are using the data sets in an integrated GIS in order to make products
that may be used by working scientists and managers. The integration of marine data in
GIS provides a means for advancing marine and coastal research, science and management,
georeferenced mapping, modeling and decision making (e.g., Wright and Bartlett 2000;
Valavanis 2002; Greene et al., 2005). The integration of the unique data sets in a GIS will
allow researchers and scientists to query the data based on any combination of all of the
available data sets. For example, a habitat for a particular fish may be indicated by depths
between 30 and 45 meters, where many small crests and depressions are interlaced at a Benthic Terrain Classification for American Samoa 107 fine scale, having high rugosity. A user can query all the data sets at one time within the
integrated GIS in order to create a new data set that includes only the habitats that meet those
criteria. Also, a user may merge data sets for adjacent areas to make a regional analysis, or
a user may choose to make a simple qualitative analysis of all the data sets in order to plan
sampling locations for in situ data collection. The data analyzed in a GIS are validated when combined with in situ data sets. Such groundtruthing (e.g., video collection, still photos, diver rugosity measurements)
was collected in the shallow waters (<30 m) around Tutuila in February/March 2002
and 2004 and along the west coast of Saipan in the Commonwealth of the Northern
Mariana Islands in December 2004. Currently, scientists at CRED are analyzing the in situ
data in order to make a more informed accuracy assessment of the data sets derived in
this study. The method and classification scheme were tested with the bathymetry from
the Saipan anchorage area (Figure 12). Resulting BPI grids were made for zones and
for structures using the classification scheme discussed in this article. The underwater
videos were classified using a scheme developed for optical validations data around coral
reef ecosystems in Pacific Island regions. The scheme does not match the one presented
in this article, but the results are promising, as they qualitatively present an immediate
association between the BPI structures and underwater video classifications. For example, Downloaded by [Professor Dawn J. Wright] at 14:01 03 November 2014 Downloaded by [Professor Dawn J. Discussion Wright] at 14:01 03 November 2 Figure 12. BPI Structures overlaid with optical validation. This is a sample from the Saipan
anchorage data set that was surveyed by CRED in 2003 and 2004. The structures were derived
from a bathymetric grid (5-m pixel size), and the optical validation represents interpretations of
videos from a towed underwater video camera-sled. A qualitative assessment validates a pattern of
associations between substrates and BPI structures. Figure 12. BPI Structures overlaid with optical validation. This is a sample from the Saipan
anchorage data set that was surveyed by CRED in 2003 and 2004. The structures were derived
from a bathymetric grid (5-m pixel size), and the optical validation represents interpretations of
videos from a towed underwater video camera-sled. A qualitative assessment validates a pattern of
associations between substrates and BPI structures. 108 E. R. Lundblad et al. where raised features outlined by open slopes (e.g., shelves) are characterized in the BPI
structures the videos were classified as hard bottom (e.g., rock). Likewise, where broad flats
are located the videos reveal unconsolidated (e.g., sand) substrate. The open slopes and
other transitional areas (e.g., narrow crests and lateral midslope features) often correspond
with mixed substrates or rubble. This analysis is continuing to find relationships between
BPI zones/structures and percentages of living cover (e.g., Coralline Algae, Scleractinian
Coral, Macroalgae), scale of relief (i.e., five categories ranging from less than 0.5 m–>
3.0 m), and number/size of cavities noted in the frame (e.g., few small and many large
cavities, many small cavities). The analysis is evolving into a product that will validate
interpolations of coral cover and substrate type across the region; it will eventually provide
a prototype habitat map and methods that may be applied to other Pacific Island regions. Additionally, during CRED’s mission in early 2004, a complete backscatter data set was
collected and is, as of August 2005, available for download at http://www.nmfs.hawaii.edu/
cred/hmapping/hmap data.php. The backscatter is an enormous asset to determining
potential benthic habitats. By incorporating it in the GIS, patterns of the types and/or
composition of substrate may be exposed within each zone, structure and rugosity class. Our scheme will be further validated by recent Pisces V submersible dives to Fagatele
Bay and Taema Bank on Hawaii Undersea Research Lab cruise KOK0510, along with
the accompanying statistical validation, July 2005 (Wright 2005; http://dusk.geo.orst.edu/
djl/samoa/ hurl ). Discussion All of the analytical procedures used for the classifications in this study have been
encapsulated into a convenient GIS desktop tool called the Benthic Terrain Modeler
(BTM; Rinehart et al. 2004; http://www.csc.noaa.gov/products/btm). This ArcGIS 8. × /
9.× extension was jointly developed by researchers at Oregon State University and the
NOAA Coastal Services Center. It should allow the user to replicate the procedures on
the bathymetry used in this study (now permanently archived and publicly available at
http://dusk.geo.orst.edu/djl/samoa), on CRED datasets, or on the user’s own dataset. The
BTM will allow users to apply the classification scheme used in this study for Tutuila on
their own coral reef bathymetry. The user may use the default classification scheme, as
described in this article, or may develop or insert other schemes in XML format (Rinehart
et al. 2004). The benthic mapping methods may potentially be applied to other study
sites around American Samoa and to coral reef ecosystems across the Pacific and in the
Caribbean. The application of the methods to extend shallow water classifications will be
fairly easy with the use of the BTM. Conclusion The study was a success in reaching its goals: (1) methods were developed for benthic
mapping and applied to three sites around American Samoa, (2) a new classification scheme
was developed introducing the concepts of BPI zones at a broad resolution (depressions,
slopes, flats, crests) and structures (finer features within zones) around the study sites
and supplemented by measures of rugosity, where complex features may be hosting high
biodiversity; and (3) visual survey information was used as initial validation for the resulting
classifications. Bathymetry, BPI, slope, and rugosity were combined with spatial analysis to develop
methods for creating a classification for deep water (>30 m) benthic zones and rugosity
around American Samoa. The methods were based on components of studies that classified
shallow water coral reef systems, terrestrial landforms, but also the satellite-based (Ikonos)
classification of NOAA Biogeography for Pacific islands. From these shallow water Benthic Terrain Classification for American Samoa 109 classifications, only the zones, at a macro habitat level (Greene et al. 1999) were suitable
for extension to deep water sites. The methods used for the deep water benthic zone
and rugosity classifications around American Samoa extend the classifications for shallow
waters around the territory. As American Samoa is an archipelago of mostly submerged volcanoes, its shoreline is
flanked by fringing reefs that plunge into deep water. This dramatic topography, combined
with a tropical climate, creates a complex coral reef ecosystem that supports thousands
of species. BPI zones, structures and rugosity provide a framework for planning scientific
surveys that will give a better understanding of species-habitat relationships and possibly
for establishing and monitoring marine protected areas. Future studies include the work of
NOAA CRED in interpreting additional extensive towed video footage around Tutuila, as
well as Saipan, to further validate our classification scheme and assess its utility to other
islands beyond Samoa, and the analysis of Pisces V submersible dive videography just
collected in July 2005 on Hawaii Undersea Reserch Lab cruise KOK0510. As the results
become available, they will provide a tool for statistical analysis along video transects
and for areas interpolated between them. The statistical results may help to define a more
automated process for using bathymetric derivatives (e.g., BPI zones, structures, rugosity,
texture) to create habitat classifications. Conclusion The classifications resulting from the methods in this study, when combined with
associated marine life information, are tools for designing management programs for the
Fagatele Bay National Marine Sanctuary, the National Park of American Samoa, and other
marine reserves in the territory. They are a baseline of information for policy makers and
managers to establish a wider and more effective network of marine protection throughout
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ESRI User Conference. San Diego, CA, August 9–13. Paper 1433. Rosenfield, G. H., K. Fitzpatrick-Lins, and H. S. Lingm. 1982. Sampling for the thematic ma
accuracy testing. Photogram. Engi. and Rem. Sens. 48:131–137. Schmal, G. P., E. L. Hickerson, D. C. Weaver, and J. V. Gardner. 2003. High-resolution multibeam
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International User Conference., San Diego, CA July 9–13. Wessel, P., and W. H. Smith. 1991. Free software helps map and display data. EOS, Transactions
American Geophysical Union 72:293–305. White, W. H., A. R. Harborne, I. S. Sotheran, R. Walton, and R. L. Foster-Smith. 2003. Using an
acoustic ground discrimination system to map coral reef benthic classes. Int. J. Rem. Sens. 24(13):2641–2660. Wright, D. 2002. Mapping the seafloor in American Samoa. Geospatial Solutions 12(7):24–25. W i h
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Undersea Research Submersible Science Program, Hawaii Undersea Research Lab, Honolulu,
HI, http://dusk.geo.orst.edu/djl/Samoa/hurl/k.kO510cruise report.pdf Accessed 5/14/06. Wright, D. J. and D. J. Bartlett, eds. 2000. Marine and coastal geographical information systems
London: Taylor & Francis. Wright, D. J., R. Wood, and B. Sylvander. 1998. ArcGMT: A suite of tools for conversion between
Arc/INFO and Generic Mapping Tools (GMT). Comp. and Geosci. 24(8):737–744. Wright, D. J., B. T. Donahue, and D. F. Naar. 2002. Seafloor mapping and GIS coordination at
America’s remotest national marine sanctuary (American Samoa). Pp. 33–63. in Dawn J. Wright
(ed.), Undersea with GIS. Redlands, CA: ESRI Press. Zajac, R. N., R. S. Lewis, L. J. Poppe, D. C. Twichell, J. Vozarik, and M. L. DiGiacomo-Cohen. 2003. Responses of infaunal populations to benthoscape structure and potential importance of
transition zones. Limnol. Oceanogr. 48(2):829–842. Zhou, X., and Y. Chen. 2005. Seafloor classification of multibeam sonar data using neural network
approach. Mar. Geod. 28:201–206.
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44. A'Kikuyu Fairy Tales (Rogano)
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57. A'Kikuyu Fairy Tales. (Rogano.)
Author(s): W. E. H. Barrett
Source: Man, Vol. 12 (1912), pp. 112-114
Published by: Royal Anthropological Institute of Great Britain and Ireland
Stable URL: http://www.jstor.org/stable/2788031
Accessed: 27-06-2016 08:15 UTC Wiley, Royal Anthropological Institute of Great Britain and Ireland are collaborating with
JSTOR to digitize, preserve and extend access to Man Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at
http://about.jstor.org/terms 57. A'Kikuyu Fairy Tales. (Rogano.)
Author(s): W. E. H. Barrett
Source: Man, Vol. 12 (1912), pp. 112-114
Published by: Royal Anthropological Institute of Great Britain and Ireland
Stable URL: http://www.jstor.org/stable/2788031
Accessed: 27-06-2016 08:15 UTC Your use of the JSTOR archive indicates your acceptance of the Terms & Conditions of Use, available at
h
// b
j
/ JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted
digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about
JSTOR, please contact support@jstor.org. Wiley, Royal Anthropological Institute of Great Britain and Ireland are collaborating with
JSTOR to digitize, preserve and extend access to Man Wiley, Royal Anthropological Institute of Great Britain and Ireland are collaborating with
JSTOR to digitize, preserve and extend access to Man This content downloaded from 128.163.2.206 on Mon, 27 Jun 2016 08:15:29 UTC
All use subject to http://about.jstor.org/terms This content downloaded from 128.163.2.206 on Mon, 27 Jun 2016 08:15:29 UTC
All use subject to http://about.jstor.org/terms 57. 19 AN the head crept softly up to his hut, pushed the door open, and having entered killed
him, and cutting out his liver and kidneys took them off with her into the forest
close by, where she lit a fire and cooked them over it. When they were well done
she took them to the hut of his mother, and leaving them lying inside near the
fire stones, betook herself off to her hiding place. The next morning the old woman woke early, and seeing some well-cooked liver
and kidneys lying in the hut ghe ate them; thinkinig that her soII, during the niglht,
had placed them there as a present for her. Shortly afterwards she heard shrieks proceeding from her son's hut, and pro-
ceeding there found her son's sweetheart (muiretu) wringinig her hands with grief,
and several other people assembled outside Entering, she found her son's dead body
lying on his bed. Everyone discussed this strange occurrence, but none could say
how it had happened. Some said God had killed him; and others, that a man had
donie the deed. As the warrior was dead, his body was taken out and thrown into
the bush. That night the head, who had been hiding in the forest, took the corpse,
and carrying it back to the hut laid it on the bed. The next morning some men
passing by, noticing that the door of this hut was open, looked in, and to their
astonishment they saw the body of the dead warrior lying inside. Everyone in the
village tried to solve the problem as to how the corpse had come to be laid in
the hut from which they had carried it the previous day, but no one was able to say
how it had returned. They again took it and threw it into the forest. That night
several warriors kept watch near the hut, determined to find out what was happenitng. Towards midnight they heard a voice singing the following song in the Masai
tongue:- "I am tired of carrying this dead man. He has not given me water, food,
blood, or milk. "I am tired of carrying this dead man. He has not given me water, food,
blood, or milk. I killed him and left him on his bed. His mother and father have thrown him away. fey. y. m, U gy
y
New Grange (Brugh na Boinne) and other Inoised Tumuli in Ireland. By George Coffey, M.R.I.A., Keeper of Irish Antiquities, National Museum, U
Dublin. Published by Hodges, Figgis, & Co., Dublin, and Williams and Norgate,
London. Royal 8vo, pp. xii + 118 with 9 plates and 95 illustrations. Price,
bound in cloth, 6s. net. This work should prove most welcome and useful to all students of Irish archoeology. It will greatly assist in arriving at an understanding of the Bronze Age culture in
Ireland and the various ornamental motives which radiated to Ireland from the Continent
and vice versa'. There has been no authoritative work dealing with New Grange
available of recent years, as this author's original Memoir, published nearly twenty
years ago, has long been out of print. Very considerable additions to our knowledge of
the archwology of the Mediterranean lands and the cultural waves which extended
from them to the west have been made during the last decade, and the author has
been led to adopt a much earlier date as the probable time when New Grange was
erected than was suggested in his previous stndy. Owing to the early plundering of
the Boyne tumuli and the consequent absence of any finds that would help in dating
them (it is not even certain what the method of interment was, though it was probably
by incineration), the incised ornament and the style of the building are the only
guides to arriving at the ages of the tombs. After giving a most detailed architectural
account of New Grange and Dowth with full descriptions of all the inscribed stones,
Mr. Coffey concentrates his attention on this point. The most vital matter in this
connection is, of course, the spiral ornament so frequent at New Grange, and in a
chapter entitled "Probable Source and Origin of the Markings" the association of the
spiral and the lozenge and the degradation of the former to conceutric circles are
dealt with. Of recent years the discovery of well-developed spiral ornament of
neolithic date in the Balkan States has caused archaeologists to discard the view
formerly so widely held, that the spiral originated in Egypt and to substitute
Europe as the starting point of this decoration. Mr. Coffey is, however, not quite
prepared to go as far as this. 57. Why do I not leave him to be eaten by the hyenas? Never mind, I will take him and leave him in his hut." They then saw the head of a Masai woman come out of the forest and enter the
hut, carrying the dead warrior's corpse with her. The warriors silently approached
the hut, and looking through the door saw her place the corpse on the bed; when
she had done this she came out and was at once seized by them. They cried out
to her, " Now you shall die," and started pulling at her ear ornaments. On this she
became very angry, and told them to kill her if they wished to do so, but that she
would not tolerate them pulling her ornaments about. Just at this moment the
deceased warrior's mother arrived on the scene and commenced weeping bitterly. Seeing her, the head laughed at her and said, "Why do you weep, you who have
" eaten your son's liver and kidneys ? " Hearing this the wretched woman wept
more than ever, but some of those standing near laughed. The head then said to
them, "Bring me a present of cattle and all will be well." Two warriors at once
went and brought her a number of cattle as a present; on the receipt of which she
entered the dead man's hut, applied medicine to his wounds, and then sewed them
up. In a short time her medicine had the desired effect, and the warrior came to life. The pair of them then came out, the latter as strong and healthy as he had
ever been. The head then asked his father and mother why they had taken him out into
the forest and then wept. She said, it was evident that as they had left him as
food for the hyeenas, they had not much affection for him. She added, "I have
" brought your son to life, and if you or any of the others mention a word of what
" has happened to a single soul I will punish you all, and in future when you are will punish y
L 113 3 will punish y
L 113 3 This content downloaded from 128.163.2.206 on Mon, 27 Jun 2016 08:15:29 UTC
All use subject to http://about.jstor.org/terms .] M Nos. 2* I2* " told not to mention a certain thing to anyone take care that you do not do so."
Having said these words, she herded up her cattle and drove them off. She has not
again made her appearance amougst the A'Kikuyu. W. E. H. BARRETT. This content downloaded from 128.163.2.206 on Mon, 27 Jun 2016 08:15:29 UTC
All use subject to http://about.jstor.org/terms fey. However, where the exact starting place of the spiral
ornament is to be eventually placed is not of vital importance in the present connection,
as it is generally admitted that the New Grange spirals are derived from the Medi-
terranean, and the important question as far as Ireland is concerned is the route by
which they travelled. While admitting that the spiral may have reached the north
by the sea route round Spain, the author, pointing out that the Irish spiral forms
incline to the Scandinavian (and in this connection the ship markings at New
Grange and Dowth are of great interest), considers the spiral followed the Elbe
route, reaching Scandinavia first and then coming to Ireland. The spirals at Gavr'inis
are later than the Irish examples. Mr. Coffey, however, says he does not wish to
insist on this point, which would not make any very considerable difference in date. The chapter on Lougherew contains several new illustrations and some very
suggestive observations with regard to the probable religious significance of many
of the markings. The incised markings on the smaller tumuli in counties Tyrone
and Sligo are treated, and a warning note is sounded against trying to connect the
New Grange spirals with those of the La Tiene period. The chapter on Lougherew contains several new illustrations and some very
suggestive observations with regard to the probable religious significance of many
of the markings. The incised markings on the smaller tumuli in counties Tyrone
and Sligo are treated, and a warning note is sounded against trying to connect the
New Grange spirals with those of the La Tiene period. g
p
p
The numerous illustrations are a feature of the book, those of the inscribed stones
being made in most cases from photographs taken from casts of the stones. The otographs t
[ 114 ]. This content downloaded from 128.163.2.206 on Mon, 27 Jun 2016 08:15:29 UTC
All use subject to http://about.jstor.org/terms
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https://zenodo.org/records/8286801/files/75.pdf
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English
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Institutional Development Management in Boarding Schools (Case Study at the Al-Mawarid Featured Dormitory at Tahfidzul Qur'an Islamic Boarding School Nurul Falah Poncol Magetan)
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Zenodo (CERN European Organization for Nuclear Research)
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I. INTRODUCTION I. INTRODUCTION
Islamic boarding school is one of the oldest educational institutions that has been involved in the history of the Indonesian people
for hundreds of years. As an Islamic educational institution, the existence of Islamic boarding schools can be categorized as a unique
institution and has its own distinctive characteristics (Rahim, 2002). Islamic boarding schools are referred to as educational
institutions that are not only identifiable with Islam but also contain the meaning of Indonesian or Indigenous origins that arise and
develop in close connection with the surrounding community. As the times develop, the problems faced by Islamic boarding schools
are increasingly complex. The problems that boarding schools face are caused by modern life. And the ability of the boarding school
to answer these problems can be used as a benchmark for how far it can be modernized. If they are able to answer these problems,
they will be qualified as modern institutions. And vice versa, if it is unable to respond to modern life, then the qualifications given
are often things that show outdated characteristics, such as old-fashioned and conservative (Majid, 2009). In improving the education system not only we need improvements in teachers and educational facilities, but there must be
improvements in management in the field of education. In Islamic educational institutions there is often a concern to always preserve
the salaf tradition or take a modern system that is in accordance with the demands of the times. Islamic educational institutions are
required to design educational systems and models that are in accordance with modern education but also maintain the salaf tradition. kyai in Indonesia have made many efforts to overcome the backwardness of education in Islamic boarding schools, one of which is
to establish Formal schools so that the quality of santri can still compete in the general public (Moh. Mahfud and Artamin Hairit,
2017). Islamic boarding school-based education is carried out by combining physical, intellectual and mental education (Hastasari Islamic boarding school is one of the oldest educational institutions that has been involved in the history of the Indonesian people
for hundreds of years. As an Islamic educational institution, the existence of Islamic boarding schools can be categorized as a unique
institution and has its own distinctive characteristics (Rahim, 2002). International Journal of Social Science And Human Research
ISSN (print): 2644-0679, ISSN (online): 2644-0695
Volume 06 Issue 08 August 2023
DOI: 10.47191/ijsshr/v6-i8-75, Impact factor- 6.686
Page No: 5185-5189 International Journal of Social Science And Human Research
ISSN (print): 2644-0679, ISSN (online): 2644-0695
Volume 06 Issue 08 August 2023
DOI: 10.47191/ijsshr/v6-i8-75, Impact factor- 6.686
Page No: 5185-5189 International Journal of Social Science A
ISSN (print): 2644-0679, ISSN (online): 2644-0695
Volume 06 Issue 08 August 2023
DOI: 10.47191/ijsshr/v6-i8-75, Impact factor- 6.686
Page No: 5185-5189 Institutional Development Management in Boarding Schools
(Case Study at the Al-Mawarid Featured Dormitory at
Tahfidzul Qur'an Islamic Boarding School Nurul Falah
Poncol Magetan) Mufti Hanafi1, Adi Pradana2, Miftahul Huda3, Nahidl Diyan Ilahiy4 Mufti Hanafi1, Adi Pradana2, Miftahul Huda3, Nahidl Diyan Ilahiy4
1,2,3 Master of Islamic Education Management, Institut Agama Islam Negeri Ponorogo, East Java,
Indonesia y
y
1,2,3 Master of Islamic Education Management, Institut Agama Islam Negeri Ponorogo, East Java
Indonesia 4 Bachelor of English Education, Institut Agama Islam Negeri Ponorogo, East Java, Indonesia ABSTRACT: The background of this research is the existence of the Al-Mawarid Excellent Dormitory program at the Tahfidzul
Qur'an Nurul Falah Islamic Boarding School, as a form of developing dormitories that already exist in the institution. At the Al-
Mawarid Featured Dormitory there are excellent programs and hostel facilities that are representative and only accommodate
students who focus on memorizing the Quran. This field research is using qualitative as the approach. The techniques used to gather
the data are through interviews, observations and documentations. The purpose of this study was to find the implementation of
institutional development in the Al-Mawarid Excellence Dormitory at Tahfidzul Qur'an Islamic Boarding School Nurul Falah
Poncol Magetan. Weaknesses and advantages of implementing institutional development in the Al-Mawarid Excellent Dormitory
and the Implications of institutional development in the Al-Mawarid Excellent Dormitory for the recitation of the students. The
findings obtained from this study are that in implementing institutional development at the Al-Mawarid Excellent Dormitory, the
management function of POAC (Planning, Organizing, Actuating, Controlling) or planning (objectives, teaching staff and costs) is
organizing (halaqoh distribution according to students' memorization, extracurricular according to the talents and interests of the
students) implementation (formal, non-formal and extracurricular learning) evaluation (quarterly meetings between administrators
and the boardss and tests for increasing students' memorization ratings). The management of institutional development in the Al-
Mawarid Featured hostel lacks social community and the advantages are 24-hour mentoring assistance and representative and
complete dormitories. The implications of institutional development at the AlMawarid Featured Dormitory are the achievement of
memorization of at least 5 juz 1 year, student achievements in academic and non-academic as well as competitions at the district
level and costs that are more expensive at this excellent Dormitory because of the facilities and programs implemented. KEYWORDS: Development Management, Institutions, Islamic Boarding Schools II.
METHOD In this study, researchers used a qualitative research approach. Qualitative research is a research procedure that produces data in the
form of words and behavior observed by researchers (Ghony, 2012). The type of research used in this qualitative research is a case
study. Case study is to analyze a phenomenon that occurs in an institution or organization to find meaning, understanding and
investigate the process of activities carried out (Prastowo, 2011). The data in this study consisted of two types of data, namely data
in the form of words obtained from informants and data in the form of activities obtained from observations (J. Moleong, 2006). Often referred to as from two sources, namely primary and secondary sources. Primary data sources are collected through in-depth
interviews with several informants related to institutional development management at Islamic boarding school Tahfidzul Qur'an
Nurul falah Poncol Magetan and through observations of the activities of the Featured Dormitory Al-Mawarid PPTQ Nurul Falah
Poncol Magetan. The secondary data sources come from data that already exists in the Excellent Dormitory Al-Mawarid PPTQ
Nurul Falah Poncol Magetan, namely in the form of profiles, achievement data, certificate documents and others. Data collection techniques are carried out using interviews where qualitative research is used to obtain data from informants through
the action of social interaction between researchers and the problem under study (Edi, 2016). The second technique is observation,
which is one of the main data collection methods in qualitative research. Observation means to watch and observe. Observation is
systematically carried out on symptoms that appear on the object of research (Sugiyono, 2009). The last data collection technique
is documentation which can be in the form of writings, audio visuals, pictures and momental works of a person. This documentation
method is a data collection method used to trace historical data (Marwadani, 2020). The data obtained through the three techniques were then evaluated inductively using a cyclical model, as proposed by Miles,
Huberman and Saldana (M.B Milles, A.M Huberman, 2014), namely data collection, data condensation, data display and
conclusion drawing. In this study, researchers used a qualitative research approach. Qualitative research is a research procedure that produces data in the
form of words and behavior observed by researchers (Ghony, 2012). The type of research used in this qualitative research is a case
study. Institutional Development Management in Boarding Schools (Case Study at the Al-Mawarid Featured Dormitory at
Tahfidzul Qur'an Islamic Boarding School Nurul Falah Poncol Magetan) et al., 2022). Educational activities must teach students related to life skills in accordance with the environment and the needs of
learners (Zaki et al., 2020). There are many Islamic boarding schools today with various backgrounds, but one of the main goals is
to portray Islamic boarding schools as role models in instilling good morals, social attitudes and noble character in the hope of
producing graduates who are competent and have excellent character (Hasanah, 2021). p
g g
p
(
)
There are five basic elements that must exist in the tradition of a traditional or classical Islamic boarding school. First, the hut or
dormitory where the students live is a distinctive feature of the Islamic boarding school tradition that distinguishes it from the
traditional education system in mosques that developed in Islamic areas in other countries. For example, in Afghanistan the students
live in the mosque. Secondly, the mosque is an important element of an Islamic boarding school, the most appropriate place to
educate students, especially in the practice of five daily congregational prayers, khutbah and Friday prayers, and the teaching of
classical Islamic books. Third, the teaching of classical books in Islamic boarding schools is usually classified into eight groups of
knowledge types: 1. Nahwu and sorof 2. Fiqh 3. Ushul Fiqh 4. Hadist 5. Tafsir 6. Tauhid 7. Tasawuf 8. Tarikh dan Balaghah. Fourth, there are 2 types of santri, namely santri mukim who live permanently in the boarding school and santri kalong who follow
the recitation at the Islamic boarding school but go back and forth from their own homes. Fifth, the kyai is the most essential element
of an Islamic boarding school. The development of an Islamic boarding school is highly dependent on the ability of a kyai (Dhofier,
2011). In addition, at the Islamic boarding school the first thing that must be instilled is related to morals, because it is closely related
to the realization of peace in society (Zakaria et al., 2022). By following the current era, the facilities or infrastructure of Islamic
boarding schools are increasingly advanced and developed, even those that used to be only non-formal education that only studied
classical books, now many Islamic boarding schools have established formal education ranging from elementary school to higher
education. This is in accordance with what was conveyed by Fathurrochman et al. written by Irwan Fathurrochman et al. Institutional Development Management in Boarding Schools (Case Study at the Al-Mawarid Featured Dormitory at
Tahfidzul Qur'an Islamic Boarding School Nurul Falah Poncol Magetan) that the
education system has shifted from a classical to a non-classical system, which is characterized by the entry of an Islamic boarding
school madrasah system that has various levels of education (Fathurrochman et al., 2021). Islamic boarding school Tahfidzul Qur'an Nurul Falah Poncol Magetan East Java founded by KH. Marhaban al-Hafidz which was
inaugurated by KH. Nawawi Abdul Aziz al-Hafidz founder of Islamic boarding school An-Nur Ngrukem Bantul Jogjakarta in 2006
M / 1427 H. And now there are 442 students consisting of 212 female students in the main dormitory 164 male students in the main
dormitory and 66 female students in the Al- Mawarid Dormitory. Pondok Islamic boarding school Nurul Falah is classified as a
mixed type Islamic boarding school because it still maintains a traditional system by still studying classic scriptures and adopting a
modern system by managing formal education, while still following the guidelines of al-muhafadhah 'ala al-Qadim al-Sholih wa
al-Ahdzu bi al-Jadid al-Ashlah maintaining good old things and taking new things that are better (Efendi, 2014). I. INTRODUCTION Islamic boarding schools are referred to as educational
institutions that are not only identifiable with Islam but also contain the meaning of Indonesian or Indigenous origins that arise and
develop in close connection with the surrounding community. As the times develop, the problems faced by Islamic boarding schools
are increasingly complex. The problems that boarding schools face are caused by modern life. And the ability of the boarding school
to answer these problems can be used as a benchmark for how far it can be modernized. If they are able to answer these problems,
they will be qualified as modern institutions. And vice versa, if it is unable to respond to modern life, then the qualifications given
are often things that show outdated characteristics, such as old-fashioned and conservative (Majid, 2009). In improving the education system not only we need improvements in teachers and educational facilities, but there must be
improvements in management in the field of education. In Islamic educational institutions there is often a concern to always preserve
the salaf tradition or take a modern system that is in accordance with the demands of the times. Islamic educational institutions are
required to design educational systems and models that are in accordance with modern education but also maintain the salaf tradition. kyai in Indonesia have made many efforts to overcome the backwardness of education in Islamic boarding schools, one of which is
to establish Formal schools so that the quality of santri can still compete in the general public (Moh. Mahfud and Artamin Hairit,
2017). Islamic boarding school-based education is carried out by combining physical, intellectual and mental education (Hastasari IJSSHR, Volume 06 Issue 08 August 2023 Page 5185 www.ijsshr.in Qur'an Nurul Falah Poncol Magetan Implementation of Institutional Development in the Featured Dormitory Al-Mawarid Pondok Islamic boarding school Tahfidzul
Qur'an Poncol Magetan in review of POAC management theory Planning, Organizing, Actuating and Evaluation according to
George R. Tery (Anwar, 2017). In terms of planning, KH. Marhaban Al-Hafidz as the the boards of Pesantren Tahfidzul Qur'an
Nurul Falah Poncol Magetan explained that institutional development in the Al-Mawarid Dormitory of Pesantren Tahfidzul Qur'an
Nurul Falah was inspired by the Islamic Boarding School Yanbu'ul Qur'an 2 Muria Kudus. So that many of the programs
implemented at the Al-Mawarid Dormitory adopted from the Yanbu'ul Qur'an 2 Muria Kudus Tahfidzul Qur'an Islamic Boarding
School. In terms of Organizing in Al-Mawarid Excellent Dormitory, the the boardss select competent senior students to become
administrators in Al-Mawarid Excellent Dormitory. In addition, in the recruitment of new students who enter the Al-Mawarid In terms of Organizing in Al-Mawarid Excellent Dormitory, the the boardss select competent senior students to become
administrators in Al-Mawarid Excellent Dormitory. In addition, in the recruitment of new students who enter the Al-Mawarid
Dormitory, a fairly strict selection is also carried out. Starting from the Psychology test and reading and writing the Quran and all In terms of Organizing in Al-Mawarid Excellent Dormitory, the the boardss select competent senior students to become
administrators in Al-Mawarid Excellent Dormitory. In addition, in the recruitment of new students who enter the Al-Mawarid
Dormitory, a fairly strict selection is also carried out. Starting from the Psychology test and reading and writing the Quran and all
students who enter the Al-Mawarid Dormitory are Marhalah Tahfidz. So as to create a conducive environmental atmosphere for
memorizing the Qur'an, this is in accordance with what was conveyed by Fia Faiqotul Muna as the Dormitory Supervisor of Al-
Mawarid. Dormitory, a fairly strict selection is also carried out. Starting from the Psychology test and reading and writing the Quran and all
students who enter the Al-Mawarid Dormitory are Marhalah Tahfidz. So as to create a conducive environmental atmosphere for
memorizing the Qur'an, this is in accordance with what was conveyed by Fia Faiqotul Muna as the Dormitory Supervisor of Al-
Mawarid. In terms of Actuating, teaching and learning activities and other Islamic activities are accompanied by female islamic teachers who
are competent in their fields. So, there is no male islamic teacher who teaches at Al-Mawarid Dormitory. II.
METHOD Case study is to analyze a phenomenon that occurs in an institution or organization to find meaning, understanding and
investigate the process of activities carried out (Prastowo, 2011). The data in this study consisted of two types of data, namely data
in the form of words obtained from informants and data in the form of activities obtained from observations (J. Moleong, 2006). Often referred to as from two sources, namely primary and secondary sources. Primary data sources are collected through in-depth
interviews with several informants related to institutional development management at Islamic boarding school Tahfidzul Qur'an
Nurul falah Poncol Magetan and through observations of the activities of the Featured Dormitory Al-Mawarid PPTQ Nurul Falah
Poncol Magetan. The secondary data sources come from data that already exists in the Excellent Dormitory Al-Mawarid PPTQ
Nurul Falah Poncol Magetan, namely in the form of profiles, achievement data, certificate documents and others. Data collection techniques are carried out using interviews where qualitative research is used to obtain data from informants through
the action of social interaction between researchers and the problem under study (Edi, 2016). The second technique is observation,
which is one of the main data collection methods in qualitative research. Observation means to watch and observe. Observation is
systematically carried out on symptoms that appear on the object of research (Sugiyono, 2009). The last data collection technique
is documentation which can be in the form of writings, audio visuals, pictures and momental works of a person. This documentation
method is a data collection method used to trace historical data (Marwadani, 2020). The data obtained through the three techniques were then evaluated inductively using a cyclical model, as proposed by Miles,
Huberman and Saldana (M.B Milles, A.M Huberman, 2014), namely data collection, data condensation, data display and
conclusion drawing IJSSHR, Volume 06 Issue 08 August 2023 Page 5186 Page 5186 www.ijsshr.in Institutional Development Management in Boarding Schools (Case Study at the Al-Mawarid Featured Dormitory at
Tahfidzul Qur'an Islamic Boarding School Nurul Falah Poncol Magetan) Institutional Development Management in Boarding Schools (Case Study at the Al-Mawarid Featured Dormitory at
Tahfidzul Qur'an Islamic Boarding School Nurul Falah Poncol Magetan) Qur'an Nurul Falah Poncol Magetan In addition, the
infrastructure in the Al-Mawarid Dormitory is equipped with a canteen, kitchen, medical room, etc. So that students of the Al-
Mawarid Dormitory do not need to leave the dormitory, all the needs of the students have been met in the dormitory. In the
implementation of memorization activities in the Al-Mawarid Dormitory, more use the Sima'i method, namely the teacher recites
the verse to be memorized then the students imitate several times until memorization, also known as guidance in the Al-Mawarid
Dormitory. and using the jama method by reciting together the verses that have been memorized together, also known as deresan. One of the things that makes the environment conducive is that in the excellent dormitory Al-Mawarid all students immediately
enter the marhalah tahfidz so that it is easy to make a halaqoh or memorization group and be accompanied by a mentor. In addition,
according to Dzarrotul Muwafiroh Irsyadi as the Head of the Featured Dormitory of Al-Mawarid, Islamic boarding school Tahfidzul
Qur'an Nurul Falah Poncol Magetan, the Talaqqi method with one-on-one report to the the boards is also still applied in the Featured
Dormitory of Al-Mawarid. In terms of Evaluation delivered by Nafi' Sa'adatul K. as the Secretary of the Al-Mawarid Dormitory of the Nurul Falah Poncol
Magetan Islamic Boarding School, there is a quarterly meeting agenda (once every 3 months) between the Management and the
board of directors. As well as evaluating the achievement of the students' memorization every semester there is a memorization test
of 2 Juz and a half, so the target is to memorize 5 Juz in 1 year. 2. The disadvantages and advantages of institutional development in the Al-Mawarid Dormitory of the Islamic boarding
school Tahfidzul Qur'an Nurul Falah Poncol Magetan. 3. Implications of institutional development at Al-Mawarid Dormitory Islamic boar The implications of institutional development in the Excellent Dormitory of Al-Mawarid Islamic boarding school Tahfidzul Qur'an
Nurul Falah Poncol Magetan explained by Naqiya Alma Humaira as the Activity Section of the The implications of institutional
development in the Excellent Dormitory of Al-Mawarid Islamic boarding school Tahfidzul Qur'an Nurul Falah Poncol Magetan
explained by Naqiya Alma Humaira as the Activity Section of the Excellent Dormitory of Al-Mawarid include, The students of the
Excellent Dormitory of Al-Mawarid become the delegation of the district-level competitions both in non-formal education between
Islamic boarding schools and formal education at the Madrasah Tsanawiyah level and have gained many achievements. The implications of institutional development in the Excellent Dormitory of Al-Mawarid Islamic boarding school Tahfidzul Qur'an
Nurul Falah Poncol Magetan explained by Naqiya Alma Humaira as the Activity Section of the The implications of institutional
development in the Excellent Dormitory of Al-Mawarid Islamic boarding school Tahfidzul Qur'an Nurul Falah Poncol Magetan
explained by Naqiya Alma Humaira as the Activity Section of the Excellent Dormitory of Al-Mawarid include, The students of the
Excellent Dormitory of Al-Mawarid become the delegation of the district-level competitions both in non-formal education between
Islamic boarding schools and formal education at the Madrasah Tsanawiyah level and have gained many achievements. In the internal in Al-Mawarid Dormitory itself, it has an impact on achieving the predetermined target of 5 juz every year as a
condition for class advancement, there are even some students who are able to exceed the target, namely being able to pass the 5
juz test for 7th grade students at the beginning of semester 2 and the 10 juz test for 8th grade students at the beginning of semester
2, this is in accordance with what was conveyed by Fia Faiqotul Muna as the Supervisor of Al-Mawarid Dormitory. However,
according to KH. Marhaban Al-Hafidz as the the boards of the Islamic boarding school there is another impact of institutional
development in the Al-Mawarid Dormitory, namely the cost which is considerably more expensive than the main dormitory of the
Islamic boarding school Tahfidzul Qur'an Nurul Falah Poncol Magetan. 3. Implications of institutional development at Al-Mawarid Dormitory Islamic boar In the external aspect, the increasing interest of the community with the existing programs in the Islamic boarding school Tahfidzul
Qur'an Nurul Falah Poncol Magetan can be an option to bring their sons and daughters to the Islamic boarding school Tahfidzul
Qur'an Nurul Falah Poncol Magetan in accordance with the desired program, this is in line with what was conveyed by Umar al-
Faruq as the Guardian of the Excellent Dormitory Students Al-Mawarid. Another impact felt by Hamim Tohari as the guardian of
the students of the Excellent Dormitory Al-Mawarid is that the children are too pampered so that they are less independent when at
home. 3. Implications of institutional development at Al-Mawarid Dormitory Islamic boar Falah Poncol Magetan
Institutional development implemented in the excellent dormitory Al-Mawarid Islamic boarding school Tahfidzul Qur'an Nurul
Falah Poncol Magetan has the following implications: Table II. Data analysis Implications of institutional development of the excellent dormitory Al-Mawarid Islamic boarding
school Tahfidzul Qur'an Nurul Falah. No
Implications of Institutional Development in the Excellent Dormitory Al-Mawarid Islamic
boarding school Tahfidzul Qur'an Nurul Falah Poncol Magetan
1. Achievements obtained by santri in various competitions both in Formal Education and Non-Formal
Islamic boarding schools which are followed at the Regency level. 2. Achievement of the minimum memorization target of 5 Juz in a year. There are even some who have
exceeded the target. 3. Costs are higher due to the development of facilities and programs. 4. Indulged in facilities so that children are less independent. 5. Increased public interest is due to several programs offered to attract the public. Table II. Data analysis Implications of institutional development of the excellent dormitory Al-Mawarid Islamic boarding
h
l T hfid
l Q
'
N
l F l h school Tahfidzul Qur an Nurul Falah. No
Implications of Institutional Development in the Excellent Dormitory Al-Mawarid Islamic
boarding school Tahfidzul Qur'an Nurul Falah Poncol Magetan
1. Achievements obtained by santri in various competitions both in Formal Education and Non-Formal
Islamic boarding schools which are followed at the Regency level. 2. Achievement of the minimum memorization target of 5 Juz in a year. There are even some who have
exceeded the target. 3. Costs are higher due to the development of facilities and programs. 4. Indulged in facilities so that children are less independent. 5. Increased public interest is due to several programs offered to attract the public. Institutional Development Management is a systematic effort in planning, organizing, leading and controlling the Educational
Institution with all its aspects to achieve goals effectively and efficiently in order for all components of the Islamic Education
Institution system to develop in a better, wider and perfect direction. This better and perfect indicator includes many things, including
the fields of learning, staffing, administration, facilities, finance and community participation as stakeholders (Makkin, 2016). 2. The disadvantages and advantages of institutional development in the Al-Mawarid Dormitory of the Islamic boarding
school Tahfidzul Qur'an Nurul Falah Poncol Magetan. In the implementation of institutional development in the superior dormitory Al-Mawarid Islamic boarding school Nurul Falah
Poncol Magetan there are some disadvantages and advantages faced. This can be described in the analyzed table as follows: Table I. Data Analysis of the shortcomings and advantages of institutional development at the excellent dormitory al
Mawarid
Disadvantages
1. Long time adaptation of new students
2. New students who do not feel at ease in the Islamic boarding school
3. Lack of motivation in memorizing the Quran
4. Lack of socializing with the community
Advantages
1. Pretest for new students
2. Tahfidz and Madrasah according to their fields
3. 24-hour guidance assistance
4. Representative and complete dormitory facilities the shortcomings and advantages of institutional development at the excellent dormitory al- Table I. Data Analysis of the shortcomings and advantages of institutional development at the e The purpose of developing educational institutions is one of them in the pedagogical aspect which plays an important role in
improving quality human resources from one period to another (Efendi, 2014). In its implementation, the excellent dormitory of Al-
Mawarid has implemented pedagogical aspects. Dzarrotul Muwafiroh Irsyadi as the Head of the Excellent Dormitory Al-Mawarid
Islamic Boarding School Tahfidzul Qur'an Nurul Falah Poncol Magetan stated that in this pedagogical aspect, it is one of the
advantages of the program implemented in the Excellent Dormitory Al-Mawarid by providing intensive assistance to students in
learning activities, memorizing the Quran and even extracurricular activities by making halaqoh according to the abilities and IJSSHR, Volume 06 Issue 08 August 2023 Page 5187 www.ijsshr.in Institutional Development Management in Boarding Schools (Case Study at the Al-Mawarid Featured Dormitory at
Tahfidzul Qur'an Islamic Boarding School Nurul Falah Poncol Magetan) achievements of the students. In addition, Aisya Indira Darmayu as a 7th grade student of Al-Mawarid Dormitory explained that
another advantage is in the aspect of complete facilities and representative dormitories so that the needs of the students have been
met in the Al-Mawarid Dormitory. This also makes it easier for the management to condition and discipline the students who live
in the Al-Mawarid Dormitory as stated by Nurul Hidayah as the Security Section of the Al-Mawarid Dormitory. achievements of the students. In addition, Aisya Indira Darmayu as a 7th grade student of Al-Mawarid Dormitory explained that
another advantage is in the aspect of complete facilities and representative dormitories so that the needs of the students have been
met in the Al-Mawarid Dormitory. This also makes it easier for the management to condition and discipline the students who live
in the Al-Mawarid Dormitory as stated by Nurul Hidayah as the Security Section of the Al-Mawarid Dormitory. The shortcomings of the Al-Mawarid Dormitory program according to Fia Faiqotul Muna as the Supervisor of the Al-Mawarid
Dormitory include that it requires a long adaptation especially for new students because of the density of activities in the Al-Mawarid
Dormitory. In addition, Lailatul Mualifah as the teacher of the Al-Mawarid Dormitory also believes that the lack of social interaction
of the students of the Al-Mawarid Dormitory with the main dormitory of the Islamic boarding school Tahfidzul Qur'an Nurul Falah
and with the surrounding community. The shortcomings of the Al-Mawarid Dormitory program according to Fia Faiqotul Muna as the Supervisor of the Al-Mawarid
Dormitory include that it requires a long adaptation especially for new students because of the density of activities in the Al-Mawarid
Dormitory. In addition, Lailatul Mualifah as the teacher of the Al-Mawarid Dormitory also believes that the lack of social interaction
of the students of the Al-Mawarid Dormitory with the main dormitory of the Islamic boarding school Tahfidzul Qur'an Nurul Falah
and with the surrounding community. 3. Implications of institutional development at Al-Mawarid Dormitory Islamic boar The implications of institutional development in the Excellent Dormitory of Al-Mawarid Islamic boarding school Tahfidzul Qur'an
Nurul Falah Poncol Magetan explained by Naqiya Alma Humaira as the Activity Section of the The implications of institutional
development in the Excellent Dormitory of Al-Mawarid Islamic boarding school Tahfidzul Qur'an Nurul Falah Poncol Magetan
explained by Naqiya Alma Humaira as the Activity Section of the Excellent Dormitory of Al-Mawarid include, The students of the
Excellent Dormitory of Al-Mawarid become the delegation of the district-level competitions both in non-formal education between
Islamic boarding schools and formal education at the Madrasah Tsanawiyah level and have gained many achievements. In the internal in Al-Mawarid Dormitory itself, it has an impact on achieving the predetermined target of 5 juz every year as a
condition for class advancement, there are even some students who are able to exceed the target, namely being able to pass the 5
juz test for 7th grade students at the beginning of semester 2 and the 10 juz test for 8th grade students at the beginning of semester
2, this is in accordance with what was conveyed by Fia Faiqotul Muna as the Supervisor of Al-Mawarid Dormitory. However,
according to KH. Marhaban Al-Hafidz as the the boards of the Islamic boarding school there is another impact of institutional
development in the Al-Mawarid Dormitory, namely the cost which is considerably more expensive than the main dormitory of the
Islamic boarding school Tahfidzul Qur'an Nurul Falah Poncol Magetan. Institutional Development Management is a systematic effort in planning, organizing, leading and controlling the Educational
Institution with all its aspects to achieve goals effectively and efficiently in order for all components of the Islamic Education
Institution system to develop in a better, wider and perfect direction. This better and perfect indicator includes many things, including
the fields of learning, staffing, administration, facilities, finance and community participation as stakeholders (Makkin, 2016). Institutional Development Management in Boarding Schools (Case Study at the Al-Mawarid Featured Dormitory at
Tahfidzul Qur'an Islamic Boarding School Nurul Falah Poncol Magetan) 2 Muria Kudus. In terms of Organizing in Al-Mawarid Dormitory, the the boardss selected competent senior students to be
administrators in Al-Mawarid Dormitory. In addition, in the recruitment of new students who enter the Al-Mawarid Dormitory, a
fairly strict selection is also conducted. Ranging from psychological tests and reading and writing the Quran and all students who
enter the Al-Mawarid Dormitory are Marhalah Tahfidz. Thereby, creating a conducive environment for the memorizers of the
Quran. In terms of Actuating, teaching and learning activities and other activities are accompanied by teachers who are competent
in their fields. In terms of Evaluation, there is an agenda for quarterly meetings between the Management and the Boards. Evaluation
of the achievement of the students' memorization every semester is held a memorization test of 2 Juz and a half so the target is 5
Juz in 1 year. The advantages in the aspect of Complete facilities and representative dormitories so that the needs of the students
are already being met in the Al-Mawarid Dormitory. This also makes it easier for the management to condition and discipline the
students who live in the Al-Mawarid Dormitory. The lack of social interaction of the students of the Al-Mawarid Dormitory with
the main dormitory of the Islamic boarding school Tahfidzul Qur'an Nurul Falah and with the surrounding community. The
implications of institutional development in the Al-Mawarid Dormitory of Pondok Islamic boarding school Tahfidzul Qur'an Nurul
Falah Poncol Magetan which are explained from various parties are, Al-Mawarid Dormitory students become the delegation of
district-level competitions both in non-formal education between Islamic boarding schools and formal education at the Madrasah
Tsanawiyah level and have received many achievements. IV. CONCLUSIONS In terms of Planning Institutional Development in the Excellent Dormitory Al-Mawarid Islamic boarding school Tahfidzul Qur'an
Nurul Falah was inspired by the Islamic boarding school Tahfidzul Qur'an Yanbu'ul Qur'an 2 Muria Kudus. Therefore, many of the
programs implemented in the Al-Mawarid Dormitory adopted from the Islamic boarding school Tahfidzul Qur'an Yanbu'ul Qur'an IJSSHR, Volume 06 Issue 08 August 2023 Page 5188 www.ijsshr.in There is an Open Access article, distributed under the term of the Creative Commons
Attribution – Non Commercial 4.0 International (CC BY-NC 4.0)
(https://creativecommons.org/licenses/by-nc/4.0/), which permits remixing, adapting and
building upon the work for non-commercial use, provided the original work is properly cited. (
)
(https://creativecommons.org/licenses/by-nc/4.0/), which permits remixing, adapting and
building upon the work for non-commercial use, provided the original work is properly cited. There is an Open Access article, distributed under the term of the Creative Commons
Attribution – Non Commercial 4.0 International (CC BY-NC 4.0) REFERENCES 1) Anwar, M. A. (2017). Manajemen Kelembagaan Pondok Pesantren. Pustaka Ilmu. 1) Anwar, M. A. (2017). Manajemen Kelembagaan Pondok Pesantren. Pustaka Ilmu. 2) Dhofier, Z. (2011). Tradisi Pesantren: Studi Pandangan Hidup Kyai dan Visinya Mengenai Masa Depan Indonesia (Cet. 8
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Sebagai Upaya Pewaris Tradisi dan Menatap Tantangan Masa Depan. Teras. 5) Fathurrochman, I., Danim, S., Anwar AB, S., Kurniah, N., & Hajja Ristianti, D. (2021). Theoretical Review of the
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Ummah Islamic Boarding School. Tafkir: Interdisciplinary Journal of Islamic Education, 2(2), 139–156. https://doi.org/10.31538/tijie.v2i2.43 7) Hasanah, M. (2021). The Role of Parents in Children Memorizing the Qur’an in Middle School Based on the Amanatul
Ummah Islamic Boarding School. Tafkir: Interdisciplinary Journal of Islamic Education, 2(2), 139–156. https://doi.org/10.31538/tijie.v2i2.43 8) Hastasari, C., Setiawan, B., & Aw, S. (2022). Students’ communication patterns of islamic boarding schools: the case of
Students in Muallimin Muhammadiyah Yogyakarta. Heliyon, 8(1), e08824. https://doi.org/10.1016/j.heliyon.2022.e08824
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J Moleong L (2006) Metodologi Peneltian Kualitatif PT Remaja Rosdakarya 8) Hastasari, C., Setiawan, B., & Aw, S. (2022). Students’ communication patterns of islamic boarding schools: the case of
Students in Muallimin Muhammadiyah Yogyakarta. Heliyon, 8(1), e08824. https://doi.org/10.1016/j.heliyon.2022.e08824
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A magyar tüdőtranszplantációs program indulása és első eredményei
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A magyar tüdőtranszplantációs program
indulása és első eredményei Gieszer Balázs dr.1, 3 ■ Radeczky Péter dr.1, 3 ■ Ghimessy Áron dr.1, 3
Farkas Attila dr.1, 3 ■ Csende Kristóf dr.1 ■ Bogyó Levente dr.1, 3
Fazekas Levente dr.3, 4 ■ Kovács Nóra3 ■ Madurka Ildikó dr.2
Kocsis Ákos dr.1, 3 ■ Agócs László dr.1, 3 ■ Török Klára dr.1, 3
Bartók Tibor dr.2 ■ Dancs Tamás dr.2 ■ Schönauer Nóra dr.2
Tóth Krisztina dr.2 ■ Szabó József dr.7 ■ Eszes Noémi dr.5
Bohács Anikó dr.5 ■ Czebe Krisztina dr.9 ■ Csiszér Eszter dr.5, 10
Mihály Sándor dr.11 ■ Kovács Lajos dr.6 ■ Müller Veronika dr.5
Elek Jenő dr.2 ■ Rényi-Vámos Ferenc dr.1, 3 ■ Lang György dr.3, 8 Országos Onkológiai Intézet, 1Mellkassebészeti Osztály,
2Aneszteziológiai és Központi Intenzív Terápiás Osztály, Budapest
Semmelweis Egyetem, Általános Orvostudományi Kar, 3Mellkassebészeti Klinika,
4Városmajori Szív- és Érgyógyászati Klinika, 5Pulmonológiai Klinika,
6I. Gyermekgyógyászati Klinika, 7Transzplantációs és Sebészeti Klinika, Budapest
8Klinische Abteilung für Thoraxchirurgie, Universitätsklinik für Chirurgie, Medizinische Universität, Wien
9Deák Jenő Kórház, Légzésrehabilitációs Osztály, Tapolca
10Országos Korányi és Pulmonológiai Intézet, Budapest
11Országos Vérellátó Szolgálat, Transzplantációs Igazgatóság, Budapest 11Országos Vérellátó Szolgálat, Transzplantációs Igazgatóság, Budapest Magyarországon az első tüdőtranszplantációt 2015. 12. 12-én végeztük el az Országos Onkológiai Intézet és a Sem
melweis Egyetem együttműködésével. Cikkünkben az elmúlt két és fél év eredményeit összegezzük. 2018 augusztu
sáig 55 tüdőtranszplantációra került sor. Az adatfeldolgozást retrospektív módszerrel végeztük. A várólistára helyezés
a Tüdő Transzplantációs Bizottság javaslatára történt. A donortüdők agyhalott donorokból származtak. A posztope
ratív gondozás a Semmelweis Egyetemen folytatódott. 2015. 12. 12. és 2018. 07. 31. között 76 szervkivételen vet
tünk részt: 45 magyar, 23 Eurotransplant-, 8 Eurotransplanton kívüli országban, ezekből 54 kétoldali és 1 egyoldali
tüdőtranszplantáció valósult meg. A műtéteket egyoldali (n = 1), kétoldali thoracotomiából (n = 1) vagy ’clamshell’
betolásból (n = 53), venoarterialis extrakorporális membránoxigenizáció-támogatással végeztük. Három esetben az
extrakorporális membránoxigenizáció-támogatást a posztoperatív szakban prolongáltuk, másik két betegnél extrakor
porális membránoxigenizáció-bridge terápiát követően végeztük el a transzplantációt. Egy kombinált tüdő-vese
transzplantáció is történt. A recipiensek alapbetegsége krónikus obstruktív tüdőbetegség (n = 28); fibrotizáló tüdő
betegség (n = 8); cystás fibrosis (n = 12); elsődleges pulmonalis hypertonia (n = 2); histiocytosis-X (n = 1); bronchiec
tasia (n = 2); lymphangioleiomyomatosis (n = 1) és bronchiolitis obliterans szindróma miatti retranszplantáció (n = 1)
volt. Átlagéletkoruk 47,5 ± 15,18 év volt. A legfiatalabb beteg 13 éves volt. A várólistán 12 beteg hunyt el. A betegek
átlagosan 24,6 ± 18,18 napot töltöttek az intenzív osztályon. ÖSSZEFOGLALÓ KÖZLEMÉNY ÖSSZEFOGLALÓ KÖZLEMÉNY Kulcsszavak: tüdőtranszplantáció, magyar tüdőtranszplantációs program Unauthenticated | Downloaded 10/24/24 03:57 AM UTC The start of the Hungarian lung transplantation program and the first results The first lung transplantation in Hungary was performed on 12th of December, 2015. It was a joint effort of the
National Institute of Oncology and the Semmelweis University. Hereby we summarise the results and experiences
from the first three years. Until August, 2018, 55 lung transplantations were performed in Hungary. This was a ret
rospective analysis. All patients were listed according to the recommendation of the Lung Transplantation Commit
tee. All implanted lungs have been procured from brain dead donors. Postoperative treatment and rehabilitation of
the patients were continued at the Semmelweis University. Between 12. 12. 2015 and 31. 07. 2018, our team per
formed 76 organ retrievals: out of 45 Hungarian offers, 23 came from Eurotransplant countries and 8 outside of the
Eurotransplant region. From these donations, 54 double and 1 single side transplantations were successfully per
formed. The surgical approach was single side thoracotomy (n = 1), bilateral thoracotomy (n = 1) and in the major
ity of the cases clamshell incision (n = 53). For the intraoperative veno-arterial extracorporeal membrane oxygenation
support was used. The extracorporeal membrane oxygenation support had to be prolonged in 3 patients into the
early postoperative period, two other recipients were bridged to transplant with extracorporeal membrane oxygena
tion. In the same time period, one combined lung-kidney transplantation was also performed. The distribution of
recipients according to the underlying disease was: chronic obstructive pulmonary disease (n = 28); idiopathic pul
monary fibrosis (n = 8); cystic fibrosis (n = 12); primary pulmonary hypertension (n = 2); hystiocytosis-X (n = 1);
bronchiectasis (n = 2); lymphangioleiomyomatosis (n = 1); and re-transplantation following bronchiolitis obliterans
syndrome (n = 1), respectively. The mean age of recipients was 47.5 ± 15.18 years. The youngest recipient was 13
years old. We unfortunately lost 12 patients on our waiting list. The mean intensive care unit stay was 24.6 ± 18.18
days. Two patients were lost in the early postoperative phase. Tracheostomy was necessary in 13 cases due to the need
of prolonged ventilation. 1-year survival of the recipients was 82.96% (until 31. 07. 2018). When looking at the first
three years of the program, the case numbers elevated quickly throughout the years which is rather unique when
compared to other centres in their starting period. Perioperative mortality and morbidity is comparable with high-
volume lung transplantation centres. A magyar tüdőtranszplantációs program
indulása és első eredményei A korai posztoperatív időszakban 2 beteget vesztettünk
el. Tartós lélegeztetési igény miatt tracheostomát 13 esetben készítettünk. A betegek 1 éves túlélése 82,96% volt. A hazai tüdőtranszplantációs programban gyorsan emelkednek az esetszámok, ami más centrumok indulásához ké
pest kivételes eredmény. A szövődmények és halálozások aránya más, nagy esetszámú centrumok számainak megfelel. A jövőben a várólista bővítését, az esetszámok további növelését, és az ’ex vivo lung perfusion’ (EVLP-) rendszer
használatának bevezetését szeretnénk megvalósítani. Kulcsszavak: tüdőtranszplantáció, magyar tüdőtranszplantációs program DOI: 10.1556/650.2018.31300 ■ © Szerző(k) 2018 ■ 159. évfolyam, 46. szám ■ 1859–1868. Unauthenticated | Downloaded 10/24/24 03:57 AM UTC ÖSSZEFOGLALÓ KÖZLEMÉNY (Beérkezett: 2018. szeptember 9.; elfogadva: 2018. október 3.) (Beérkezett: 2018. szeptember 9.; elfogadva: 2018. október 3.) Országos Vérellátó Szolgálat, Szervkoordinációs Iroda; PGD =
(primary graft dysfunction) korai graftelégtelenség; PPH =
(primary pulmonary hypertension) primer pulmonalis hyper
tonia; PRA = (panel-reactive antibody) panelreaktív antitest;
PRES = (posterior reversible encephalopathy syndrome) poste
rior reverzibilis encephalopathia szindróma; SLTx = (single
lung transplantation) egyoldali tüdőtranszplantáció; TTB =
Tüdő Transzplantációs Bizottság, Tx = (transplantation)
transzplantáció; VA = (veno-arterial) venoarteriosus; VAC =
(vacuum assisted closure) vákuumasszisztált sebkezelés; VV =
venovenosus; VVT = (red blood cell concentrates) vörösvér
test-koncentrátum The start of the Hungarian lung transplantation program and the first results In the future we would like to increase the number of patients on the waiting
list, thus increasing the total number of transplantations performed, and we are also planning to implement the use
of the ex vivo lung perfusion system (EVLP) in our program. Keywords: lung transplantation, Hungarian lung transplantation program Gieszer B, Radeczky P, Ghimessy Á, Farkas A, Csende K, Bogyó L, Fazekas L, Kovács N, Madurka I, Kocsis Á,
Agócs L, Török K, Bartók T, Dancs T, Schönauer N, Tóth K, Szabó J, Eszes N, Bohács A, Czebe K, Csiszér E,
Mihály S, Kovács L, Müller V, Elek J, Rényi-Vámos F, Lang Gy. [The start of the Hungarian lung transplantation
program and the first results]. Orv Hetil. 2018; 159(46): 1859–1868. (Beérkezett: 2018. szeptember 9.; elfogadva: 2018. október 3.) Rövidítések Országos Vérellátó Szolgálat, Szervkoordinációs Iroda; PGD =
(primary graft dysfunction) korai graftelégtelenség; PPH =
(primary pulmonary hypertension) primer pulmonalis hyper
tonia; PRA = (panel-reactive antibody) panelreaktív antitest;
PRES = (posterior reversible encephalopathy syndrome) poste
rior reverzibilis encephalopathia szindróma; SLTx = (single
lung transplantation) egyoldali tüdőtranszplantáció; TTB =
Tüdő Transzplantációs Bizottság, Tx = (transplantation)
transzplantáció; VA = (veno-arterial) venoarteriosus; VAC =
(vacuum assisted closure) vákuumasszisztált sebkezelés; VV =
venovenosus; VVT = (red blood cell concentrates) vörösvér
test-koncentrátum ACT = (activated clotting time) aktivált alvadási idő; AITO =
(Anesthesiology and Intensive Care Unit) Aneszteziológiai és
Intenzív Terápiás Osztály; ATG = antithymocytaglobulin;
BLTx = (bilateral lung transplantation) kétoldali tüdőtransz
plantáció; BOS = (bronchiolitis obliterans syndrome) bronchi
olitis obliterans szindróma; CF = (cystic fibrosis) cystás fibrosis;
CIT = (cold ischemic time) hidegischaemiás idő; COPD =
(chronic obstructive pulmonary disease) krónikus obstruktív
tüdőbetegség; DBD = (donor after brain death) agyhalott do
nor; DSA = (donor-specific antibodies) donorspecifikus anti
test; ECMO = (extracorporeal membrane oxygenation) extra
korporális membránoxigenizáció; ET = Eurotransplant; EVLP
= (ex vivo lung perfusion) extrakorporális tüdőperfúzió; HLA =
humán leukocytaantigén; IPF = (idiopathic lung fibrosis) idio
pathiás tüdőfibrosis; ISHLT = (International Society for Heart
and Lung Transplantation) Nemzetközi Szív- és Tüdőtransz
plantációs Társaság; LAM = lymphangioleiomyomatosis; LAS
= Lung Allocation Score ; LuTx = (lung transplantation) tüdő
transzplantáció; OOI = (National Institute of Oncology) Or
szágos Onkológiai Intézet; OVSZ SZKI = (Hungarian Natio
nal Blood Transfusion Service, Organ Coordination Office) Az első tüdőtranszplantációt 1963-ban végezte James
Hardy, a beteg azonban immunszuppresszív szerek hiá
nyában csak 18 napig élt [1]. Az első tartósan sikeres
egyoldali tüdőtranszplantációt Joel Cooper végezte To
rontóban 1983-ban, majd 1986-ban szintén ő hajtotta
végre az első kétoldali tüdőtranszplantációt is [1, 2]. Az International Society for Heart and Lung Trans
plantation (ISHLT) legutóbb közzétett statisztikája sze 1860 2018 ■ 159. évfolyam, 46. szám ORVOSI HETILAP Unauthenticated | Downloaded 10/24/24 03:57 AM UTC ÖSSZEFOGLALÓ KÖZLEMÉNY rint 2017-ben a világon több mint 3800 tüdőtranszplan
tációt végeztek [3]. plantáció speciális igényeit kielégítő infrastrukturális be
ruházásokat. A Bécsi Tüdőtranszplantációs Centrummal továbbra
is megmaradt a kapcsolat, és ez hazánk ET-szerződésé
nek is nevesített része. Ennek értelmében bizonyos ma
gas rizikójú betegségcsoportba tartozó recipiensek (pél
dául kis súlyú gyermek, kombinált szervtranszplantáció)
elsődlegesen továbbra is Bécsben kerülnek ellátásra. Anyag és módszer Indikáció felállítása, a beteg kivizsgálása,
felkészítése és listára helyezése 2001: A transzplantált betegek gondozását 2001 ja
nuárjától centralizáltan az Országos Korányi és Pulmo
nológiai Intézet, a kelet-magyarországi betegek esetében
pedig a Debreceni Egyetem Pulmonológiai Klinikája tar
totta kézben. 2008 októberétől valamennyi új beteg
posztoperatív ellátását, majd 2010-től az összes transz
plantált beteg kontrollját a Semmelweis Egyetem Pul
monológiai Klinikája, illetve gyermekek esetében az I. Gyermekgyógyászati Klinika vette át. Amennyiben a gondozó pulmonológus úgy ítéli meg,
hogy a beteg esetében tüdőátültetés merül fel, úgy néhány
alapvizsgálat elvégzését követően (labor, légzésfunkció,
vérgázvizsgálat, mellkas-CT) a TTB ülésén a beteg bemu
tatásra kerül. A referálás az erre a célra létrehozott interne
tes felületen lehetséges. A bizottsági ülések helyszíne a
Semmelweis Egyetem Pulmonológiai Klinikája. y
gy gy
2002: A magyarországi tüdőkivételek 2002-ben kez
dődtek el az akkor létrehozott, transzplantációs centru
moktól független donorkoordinátor szervezet (Hunga
rotransplant Kht.) koordinációjával. 2007 óta a
szervriadókat országos szinten az Országos Vérellátó
Szolgálat Szervkoordinációs Irodája (OVSZ SZKI) szer
vezi. A hazai tüdőtranszplantációs program indulását
megelőzően, kétoldalú együttműködési megállapodás
keretében, az itthon kiemelt donortüdőket lehetőség
szerint magyar recipiensekbe ültették be Bécsben. 2002: A magyarországi tüdőkivételek 2002-ben kez
dődtek el az akkor létrehozott, transzplantációs centru
moktól független donorkoordinátor szervezet (Hunga
rotransplant Kht.) koordinációjával. 2007 óta a
szervriadókat országos szinten az Országos Vérellátó
Szolgálat Szervkoordinációs Irodája (OVSZ SZKI) szer
vezi. A hazai tüdőtranszplantációs program indulását
megelőzően, kétoldalú együttműködési megállapodás
keretében, az itthon kiemelt donortüdőket lehetőség
szerint magyar recipiensekbe ültették be Bécsben. j
A betegek szelekciója, kivizsgálása és végül a listára he
lyezése a szakmai bizottság javaslatára és ellenőrzése
alatt, egységes irányelvek szerint zajlik. Rövidítések Mi
után a hazai program infrastruktúrája és műtéti teamje
jelenleg még korlátozott kapacitású, a bécsi transzplantá
ció lehetősége továbbra is nyitott minden magyar beteg
számára, amennyiben ezt a hazai ellátási lehetőség korlá
tai szükségessé teszik. A Bécsi Orvostudományi Egyetem Sebészeti Kliniká
jának Mellkassebészeti Osztálya (Medizinische Universi
tät Wien, Chirurgische Universitätsklinik, Abteilung für
Thoraxchirurgie) jelenleg a világ egyik vezető tüdő
transzplantációs centruma, ahol 1989. 11. 08-án történt
meg az első tüdőátültetés [4]. Itt végezték 1996-ban az
első magyar beteg tüdőtranszplantációját, és a magyar
program indulásáig további 187 magyar beteg tüdőátül
tetését is. A bécsi centrum hosszú éveken keresztül tá
mogatta a tüdőtranszplantációval foglalkozó hazai szak
emberek képzését. A transzplantációs folyamat egyes
elemei fokozatosan kerültek bevezetésre: 1996: Az első magyar beteg tüdőtranszplantációja
1996. március 5-én, Bécsben történt meg. Allokáció, transzplantációs riadó megszervezése,
donorszerv kiemelése és szállítása Ezért az ET egyéb tagországaiból elsősorban magas
LAS alapján vagy úgynevezett „rescue” allokáció során
kapunk szerveket. )
A tüdőfelek beültetésekor a három fő képlet – sor
rendben a hörgő, a bal pitvar, majd az arteria pulmona
lis – vég a véghez anasztomózisát tovafutó varratsorral
készítjük. A főhörgő-anasztomózist 4-0 PDS tovafutó, a
bal pitvari anasztomózist 4-0 Prolene tovafutó, az arteria
pulmonalis végeket 5-0 Prolene tovafutó varrattal egyez
tetjük. p
A veseátültetéstől eltérően a tüdőátültetés során nem
törekszünk a HLA-kompatibilitásra. A műtét előtt ke
resztpróba-vizsgálatra logisztikai okok miatt rutinszerű
en nincs lehetőség. Ha a műtét után megküldött kereszt
próba pozitív, akkor emelt immunszuppressziót, szükség
esetén plazmaferézist alkalmazunk. Retranszplantáció,
magas HLA-ellenes antitesttiter esetén törekszünk az
előzetes keresztpróba elvégzésére, ez azonban általában
csak hazai donáció esetén szervezhető meg. Donor/recipiens méretdiszkrepancia esetén a na
gyobb tüdő mintegy 20%-os megkisebbítését a leggyak
rabban a középlebeny és/vagy a lingula atípusos gépi
tüdőállomány reszekciójával érjük el. Jelentősebb ’size-
mismatch’ esetén lebenyátültetést (lobar-TX) végzünk. Ilyenkor az implantáció előtt extracorporalis lobectomiát
követően végezzük a mérete, illetve alakja szerint kivá
lasztott lebenyek oldalazonos beültetését. Magyarországi, de főleg külföldi szervkivételek esetén
a szervezés során az egyik legfontosabb szempont a hi
degischaemiás idő (cold ischemic time – CIT), mely ide
ális esetben nem haladja meg a 6–8 órát. CIT-en tüdőát
ültetés esetében a donoraorta lefogása és a – bilaterális
transzplantáció esetén másodikként – beültetett tüdő
reperfúziója között eltelt időt értjük, eközben ugyanis
nincs keringése a tüdőnek, és nincs lélegeztetve. A transzplantációkat saját gyakorlatunkban venoarteri
osus (VA) ECMO (Cardiohelp System, Maquet GmbH,
Rastatt, Németország) támogatással operáljuk. A vénás
kanült a jobb pitvarba, az artériás kanült az aorta ascen
densbe helyezzük be. A kanüláláshoz heparinbevonatos
(vénás:
CB96825;
CB6570;
CB667;
CB67636;
CB91437C; artériás: CB77618; CB77620; Medtronic
Kft., Budapest) kanülöket alkalmazunk. A kanüláláshoz
csak részleges antikoagulálás szükséges, melyet iv. adott
heparinnal (60–70 NE/ttkg) végzünk. Az aktivált alva
dási idő (activated clotting time – ACT) célértéke gya
korlatunkban 180 és 220 s közötti. Súlyos primer graft
elégtelenség (primary graft dysfunction – PGD) esetén,
illetve PPH-s recipiensek esetén a VA ECMO-támoga
tást a posztoperatív szakban is folytatjuk. Magyarországon jelenleg csak agyhalott (donor after
brain death – DBD) donorból végzünk szervkivételt. A szervkivétel során az arteria pulmonalis törzsön ke
resztül 4 °C-os, alacsony káliumtartalmú, hűtött dextrán
oldattal (Perfadex®, Vitrolife, Göteborg, Svédország)
perfundálva konzerváljuk a tüdőt. A hideg okozta vaso
constrictio ellensúlyozása érdekében közvetlenül az
aortalefogás és a perfúzió indítása előtt potens vasodila
tatort alkalmazunk (epoprosztenol). Allokáció, transzplantációs riadó megszervezése,
donorszerv kiemelése és szállítása A szervriadókat országos szinten az OVSZ SZKI szer
vezi a tüdőtranszplantációs centrum koordinátoraival
együttműködve. 2004: Megalakult a Tüdő Transzplantációs Bizottság
(TTB), amely havonta/kéthetente (de szükség esetén
soron kívül is) véleményezi és vizsgálja az ország terüle
téről érkező, tüdőtranszplantációra javasolt betegeket. A betegek a TTB javaslata alapján kerülnek a magyar és
2013-tól egyben az Eurotransplant (ET) tüdőtransz
plantációs várólistára. A listára került betegeknek az elvégzett vizsgálatok
eredményei, a betegség súlyossága alapján az ET interne
tes felületén kiszámoljuk a Lung Allocation Score (LAS)
értékét. A LAS olyan numerikus pontrendszer, mely a
donorszervek allokációját hivatott segíteni. Minél na
gyobb ez az érték, annál sürgősebb a tüdőátültetés. A LAS maximumértéke 100 lehet. A gyermekek (12 év
alatt) automatikusan 100 pontot kapnak, mivel kis test
méretük miatt csak nehezen jutnak új tüdőhöz. 2012: Magyarország 2012. január 1-től előzetes
együttműködési megállapodás keretein belül, majd
2013. július 1-től teljes jogú Eurotransplant-tag lett,
aminek köszönhetően a program indulásakor már kül
földről is hozhattunk haza tüdőket. Az allokáció az Eurotransplant tagországain belül,
Németországban és Hollandiában a LAS alapján törté
nik. Más országokban a centrum azonban saját maga
döntheti el, hogy kit transzplantál, a bécsi centrummal
együtt mi is ezt a gyakorlatot követjük. Saját donorterü
letről érkezett szervfelajánlás esetén a tüdőallokáció
AB0-vércsoport-kompatibilitás és méretegyezés (size-
match) figyelembevételével, interdiszciplináris, egyedi
szakmai döntés alapján történik (centrumallokáció). Az
érvényes ET-szerződések szerint jelenleg Ausztria, Szlo
vénia, Horvátország és Magyarország területe számunk 2015: Az első tüdőbeültetést 2015. 12. 12-én végez
tük el az Országos Onkológiai Intézetben (OOI), az Or
szágos Onkológiai Intézet és a Semmelweis Egyetem
együttműködésében. Az első hazai tüdőátültetést a köz
vetlen perioperatív tevékenységben részt vevő szakembe
rek (sebészek, aneszteziológusok, koordinátorok, műtő
asszisztensek és gyógytornászok) intenzív felkészítése
előzte meg. Magyarország kormánya által nyújtott közel
1 milliárd forintos céltámogatás fedezte a szervtransz 1861 2018 ■ 159. évfolyam, 46. szám ORVOSI HETILAP Unauthenticated | Downloaded 10/24/24 03:57 AM UTC ÖSSZEFOGLALÓ KÖZLEMÉNY tetés ugyanennek a technikának a szekvenciális formája,
azaz két izolált („egymás után két egyoldali”) tüdő
transzplantáció (bilateral lung transplantation – BLTx). A műtéteket egy- vagy kétoldali anterolateralis thoraco
tomiából vagy ’clamshell’ metszésből végezzük (ez
utóbbi két, általában a negyedik bordaközben végzett
anterolateralis thoracotomia haránt sternotomiával ös�
szekötve). ra lokális donor ’pool’-nak számít. Miután az ET-térben
tüdő esetében 50 feletti, úgynevezett „high-LAS”-érték
esetén nemzetközi allokáció is lehetséges, a LAS-értéket
minden esetben kiszámoljuk, ezzel is javítva a súlyosabb
állapotú, sürgős transzplantációt igénylő betegek esélye
it. Allokáció, transzplantációs riadó megszervezése,
donorszerv kiemelése és szállítása A két tüdőfelet egy
ben, közepesen felfújt állapotban, a bal pitvar hátsó falá
val együtt távolítjuk el. A két tüdőfelet csak akkor
választjuk szét szállítás előtt, ha a tüdők alkalmasak beül
tetésre. Úgynevezett marginális (határeseti) tüdők eseté
ben, a végső funkcionális megítéléshez a kiemelést köve
tően ex vivo perfúziót (ex vivo lung perfusion – EVLP)
alkalmazunk. Az EVLP használata során a közös kétol
dali tüdőblokkon a bal pitvar visszahagyott hátsó falára
és az arteria pulmonalis főtörzsre szájaztatjuk a kanülö
ket, valamint a tracheán keresztül lélegeztetjük a tüdőt. A módszer segítségével rekondicionálhatók, jobban
megítélhetők és bizonyos esetekben biztonságosan beül
tetésre alkalmassá tehetők a marginális tüdők. Ha EVLP
mellett döntünk, a tüdőket Bécsbe szállítjuk, mert jelen
leg ez az eljárás csak a fennálló együttműködésünk kere
tében férhető hozzá számunkra. Bridge-to-TX indikációval alkalmazott ECMO-terápia
esetén a venovenosus (VV) ECMO-eljárásokat preferál
juk, a kanülálást Seldinger-technikával végezzük jugula
ris, illetve inguinalis behatolásból (femorojugularis VV
ECMO). A korai graftdiszfunkció (PGD) nemzetközi adatok
szerint a tüdőtranszplantációk 10–25%-át érinti, és a ko
rai poszttranszplantációs morbiditás és mortalitás fő oka ORVOSI HETILAP
62
1. táblázat
A PGD-fokozatok klasszifikációja
Fokozat
PaO2/FiO2
A tüdőödéma radiológiai jelei
0. >300
Nem észlelhető
1. >300
Észlelhető
2. 200–300
Észlelhető
3. <200
Észlelhető
PaO2 = parciális oxigénnyomás; PGD = korai graftelégtelenség; FiO2
= a belélegzett oxigén aránya 1. táblázat
A PGD-fokozatok klasszifikációja 1. táblázat
A PGD-fokozatok klasszifikációja
Fokozat
PaO2/FiO2
A tüdőödéma radiológiai jelei
0. >300
Nem észlelhető
1. >300
Észlelhető
2. 200–300
Észlelhető
3. <200
Észlelhető
PaO2 = parciális oxigénnyomás; PGD = korai graftelégtelenség; FiO2
= a belélegzett oxigén aránya 1. táblázat
A PGD-fokozatok klasszifikációja 2018 ■ 159. évfolyam, 46. szám Szervkivételek 2015. 12. 12. és 2018. 07. 31. között összesen 76 szerv
kivételen vettünk részt: 45 magyar, 23 Eurotransplant-,
8 Eurotransplanton kívüli országban. A LAS alapján • Szteroid: prednizolont vagy metilprednizolont al
kalmazunk. Fél–egy órával a donortüdő reperfúzió
ja előtt 500–1000 mg metilprednizolont adunk. A
reperfúziót követő 8., 16., és 24. órában 125 mg,
majd 24 óra után 1 mg/ttkg napi dózist alkalma
zunk, amelyet fokozatosan csökkentünk. 63
2018 ■ 159. évfolyam, 46. szám
1. ábra
Várólistán lévő betegek száma az adott év december 31-én 1. ábra
Várólistán lévő betegek száma az adott év december 31-én A per os immunszuppressziót csak akkor indítjuk, ami
kor már rendeződött a betegek emésztése, megindult a
bélműködésük. Várólistaadatok 2017. december 31-én a tüdőtranszplantációs várólistán
12 aktív beteg volt, ez a szám lassan, de folyamatosan
növekedett a vizsgált időszakban (1. ábra). A transzplan
tált betegek várólistán eltöltött medián ideje 50 (range
1–427) nap volt. A tüdőtranszplantációs várólistán ös�
szesen 12 beteg halt meg a program indulása és 2018. 07. 31. között, a várólista mortalitása 15%-os. A listán
eltöltött medián idő az elhunytak esetében 106 (range
4–481) nap volt. Az immunszuppresszió részeként betegeink indukciós
terápiát kapnak, amennyiben ennek speciális kontraindi
kációja nincs. Ezt követően kettős, később hármas kom
binációjú bázisterápiát állítunk be. 1. Indukciós kezelés: alemtuzumab (Campath) 0,4–
0,5 mg/ttkg vagy polyclonalis antithymocytaglobulin
(ATG) 2 mg/ttkg. 2. Bázisterápia: Korai posztoperatív időszak, intenzíves gondozás A betegek gondozása a korai időszakban az AITO külön
transzplantációs célra elkülönített részlegén, a transz
plantációs őrzőben történik. Az elsődleges cél a betegek
keringési, légzési, hasi és pszichés statusának rendezése,
a lélegeztetőgépről való leszoktatása, az immunszupres�
szív szerek beállítása, a beteg mobilizálása. Korán elkezd
jük az ágyban használható, fekvő kerékpározás passzív és
aktív formáját. A kerékpározás stimulálja a bélmozgáso
kat, növeli az izomerőt és a cardiopulmonalis terhelhető
séget. Szervátültetés Gyakorlatunkban izolált egyoldali tüdőtranszplantáció
(single lung transplantation – SLTx) során ellenoldali lé
legeztetés mellett, az operált oldali tüdő eltávolítását kö
vetően kerül beültetésre az új tüdő. A kétoldali tüdőátül 2018 ■ 159. évfolyam, 46. szám 1862 ORVOSI HETILAP Unauthenticated | Downloaded 10/24/24 03:57 AM UTC Unauthenticated | Downloaded 10/24/24 03:57 AM UTC ÖSSZEFOGLALÓ KÖZLEMÉNY [5, 6]. A PGD mértékét az érvényes ISHLT-klasszifiká
ció alapján értékeljük (1. táblázat). ellenőrzés mellett rendszeres, gyógytornász felügyeleté
vel végzett edzést, izomépítést, valamint gyógyszerbeál
lítást, az otthoni, mindennapi életre való felkészülést vé
geznek. Itt történik a javasolt gyógyszerek beszerzése,
szükség esetén engedélyeztetése és a segédeszközökkel
való ellátás. A műtét utáni, kórházi rehabilitációval eltöl
tött idő általában 3–4 hét. A betegek állapotát a funkcio
nális paraméterek mérésével, bronchoszkópos szövettani
mintavétellel, illetve CT-vizsgálattal ellenőrizzük. A
transzplantáltak hazabocsátásukat követően meghatáro
zott időrendben rendszeresen kontrollra jelentkeznek
tüdőgyógyász teamnél. Egyes esetekben például előrehaladott PPH-s bete
geknél, szupraszisztémás pulmonalis tensiónál az intubá
ciót követően indított, pozitív nyomású lélegeztetés
okozta mellkasi nyomásemelkedés hemodinamikai ös�
szeomlást okozhat. Ezekben az esetekben lokális érzés
telenítésben, már altatás előtt perifériás behatolásból ve
zetjük be a (VA) ECMO-t, vagy JET-lélegeztetést
(Monsoon JET, Acutronic Medical Systems, Hirzel,
Svájc) alkalmazunk a centrális ECMO elindításáig. A hazai gondozás centruma a Semmelweis Egyetem:
a felnőtt transzplantáltakat a Pulmonológiai Klinika,
a gyermekeket az I. Gyermekgyógyászati Klinika gon
dozza. Tüdő Transzplantációs Bizottság • Kalcineurininhibitorok: saját gyakorlatunkban tak
rolimuszt (Prograf) 0,01 mg/ttkg/24ó alkalma
zunk rutinszerűen. A kívánt vérszint: 8–10 ng/ml. A megjelenő betegek száma 2013-tól folyamatosan
emelkedik, az utolsó évben összesen 85 beteg került be
mutatásra az üléseken. A beutaló diagnózisok megoszlá
sának aránya közel állandó (2. ábra). • Purinszintézis-gátlók: rutinszerűen mikofenolát-
mofetilt (CellCept) alkalmazunk. Ha a transzplan
tációt követően Campathtal történt indukciós keze
lés, akkor 1 évvel a műtétet követően vezetjük be
1–2 × 500 mg/napi dózisban adva. Ha ATG-vel
történt az indukció, akkor a Tx-t követően rögtön
adjuk, 3 × 1 g-ot naponta. 18 ■ 159. évfolyam, 46. szám Utógondozás/rehabilitáció A betegek optimális esetben kb. 2–4 hetet töltenek inté
zetünkben. Rehabilitáció és utógondozás céljából pul
monológiai osztályra kerülnek áthelyezésre, ahol orvosi 1. ábra Várólistán lévő betegek száma az adott év december 31-én 1863 ORVOSI HETILAP ■ 159. évfolyam, 46. szám Unauthenticated | Downloaded 10/24/24 03:57 AM UTC ÖSSZEFOGLAL
2. ábra
A Tüdő Transzplantációs Bizottság ülésein megjelenő betegek
száma és beutaló diagnózis szerinti megoszlásuk az adott évben
BOS = bronchiolitis obliterans szindróma; CF = cystás fibrosis;
COPD = krónikus obstruktív tüdőbetegség; IPF = idiopathiás
tüdőfibrosis ÖSSZEFOGLAL
2. ábra
A Tüdő Transzplantációs Bizottság ülésein megjelenő betegek
száma és beutaló diagnózis szerinti megoszlásuk az adott évben
BOS = bronchiolitis obliterans szindróma; CF = cystás fibrosis;
COPD = krónikus obstruktív tüdőbetegség; IPF = idiopathiás
tüdőfibrosis ÖSSZEFOGLALÓ KÖZLEMÉNY 2. táblázat
A magyar betegek tüdőtranszplantációs diagnózisának megosz
lása (2015. december–2018. július)
Alapbetegség
LuTx Bécs (n = 9)
LuTx Budapest (n = 55)
Felnőtt
COPD
0
28
IPF
1
7
IPF/ECMO-bridge
0
1
CF
0
9
CF/Tüdő-vese Tx
0
1
CF/Tüdő-máj Tx
1
0
CF/ECMO-bridge
0
1
PPH
2
2
PPH/ECMO-bridge
1
0
Histiocytosis
0
1
Bronchiectasia
0
2
Sarcoidosis
1
0
LAM
0
1
BOS (re-Tx)
0
1
Gyermek
CF
1
1
CF/ECMO-bridge
1
0
PPH/ECMO-bridge
1
0
BOS = bronchiolitis obliterans szindróma; CF = cystás fibrosis; COPD
= krónikus obstruktív tüdőbetegség; ECMO = extrakorporális memb
ránoxigenizáció; IPF = idiopathiás tüdőfibrosis; LAM = lymphangiole
iomyomatosis; LuTx = tüdőtranszplantáció; PPH = primer pulmonalis
hypertonia; Tx = transzplantáció meghatározott direkt allokáció 4 esetben volt, a többi 72
esetben centrumallokáció szerint történt a recipiens ki
választása. 21 esetben sajnos nem volt alkalmas beülte
tésre a donortüdő, így a transzplantáció meghiúsult. A
recipiens magas anti-HLA-titere és retranszplantáció mi
att 3 alkalommal volt szükség preoperatív ’crossmatch’
vizsgálatra. Betegek BOS = bronchiolitis obliterans szindróma; CF = cystás fibrosis; COPD
= krónikus obstruktív tüdőbetegség; ECMO = extrakorporális memb
ránoxigenizáció; IPF = idiopathiás tüdőfibrosis; LAM = lymphangiole
iomyomatosis; LuTx = tüdőtranszplantáció; PPH = primer pulmonalis
hypertonia; Tx = transzplantáció BOS = bronchiolitis obliterans szindróma; CF = cystás fibrosis; COPD
= krónikus obstruktív tüdőbetegség; ECMO = extrakorporális memb
ránoxigenizáció; IPF = idiopathiás tüdőfibrosis; LAM = lymphangiole
iomyomatosis; LuTx = tüdőtranszplantáció; PPH = primer pulmonalis
hypertonia; Tx = transzplantáció Az első magyar beteg bécsi tüdőtranszplantációja óta
2018 augusztusáig összesen 187 bécsi és 55 budapesti
tüdőátültetés történt magyar betegbe (3. ábra). A Bécsben, illetve a Budapesten transzplantált betegek
diagnózisának megoszlását a 2. táblázat tartalmazza. A
legtöbb transzplantációt krónikus obstruktív tüdőbeteg
ség (COPD) miatt végeztük. első kombinált (tüdő-vese) transzplantáció is, a Transz
plantációs és Sebészeti Klinikával együttműködve. 2018-
ban elvégeztük az első gyermek- és az első retranszplan
tációt. Két alkalommal ECMO-bridge terápiát követően
végeztük el a műtétet. Budapesten 2017-ben volt az első primer pulmonalis
hypertoniás (PPH-) beteg hazai szervátültetése és az 3. ábra
Magyar betegek tüdőtranszplantációi Bécsben és Budapesten
LuTx = tüdőtranszplantáció 3. ábra
Magyar betegek tüdőtranszplantációi Bécsben és Budapesten
LuTx = tüdőtranszplantáció 3. ábra
Magyar betegek tüdőtranszplantációi Bécsben és Budapesten 2018 ■ 159. évfolyam, 46. szám 1864 ORVOSI HETILAP Unauthenticated | Downloaded 10/24/24 03:57 AM UTC Ó KÖZLEMÉNY
4. ábra
A Budapesten transzplantált betegek túlélése ÖSSZEFOGLALÓ KÖZLEMÉNY 4. ábra
A Budapesten transzplantált betegek túlélése A LAS értéke átlagosan 37,46 ± 11,81 volt a transz
plantáció időpontjában. A betegek átlagéletkora a transz
plantáció időpontjában 47,5 ± 15,18 év volt. A legfiata
labb 13 éves CF-es, a legidősebb, 65 éves COPD-s beteg
volt. A nemek aránya: 53,7% férfi, 46,3% nő. Az utólagos HLA ’crossmatch’ vizsgálat egy beteg
esetében lett pozitív. 3 betegnél volt szükség előzetes
’crossmatch’ vizsgálatra: 1 esetben magas PRA (21%)
miatt; 2 esetben retranszplantáció miatt. Sajnos az egyik
retranszplantációra váró beteg az életmentő tüdő megér
kezése előtt elhunyt. A magas panelreaktív antitest (pan
el-reactive antibody – PRA) szinttel rendelkező betegnél
közvetlenül a transzplantációs műtét előtt egy és a műtét
után több alkalommal végeztünk plazmaferézist. Korai szövődmények Magas pulmonalis nyomás miatt 5 esetben JET-léle
geztetéssel, egy esetben perifériás ECMO védelmében
történt az anesztézia bevezetése. Intraoperatív halálozás nem történt egy esetben sem. A korai posztoperatív időszakban, 30 napon belül 2 be
teget veszítettünk el. Az egyik beteg passenger lympho
cyta szindróma okozta sokk miatt, a másik beteg fulmi
náns szepszis miatt hunyt el a posztoperatív 26., illetve 5. napon. További 2 beteget vesztettünk el 30 napon túl az
intézetben történő ápolási időszak alatt uralhatatlan
szepszisben. Negyven esetben a donortüdők reszekció nélkül ke
rültek beültetésre, 9 esetben kétoldali, 4 esetben egyol
dali atípusos reszekciót végeztünk. 2 kritikus beteg ese
tében nem volt idő megvárni a méretben megfelelő
tüdőt, ezért a donor–recipiens méretaránytalanság miatt
az egyik oldalra csak egy lebeny beültetését végeztük. Három beteget kivéve az összes műtétet centrális veno
arterialis ECMO védelmében végeztük. 1 beteg esetében
egyáltalán nem alkalmaztunk, 2 betegnél perifériás beha
tolásból került bevezetésre ECMO. Az átlagos ECMO-idő
201,9 ± 36,51 perc volt. Prolongált, posztoperatív peri
fériás ECMO-kezelést 3 betegnél alkalmaztunk, melyet
két esetben 4, egy esetben 3 napig folytattunk. A PGD incidenciája alacsony volt, súlyos formáját nem
észleltük. 3 beteg esetében a PGD grade 1, egy beteg
esetében grade 2 volt. Posztoperatív vérzés miatt 5 be
tegnek végeztünk haematomaevakuációt, két esetben
korai dekortikációt. A nervus phrenicus egyoldali sérülé
se két esetben fordult elő, egy esetben rekeszduplikációt
végeztünk. Egy betegünknél nervus hypoglossus paresis
miatt a nyelv mozgása szinte teljesen kiesett, aminek
okát biztosan nem tudtuk meghatározni, gyógyszertoxi
citást feltételeztünk. Posterior reverzibilis encephalopa
thia szindróma (PRES) 5 betegnél fordult elő. Vesepótló
kezelésre egy esetben sem volt szükség. A műtétek során átlagosan 5 ± 0,57 egység (E) szűrt,
sugarazott vörösvértest-koncentrátumot (VVT) és 10 ±
2,6 E Octaplas (Octapharma AG, Lachen, Svájc) készít
ményt használtunk fel. Az átlag CIT-idő az elsőként beültetett tüdők eseté
ben 322,44 ± 41,13 perc, a másodikként beültetett tü
dők esetében 403,54 ± 42,61 perc volt. Transzplantációs műtét A Budapesten elvégzett 55 tüdőtranszplantációból 54
kétoldali és 1 egyoldali átültetést végeztünk. 53 esetben
’clamshell’ behatolásból, 1 esetben kétoldali anterola
teralis thoracotomiából, 1 esetben egyoldali anterola
teralis thoracotomiából végeztük a műtétet. A Budapesten transzplantált betegek túlélése 4. ábra
A Budapesten transzplantált betegek túlélése Megbeszélés A tüdőtranszplantációs várólistán regisztrált betegek szá
ma a vizsgált időszakban lassan, de folyamatosan növeke
dett. Ha azonban az Eurotransplant-adatokat vesszük
alapul, akkor a magyar tüdővárólista feltöltöttsége
20,3%-os. Ehhez viszonyítva a tüdőtranszplantációs vá
rólistánkon mintegy 59 recipiensnek kellene folyamato
san lennie. Ez a különbség azt jelenti, hogy sok hazai
beteg még nem jut el a tüdőtranszplantációig, holott az
életük megmenthető lenne. A tüdőtranszplantációs várólistán összesen 12 beteg
halt meg a program indulása óta: 2016-ban 7 beteget,
2017-ben 2 beteget, 2018-ban (2018. július 31-ig) 3
beteget vesztettünk el, a várólista mortalitása 15%-os. y
p
Az 55 tüdőtranszplantációból 54 kétoldali és 1 egyol
dali átültetést végeztünk. A nem infektív parenchymás
tüdőbetegségek (például IPF, COPD) esetében egyolda
li átültetés is végezhető, míg az infektív (például CF-)
eredetű esetekben mindig bilaterális transzplantáció
szükséges, és a vascularis csoportban (például PPH) is
ezt preferáljuk. Ugyanis a fertőzéses csoportban megha
gyott saját tüdő az immunszupprimált betegben infektív
rezervoárként működhet, a vascularis csoportban pedig
az egyoldali átültetés után instabil és nehezen uralható
hemodinamikai helyzet alakulhat ki. Miután a hazai
programban a szervdonációs aktivitás és a kielégítő tüdő
donációs potenciál nem kényszerített az egyoldali
transzplantációk irányába, így a nemzetközi gyakorlat
nak megfelelően, lehetőség szerint törekedtünk a kétol
dali átültetésre, mivel ebben a csoportban jobbak a hos�
szú távú túlélési eredmények [7, 8]. Egyoldali átültetést
egy esetben végeztünk, ennek a betegnek korábban tal
kumos pleurodesist végeztek az ellenoldali mellkasfél
ben. A 2016-os évet kiemelve jól azonosíthatók ennek
egyes meghatározó tényezői: egy elvesztett betegünk 50
napnál rövidebb ideig volt a várólistán, vagyis nagyon
előrehaladott állapotban került a látóterünkbe. Egy fiatal
CF-es beteg esetében Burkholderia cepacia miatt húzó
dott el a listázás, és többször inaktiválni kellett szeptikus
problémák miatt; másik 2 beteg esetében a várólistán el
töltött idő volt hosszú (481, 193 nap), egyiküket (bridge
to transplant) ECMO-ra tettük, de az életmentő tüdő
érkezése előtt elvesztettük. Különösen tragikus kis súlyú,
de a 12. életévét már betöltött gyermek halála. Sajnos a
LAS-rendszer csak a 12 év alatti gyermekeknek biztosít
prioritást a várólistán, a gyermek donorok száma pedig
alacsony. 2 további elvesztett beteg alapbetegsége PPH
volt: a hirtelen halál ebben a betegségben közismerten
gyakori, nincs ismert várólista-mortalitást jelző prog
nosztikai tényező, ezért a LAS-rendszerben sem sikerült
leképezni a sürgősségüket. Bár az összesített várólista-mortalitásunk magasnak
tűnik, az átlagot csak kismértékben haladja meg: az ET-
várólista mortalitása 2017-ben 12,4% volt. Posztoperatív időszak és túlélés A ’clamshell’ metszés sebének gyógyulási zavara miatt 3
alkalommal kellett VAC-kezelést alkalmaznunk, 2 eset
ben pedig musculus latissimus dorsi lebeny beforgatással
sikerült a végleges sebgyógyulást elérni. Hörgőanaszto
mózis-szűkület miatt 4 esetben volt szükség broncho
szkópos intervencióra (ballonos tágítás, sztentelés). Kró
nikus rejekció 2 esetben fordult elő, az egyik beteget 8
hónappal az első transzplantációt követően retranszplan
táltuk, a másikat sajnos 14 hónappal a Tx-műtét után
elvesztettük. Tartós lélegeztetés szükségessége miatt a betegek 23,6%-
ánál tracheostomát készítettünk. A betegek áltagosan
24,6 ± 18,18 napot töltöttek az intenzív osztályon. A
Semmelweis Egyetem Pulmonológiai Klinikájára történő
áthelyezést megelőzően 18 beteget ápoltunk Mellkasse
bészeti Osztályunkon, ahol az átlagosan eltöltött idő 6,8
± 3,19 nap volt. A betegek Kaplan–Meier szerinti 1 éves túlélése (2018. 07. 31-ig bezárólag) 82,96% volt (4. ábra). 2018 ■ 159. évfolyam, 46. szám 1865 ORVOSI HETILAP Unauthenticated | Downloaded 10/24/24 03:57 AM UTC ÖSSZEFOGLALÓ KÖZLEMÉNY PPH-s beteg szervátültetését, az első kombinált (tüdő-
vese) és az első retranszplantációt, illetve két alkalommal
ECMO-bridge terápiát követően végeztünk tüdőátülte
tést. Ezeket a nagy rizikójú műtéteket nemzetközi össze
hasonlításban is csak a legnagyobb felkészültségű centru
mok tudják vállalni. Jelenleg csak a kis súlyú (25 kg
alatti) gyermekek, bizonyos kombinált tüdőtranszplan
tációk (tüdő-máj Tx, tüdő-szív Tx) és kapacitáshiány
esetén történik magyar recipiens transzplantációja a bécsi
centrumban. A legtöbb transzplantációt, 28 esetben
COPD miatt, 12 esetben CF miatt és 8 esetben IPF mi
att végeztük. A nemzetközi, ISHLT-adatok szerint is
COPD miatt végzik a legtöbb tüdőátültetést, azonban a
második helyen az IPF szerepel [3]. 2018 ■ 159. évfolyam, 46. szám Megbeszélés Ezekben az esetekben a betegek már nem igé
nyeltek intenzív osztályos ellátást, de sebészeti (mellkasi
szívódrén, sebgyógyulási zavar stb.) vagy egyéb okból
nem voltak még átadhatók. A betegek 1 éves túlélése 82,96%-volt, ami az ISHLT
adataival összehasonlítva jobb, mint a nemzetközi 78%-os
átlag [3]. Magas pulmonalis nyomás esetén több esetben JET-
lélegeztetést alkalmaztunk a centrális ECMO elindítá
sáig. A nyílt rendszerű JET-lélegeztetés minimális volu
mennel és nagy frekvenciával biztosítja a légcseréhez
szükséges gázáramlást, így nem emeli jelentősen az int
rathoracalis nyomást. Tapasztalataink alapján ezzel a
technikával biztonsággal kivédhető a fenyegető keringés-
összeomlás. A tüdőtranszplantációs programunk egyik erőssége,
hogy olyan centrumban kapott helyet, ahol évente több
mint ezer mellkassebészeti nagyműtétet végzünk, ennek
megfelelően az intenzív osztálynak kiemelkedő rutinja
van az általános mellkassebészeti posztoperatív betegel
látásban, beleértve a szövődmények kezelését is. A prog
ram indításakor további előny volt, hogy a transzplan
tációs folyamatnak több eleme is – donorprogram,
kivizsgálás és előkészítés, utógondozás – már bejáratot
tan működött Magyarországon [10, 11]. Jelenleg a bécsi centrummal közös a várólistánk és a
donor ’pool’-unk is. A közös várólista egyik előnye, hogy
szükség esetén a tüdőtranszplantációs műtétek Bécsben
is elvégezhetők. A közös donor ’pool’-ból is centrumal
lokáció alapján határozhatjuk meg a recipienst, ami sok
esetben lerövidíti a várólistán a várakozási időt (például
gyermek, kis mellkas, immunológiai problémák esetén). A LAS bevezetése az Egyesült Államokban kezdődött; az
allokációs eszköz fő célja az volt, hogy csökkentse a váró
lista-mortalitást, és javítsa a transzplantációt követő 1
éves túlélést, ami ott sikerült is, de ez még ma sem éri el
a centrumallokáció által biztosított indikátorokat, ezért
több ET-tagországgal együtt jelenleg mi is ez utóbbi al
lokációs rendszert preferáljuk. Az ET-tagságnak köszön
hetően az ET-tagországokból is számos tüdő kerül Ma
gyarországon beültetésre. 2015. 12. 12. és 2018. 07. 31. között összesen 76 szervkivételen vettünk részt, me
lyeknek kicsit kevesebb, mint a fele (31) külföldi szervki
vétel volt. gy
g
Szeretnénk a transzplantációk számát ugyanakkor to
vább növelni. Nemzetközi adatok alapján (9,8 milliós
magyar népességgel számolva) évente 30–40 tüdő
transzplantáció lenne szükséges hazánkban. Az elvégez
hető transzplantációk számát a világon szinte mindenhol
a rendelkezésre álló donorszervek határozzák meg. Ma
gyarországon az elmúlt években körülbelül 30–40 tüdő
donáció valósult meg. Itthon a beültetések számát jelen
leg a várólistán lévő betegek száma korlátozza a
leginkább, ami szakmailag tarthatatlan. Sajnos nehézsé
get jelent a recipiensek transzplantáció előtti kivizsgálása. Ez komplex folyamat, a rutin-betegellátás mellett nagy
terhet ró a kivizsgálóintézmények személyzetére, és je
lenleg súlyosan alulfinanszírozott. Megbeszélés VA ECMO-t vezettünk be inguinalis behatolásból, így
stabilizálva a beteg keringését, és a műtétet a perifériás
támogatással végeztük el. A másik beteg esetében csak
bal oldali tüdőátültetés történt, és bal oldali anterola
teralis thoracotomiából végeztük a műtétet. Mivel a vé
nás kanül behelyezése ebből a feltárásból nem kivitelez
hető, inguinalis behatolásból történt a VA ECMO
bevezetése. VA ECMO-t vezettünk be inguinalis behatolásból, így
stabilizálva a beteg keringését, és a műtétet a perifériás
támogatással végeztük el. A másik beteg esetében csak
bal oldali tüdőátültetés történt, és bal oldali anterola
teralis thoracotomiából végeztük a műtétet. Mivel a vé
nás kanül behelyezése ebből a feltárásból nem kivitelez
hető, inguinalis behatolásból történt a VA ECMO
bevezetése. A nemzetközi összehasonlításban kiemelkedően ala
csony PGD-incidencia valószínűleg az intraoperatív
ECMO konzekvens használatával függ össze. Nem volt
egy esetben sem szükség vesepótló kezelésre, ami azért
kimagasló eredmény, mert a korai posztoperatív szakban
a tüdőt „szárazon”, a betegek folyadékegyensúlyát nega
tív oldalon kell tartani. Az érzékeny egyensúly megtalá
lása az intenzív osztályos kezelés színvonalát minősíti. BLTx esetén a második oldal műtéte alatt a teljes perc
térfogat az elsőként beültetett új tüdőn halad át, ami a
rövidebb CIT-idő ellenére is jelentős károsító tényező,
és mindig kisebb-nagyobb reperfúziós ödéma képében
észlelhető. A műtét végén az elsőként beültetett tüdő
nehezebb, compliance-e rosszabb, a korai mellkas-rönt
genfelvételeken ez az oldal jellegzetesen fedettebb (’first
lung syndrome’). A VA ECMO előnye, hogy csökkent
hetjük a kis vérkör keringő vértérfogatát (’bypass’), így
mérsékelhetjük az új tüdőre eső keringési volument,
ugyanakkor a gázcsere is biztosítva van. A nemzetközi
gyakorlatban sok centrumban szívmotoron végzik eze
ket a műtéteket. Az ECMO előnye a szívmotorhoz ké
pest, hogy csak részleges antikoagulálás szükséges, mivel
zárt rendszerben történik a vér keringetése, gázcseréje,
így jelentősen csökken az intraoperatív vérzés mennyisé
ge. További pozitívum, hogy szükség esetén műtét után
az extracorporalis támogatás prolongálható perifériás (ál
talában inguinalis) behatolásból. Intraoperatív halálozás nem volt, ugyanakkor a korai
időszakban 2 beteget vesztettünk el. Az egyik beteg va
lószínűleg passenger lymphocyta szindróma okozta ural
hatatlan sokk miatt hunyt el a posztoperatív 26. napon, a
másik beteg már a transzplantáció időpontjában kritikus
állapotban volt, és végül befolyásolhatatlan szepszisben
vesztettük el a posztoperatív 5. napon. További 2 bete
get vesztettünk el 30 napon túl az intézetben történő
ápolási időszak alatt szepszisben. A betegeket általában közvetlenül az AITO-ról he
lyeztük át Pulmonológiai Osztályra, és csak 18 beteg
esetében volt köztes, Mellkassebészeti Osztályon törté
nő ápolás. Megbeszélés Ugyanakkor
a várólistán eltöltött medián idő az ET-ben 152 nap
(≈5 hónap), ami jóval több, mint Magyarországon, ahol
a transzplantált betegeknek 50 napos (≈2 hónapos), míg
az elhunyt betegeknek 106 napos (≈3,5 hónapos) a me
dián várakozási ideje. Az alacsony várakozási idő a jó do
norellátottságnak és a relatíve alacsony várólista-feltöl
töttségnek köszönhető. Ugyanakkor sajnos a betegek
még mindig sok esetben csak későn, súlyos állapotban
kerülnek a transzplantációs program látókörébe, és ki
csúsznak a transzplantációs időablakból. A Budapesten elvégzett 55 tüdőtranszplantációból 2
beteg kivételével az összes műtétet ’clamshell’ behatolás
ból végeztük. Az egyik esetben csak egyoldali tüdőátül
tetésre került sor a már fentebb említett ellenoldali pleu
rodesis miatt. A másik esetben, a beteg köpetéből
kitenyészett Mycobacterium abscessus-törzs miatt, kétol
dali anterolateralis thoracotomiából végeztük el a műté
tet a sternum átvágása nélkül, ugyanis ezekben az esetek
ben rendkívül magas a sternum gyógyulási zavarainak
kockázata. A korai posztoperatív szakban mért funkciós
paraméterek alapján a kétoldali thoracotomia ugyan kí
méletesebb a ’clamshell’ behatolásnál, de sok esetben
rosszabb feltárást biztosít [9]. Centrális keringéstámoga
tás igénye (például ECMO, szívmotor) esetén a ’clam
shell’ behatolás kiváló hozzáférést ad mind a kanülálás
hoz, mind a tüdőhilusokhoz. Az első magyar beteg bécsi tüdőtranszplantációja óta
2018 augusztusáig összesen 187 bécsi és 55 budapesti
tüdőátültetés történt magyar betegbe. Ha az induló
transzplantációs programunk esetszámait nézzük, akkor
más transzplantáló centrumokkal összehasonlításban ki
emelkedő teljesítménynek számít, hogy az első években
elértük a 20 körüli esetszámot. A magyar program indu
lása után 8 esetben magas rizikójú betegség miatt, 2 eset
ben kapacitáshiány miatt történt a bécsi centrumban a
beteg tüdőátültetése, ugyanakkor 2,5 évvel az indulá
sunk után elvégeztük már az első gyermek, és az első Az összes műtétet centrális, venoarterialis ECMO vé
delmében végeztük három esetet leszámítva. Az egyik
beteg esetében a lap szerint kitapadt tüdő leválasztásából
származó diffúz vérzés miatt tekintettünk el az ECMO
és a következményes antikoagulálás alkalmazásától. A
másik két esetben perifériás VA ECMO-t alkalmaztunk. Az egyik előrehaladott, szupraszisztémás pulmonalis 1866 ORVOSI HETILAP Unauthenticated | Downloaded 10/24/24 03:57 AM UTC Unauthenticated | Downloaded 10/24/24 03:57 AM UTC ÖSSZEFOGLALÓ KÖZLEMÉNY Az átlag CIT-idő a másodikként beültetett tüdők ese
tében 403,54 ± 42,61 perc volt, ami az elvárt 8 órán
belül van. nyomással bíró, PPH-s betegnél már altatás előtt, lokális
érzéstelenítésben szükség volt az ECMO használatára. nyomással bíró, PPH-s betegnél már altatás előtt, lokális
érzéstelenítésben szükség volt az ECMO használatára. Irodalom [1] Toronto Lung Transplant Group. Unilateral lung transplanta
tion for pulmonary fibrosis. N Engl J Med. 1986; 314: 1140–
1145. A tüdőtranszplantáció utáni első évet túlélő betegek
medián túlélése 8,1 év [3]. Az új tüdővel a betegek vis�
szaállhatnak a munkájukba, elkezdhetnek sportolni,
utazni, aktívan élni. Beszédes teljesítmény, hogy a cent
rumunk részvételével 2017 nyarán, egy nemzetközi ex
pedíció keretein belül, 8 tüdőtranszplantált (2 magyar)
megmászta a Kilimandzsáró 5895 m magas csúcsát. [2] Patterson GA, Cooper JD, Dark JH, et al. Experimental and
clinical double lung transplantation. J Thorac Cardiovasc Surg. 1988; 95: 70–74. [3] Lund LH, Khush KK, Cherikh WS, et al. The Registry of the
International Society for Heart and Lung Transplantation: thir
ty-fourth adult heart transplantation report – 2017. J Heart
Lung Transplant. 2017; 36: 1037–1046. A jövőben azt szeretnénk elérni, hogy a várólista fel
töltöttsége megfeleljen a nemzetközi átlagnak, valamint
célunk, hogy növeljük az átültetések számát, és emellett
új technikákat (például EVLP) is bevezessünk. Megvaló
sításra vár a kombinált szív-tüdő és tüdő-máj átültetések
elindítása is Magyarországon. [4] Klepetko W, Grimm M, Laufer G, et al. Uni- and bilateral lung
transplantation: surgical results and experiences of the 1st year. Vienna Lung Transplant Group. Wien Klin Wochenschr. 1991;
103: 728–733. [5] Christie JD, Kotloff RM, Ahya VN, et al. The effect of primary
graft dysfunction on survival after lung transplantation. Am J
Respir Crit Care Med. 2005; 171: 1312–1316. [6] Daud SA, Yusen RD, Meyers BF, et al. Impact of immediate pri
mary lung allograft dysfunction on bronchiolitis obliterans syn
drome. Am J Respir Crit Care Med. 2007; 175: 507–513. Anyagi támogatás: A közlemény megírása anyagi támo
gatásban nem részesült. [7] Black MC, Trivedi J, Schumer EM, et al. Double lung trans
plants have significantly improved survival compared with single
lung transplants in high lung allocation score patients. Ann
Thorac Surg. 2014; 98: 1737–1741. Szerzői munkamegosztás: Az irodalomkutatásban, a kéz
irat összeállításában és megírásában a szerzők egyenlő
mértékben tevékenykedtek. A cikk végleges változatát
valamennyi szerző elolvasta és jóváhagyta. [8] Force SD, Kilgo P, Neujahr DC, et al. Bilateral lung transplanta
tion offers better long-term survival, compared with single-lung
transplantation, for younger patients with idiopathic pulmonary
fibrosis. Ann Thorac Surg. 2011; 91: 244–249. Érdekeltségek: A szerzőknek a cikk megírásával kapcsolat
ban nincsenek érdekeltségeik. [9] Marczin N, Popov AF, Zych B, et al. Megbeszélés Megoldást csak a ki
vizsgálás jobb finanszírozása és centralizálása jelentene. Az erre vonatkozó javaslatot a Szakmai Kollégium
Transzplantációs, Tüdőgyógyászati és Mellkassebészeti 1867 2018 ■ 159. évfolyam, 46. szám ORVOSI HETILAP Unauthenticated | Downloaded 10/24/24 03:57 AM UTC ÖSSZEFOGLALÓ KÖZLEMÉNY Tagozata közösen dolgozta ki és terjesztette a döntésho
zók elé. Irodalom Outcomes of minimally
invasive lung transplantation in a single centre: the routine ap
proach for the future or do we still need clamshell incision? In
teract Cardiovasc Thorac Surg. 2016; 22: 537–545. Köszönetnyilvánítás [10] Lang G, Czebe K, Gieszer B, et al. Lung transplantation pro
gram for Hungarian patients. [Tüdőtranszplantáció magyar
betegek számára.] Orv Hetil. 2013; 154: 868–871. [Hungarian]i A magyar tüdőtranszplantációs program nevében ezúton is köszönjük
a Bécsi Tüdőtranszplantációs Centrum segítségét és Walter Klepetko
professzor úr támogatását. [11] Czebe K, Csiszér E, Lang G, et al. The first 12 years of history of
Hungarian lung transplantations. [A tüdőtranszplantáció magyar
történetének első 12 éve.] Orv Hetil. 2008; 149: 1635–1644. [Hungarian] A cikk a Creative Commons Attribution-NonCommercial 4.0 International License (https://creativecommons.org/licenses/by-nc/4.0) feltételei szerint publikált
Open Access közlemény, melynek szellemében a cikk nem kereskedelmi célból bármilyen médiumban szabadon felhasználható, megosztható és újraközölhető,
feltéve, hogy az eredeti szerző és a közlés helye, illetve a CC License linkje és az esetlegesen végrehajtott módosítások feltüntetésre kerülnek. Hasznos linkek (Gieszer Balázs dr.,
Budapest, Ráth Gy. u. 7–9., 1122
e-mail: dr.gieszer@gmail.com) http://www.onkol.hu/hu/tudotranszplant
http://semmelweis.hu/mellkassebeszet/tudotranszplantacio
https://tudogyogyasz.hu/Page/Index/16919 1868 2018 ■ 159. évfolyam, 46. szám ORVOSI HETILAP ORVOSI HETILAP Unauthenticated | Downloaded 10/24/24 03:57 AM UTC
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https://openalex.org/W2618723485
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https://zenodo.org/records/4676955/files/source.pdf
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English
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Notes on the ant-mimic genus Anatea Berland (Araneae: Theridiidae) and two new species from tropical Australia
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Records of the Australian Museum
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cc-by
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© The Authors, 2017. Journal compilation © Australian Museum, Sydney, 2017
Records of the Australian Museum (2017) Vol. 69, issue number 1, pp. 1–13.
ISSN 0067-1975 (print), ISSN 2201-4349 (online)
https://doi.org/10.3853/j.2201-4349.69.2017.1672 © The Authors, 2017. Journal compilation © Australian Museum, Sydney, 2017
Records of the Australian Museum (2017) Vol. 69, issue number 1, pp. 1–13. ISSN 0067-1975 (print), ISSN 2201-4349 (online)
https://doi.org/10.3853/j.2201-4349.69.2017.1672 * Corresponding author: helen.smith@austmus.gov.au Helen M. Smith,1* Mark S. Harvey,2 Ingi Agnarsson3 and Gregory J. Anderson4 Helen M. Smith,1* Mark S. Harvey,2 Ingi Agnarsson3 and Gregory J. Anderson4 1 Australian Museum Research Institute, Australian Museum, 1 William Street, Sydney NSW 2010, Australia 2 Department of Terrestrial Zoology, Western Australian Museum,
Locked Bag 49, Welshpool DC WA 6986; School of Animal Biology, University of Western Australia WA 6009;
School of Natural Sciences, Edith Cowan University, Joondalup WA 6009, Australia 3 University of Vermont, Department of Biology, 109 Carrigan Drive, Burlington, VT, 05405-0086;
Department of Entomology, National Museum of Natural History,
Smithsonian Institution, Washington, DC, United States of America 4 Iron Metabolism Laboratory, QIMR Berghofer Medical Research Institute,
300 Herston Road, Herston QLD 4006, Australia 4 Iron Metabolism Laboratory, QIMR Berghofer Medical Research Institute,
300 Herston Road, Herston QLD 4006, Australia 300 Herston Road, Herston QLD 4006, Australia Abstract. The taxonomic history of the New Caledonian myrmecomorph spider, Anatea formicaria
Berland (Hadrotarsinae: Theridiidae) is summarized, new records are presented and the female is figured
for the first time. Two new species provisionally assigned to the genus are described from north-eastern
Australia, A. monteithi Smith sp. nov. and A. elongata Smith sp. nov. Some undescribed Anatea species
occurring on New Caledonia are shown, and aspects of hadrotarsine anatomy and ant specialization are
discussed. Keywords. Anatea formicaria; myrmecomorphy; myrmecophagy; New Caledonia; Queensland; taxonomy Keywords. Anatea formicaria; myrmecomorphy; myrmecophagy; New Caledonia; Queensland; taxonom Smith, Helen M., Mark S. Harvey, Ingi Agnarsson, and Gregory J. Anderson. 2017. Notes on the ant-mimic
genus Anatea Berland (Araneae: Theridiidae) and two new species from tropical Australia. Records of the Australian
Museum 69(1): 1–13. https://doi.org/10.3853/j.2201-4349.69.2017.1672 The New Caledonian hadrotarsine spider, Anatea formicaria
Berland, 1927, is thought to be a morphological ant mimic,
or myrmecomorph (Berland, 1927; Reiskind & Levi, 1967;
Cushing, 1997). Myrmecomorphs are especially prevalent in
families such as Salticidae and Corinnidae (Cushing, 1997)
but there are few in Theridiidae, with only five species in four
genera listed by Cushing (1997; the sixth species listed appears
to be a lapsus). Whilst most myrmecomorph spiders gain The New Caledonian hadrotarsine spider, Anatea formicaria
Berland, 1927, is thought to be a morphological ant mimic,
or myrmecomorph (Berland, 1927; Reiskind & Levi, 1967;
Cushing, 1997). Myrmecomorphs are especially prevalent in
families such as Salticidae and Corinnidae (Cushing, 1997)
but there are few in Theridiidae, with only five species in four
genera listed by Cushing (1997; the sixth species listed appears
to be a lapsus). Whilst most myrmecomorph spiders gain the “extra” body section (a “petiole”) as well as sometimes
a postpetiole from constrictions in the cephalothorax or
abdomen and / or colour (Reiskind, 1972), A. formicaria is
unusual in that the primary modification is to the pedicel,
which is not only elongated, but has an ant-like “node”
(Figs 1, 5). Most hadrotarsines have not been thought of as
particularly ant-like, although there are certain aspects of the
anatomy of many species that could aid them hiding among * Corresponding author: helen.smith@austmus.gov.au Records of the Australian Museum (2017) Vol. 69 2 ant corpses—as recorded for a “Dipoena sp.” regarded as
a myrmecophile (Cushing, 1997). The adaptations of A. formicaria are therefore particularly striking. Associated
behavioural traits that enhance an ant-like appearance, such
as running on six legs and holding the front legs aloft like
antennae, are common among better known myrmecomorph
taxa, but we have found no field observations of the behaviour
of A. formicaria in the literature. web-building non-mimic by McIver and Stonedahl (1993),
and Cushing (1997) overlooked Berland’s description and
listed the female as unknown. But as described by Berland
(1927), and shown here (Figs 5–7), it is similar to the male
but with reduced scuta. Methods Specimen examinations, measurements and drawings
were made using an Olympus SZ16 microscope, fitted
with graticule and camera lucida. All measurements are
in millimetres. Multiple photographs in different focal
planes were taken using a Leica MZ16 microscope fitted
with Spot Flex 15.2 Mp camera and layers combined using
HeliconFocus 5.3 software. Plates were composed using
Adobe Photoshop Elements. Maps were created using
ArcGISt software by Esri (www.esri.com). Square brackets
enclose notes added to original specimen record information. Specimen preparations for scanning electron microscopy
were passed through an alcohol series (75–100 %), critical
point dried and mounted on wire or pins before coating with
gold and viewing using a Zeiss Evo LS15 SEM incorporating
a Robinson backscatter detector. Details of female genitalia
were usually drawn from an excised epigynum, briefly
cleared in lactic acid if necessary. Berland came across the specimens he described as
Anatea among ants along with some other ant-mimicking
spider taxa in the collection of myrmecologist Ernest André
(1838–1911). He surmised that although no doubt André
knew these specimens were spiders, the original collector had
probably mistaken them for ants whilst collecting. Then, as
now, arachnologists recognized that ant mimicking spiders
occur in several families (and are especially prevalent in
Salticidae; Cushing, 1997). However, spiders of several
genera that we now recognize as being unrelated were then
commonly considered to belong together in the Clubionidae. Berland accordingly placed his monotypic genus Anatea
there, in subfamily Micariinae, and suggested a relationship
with Micaria Westring, 1851, a genus now included in the
Gnaphosidae (World Spider Catalog, 2017). fi Keywords. Anatea formicaria; myrmecomorphy; myrmecophagy; New Caledonia; Queensland; taxonom An ongoing review of Australian Hadrotarsinae has
found two new species in northern Queensland that possess
certain characters of Anatea, and in particular, they have an
elongate pedicel that is currently diagnostic for the genus. The new species we describe are, however, somewhat
tentatively placed here until further analyses are completed
and, hopefully, the unknown male of one species is found. Here we figure the correct male of the New Caledonian
Anatea formicaria, and also figure a female of that species
for the first time. We describe the two new species from
north-eastern Australia as Anatea monteithi sp. nov. and
Anatea elongata sp. nov. We also provide a brief introduction
to some of the New Caledonian Anatea diversity awaiting
researchers. Many spiders in the subfamily Hadrotarsinae have been
noted to feed on ants. Cushing (2012) lists 10 hadrotarsine
taxa as myrmecophages (plus “other Euryopis spp”). Indeed,
Liu et al. (2016) based on a phylogenetic analysis, found
that ant feeding is ancestral for the subfamily, though based
on relatively sparse taxon sampling. They also found that:
(a) subfamily Hadrotarsinae originates about the same time
that ants become noticeably abundant in the fossil record (c. 55–45 Mya); (b) there is a strong correspondence between
the abundance of ants and the diversity and abundance of
hadrotarsines through evolutionary time; and (c) there is a
similar correspondence in modern biodiversity inventories
(Liu et al., 2016). Finally, the small, flexible chelicerae with
sickle-shaped fangs, suggested by Reiskind & Levi (1967) to
possibly indicate an ant-specialized diet, are found in most
hadrotarsine taxa. Nevertheless, for the majority of species,
like A. formicaria, we have no field observations. Our own
observations of Australian hadrotarsines hunting, and with
prey support the suggestion that many, if not all taxa are
primarily myrmecophagous. Since species of Anatea possess
similar cheliceral modifications to the known hadrotarsine
myrmecophages, they may also feed on ants, or similar prey. Abbreviations All examined specimens are from Australian collections
as indicated by the following: AM, Australian Museum,
Sydney; ANIC, Australian National Insect Collection,
Canberra; QM, Queensland Museum, Brisbane; WAM,
Western Australian Museum, Perth. Anatea remained in the Clubionidae until its affiliations
with Euryopis Menge and Dipoena Thorell were recognized
and it was transferred to Theridiidae (Reiskind & Levi,
1967). In this paper Reiskind & Levi described a male, which
they attributed to A. formicaria, although we now realise
this identification was incorrect (see below). However, no
female Anatea has ever been figured despite being included
in the original description by Berland (1927). The female
of A. formicaria was erroneously characterized as being a Morphological abbreviations: AME anterior median
eye; ALE anterior lateral eye; Co conductor; Cy cymbium;
E embolus; ES embolus spire; FD fertilisation duct; ID
insemination duct; MA median apophysis; PME posterior
median eye; PLE posterior lateral eye; Sp spermathecae;
ST subtegulum; T tegulum; TR retrolateral tegulum rim; and
TTA theridiid tegular apophysis. Smith et al.: The ant-mimic spider genus Anatea 3 Figures 1–7. Anatea formicaria Berland, (1–4) male, habitus and eyes (AM KS.64090): (1) dorsal; (2) lateral; (3) ventral; (4) prosoma,
frontal. (5–7) Female, habitus and opisthosoma (QM S34107): (5) dorsal; (6) lateral; (7) ventral. Scale bars (1, 5) 0.5 mm. Figures 1–7. Anatea formicaria Berland, (1–4) male, habitus and eyes (AM KS.64090): (1) dorsal; (2) lateral; (3) ventral; (4) prosoma,
frontal. (5–7) Female, habitus and opisthosoma (QM S34107): (5) dorsal; (6) lateral; (7) ventral. Scale bars (1, 5) 0.5 mm. (1) dorsal; (2) lateral; (3) ventral; (4) prosoma,
ventral. Scale bars (1, 5) 0.5 mm. Figures 1–7. Anatea formicaria Berland, (1–4) male, habitus and eyes (AM KS.64090): (1) dors
frontal. (5–7) Female, habitus and opisthosoma (QM S34107): (5) dorsal; (6) lateral; (7) ventral (1) dorsal; (2) lateral; (3) ventral; (4) prosoma,
ventral. Scale bars (1, 5) 0.5 mm. Figures 1–7. Anatea formicaria Berland, (1–4) male, habitus and eyes (AM KS.64090): (1) dorsal; (2) lateral; (3) ventral; (4) prosoma,
frontal. (5–7) Female, habitus and opisthosoma (QM S34107): (5) dorsal; (6) lateral; (7) ventral. Scale bars (1, 5) 0.5 mm. Records of the Australian Museum (2017) Vol. 69 4 69
Figures 8–11. Anatea formicaria Berland, genitalia, (8, 9) male palpus (AM
KS.64090): (8) prolateral; (9) retrolateral. (10. 11) Female (QM S107230): (10)
epigynum, ventral (cleared); (11) internal genitalia, dorsal. Scale bars 0.1 mm. Figures 8–11. Abbreviations Anatea formicaria Berland, genitalia, (8, 9) male palpus (AM
KS.64090): (8) prolateral; (9) retrolateral. (10. 11) Female (QM S107230): (10)
epigynum, ventral (cleared); (11) internal genitalia, dorsal. Scale bars 0.1 mm. Anatea Berland, 1927 Anatea Berland, 1927: 53–55. Type species Anatea
formicaria Berland by monotypy. Anatea Berland, 1927: 53–55. Type species Anatea
formicaria Berland by monotypy. Anatea formicaria Berland
Figs 1–12, 42 All three ants are of similar appearance (but may not be
conspecific) and are from the genus Pheidole. The Pheidole
specimens are of pale golden colour, similar to A. formicaria
or our species “J5” (and possibly similar to juveniles of species
“J1”). All these three Anatea species occur together: QM
S10730 and QM S10732 that now contain A. fomicaria and
species “J5”, respectively, were separated from QM S88025,
which now contains species “J1”; QM S88026 contains adults
and a juvenile of species “J1” and a subadult A. ?formicaria. Of note is the size of the ants, which at c. 1.6 mm total length
are considerably smaller than adult Anatea of either similarly
coloured species, and slightly smaller than the sub adult male
Anatea spp. included in two of the samples. Other material examined. NEW CALEDONIA: 1♀ QM
S34107, Rivière Bleu, Parc.6, 22°05'S 166°40'E, 160 m, 9–22. iv.1987, L. Bonnet de Larbogne, J. Chazeau & A. & S. Tillier,
Malaise trap, rainforest sassafras; 1♂ QM S34113, Rivière
Bleu, Parc.7, 22°05'S 166°40'E, 170 m, 25.xi–8.xii.1986, L. Bonnet de Larbogne, J. Chazeau & A. & S. Tillier, Malaise
trap; 1♂ 1♀ QM S20728, Mt Koghis, 22°11'S 166°32'E, 500
m, 23–24.v.1987, R. Raven, rainforest, general and night
collection; 1♀, QM S107230, Pic d’Amoa, N slopes, 20°58'S
165°17'E, 500 m, 27.xi.2003–30.1.2004, G. Monteith, with
Pheidole sp. ant, flight intercept trap, SC11482; 1♂ KS.64090,
Mt Panie, 20°38'S 164°46'E, 6.vi.1996, M. Moulds; 1♀ WAM
T86527, Col de Rossettes, 21°27'S, 165°28'E, 11.ii.1993,
Harvey, M.S., Platnick, N. I., Raven, R.J., rainforest litter. Other material examined. NEW CALEDONIA: 1♀ QM
S34107, Rivière Bleu, Parc.6, 22°05'S 166°40'E, 160 m, 9–22. iv.1987, L. Bonnet de Larbogne, J. Chazeau & A. & S. Tillier,
Malaise trap, rainforest sassafras; 1♂ QM S34113, Rivière
Bleu, Parc.7, 22°05'S 166°40'E, 170 m, 25.xi–8.xii.1986, L. Bonnet de Larbogne, J. Chazeau & A. & S. Tillier, Malaise
trap; 1♂ 1♀ QM S20728, Mt Koghis, 22°11'S 166°32'E, 500
m, 23–24.v.1987, R. Raven, rainforest, general and night
collection; 1♀, QM S107230, Pic d’Amoa, N slopes, 20°58'S
165°17'E, 500 m, 27.xi.2003–30.1.2004, G. Monteith, with
Pheidole sp. ant, flight intercept trap, SC11482; 1♂ KS.64090,
Mt Panie, 20°38'S 164°46'E, 6.vi.1996, M. Moulds; 1♀ WAM
T86527, Col de Rossettes, 21°27'S, 165°28'E, 11.ii.1993,
Harvey, M.S., Platnick, N. I., Raven, R.J., rainforest litter. Figure 12. Recorded localities of Anatea formicaria Berland in
New Caledonia. Size range. Male (n = 3). Carapace range, 0.75–0.80. Total
length 2.05–2.15. Anatea formicaria Berland
Figs 1–12, 42 (which we have re-identified as a closely related species, “J5”,
see Figs 41, 42), they write: “Anatea formicaria is an accurate
mimic of the small myrmicine ant Chelaner croceiventre
(Emery), 2.6 mm long, which has been collected at the same
locality as the spider. The color pattern (dark brown anterior
and light, yellow-brown posterior) of both is quite rare in ants
and is also found in specimens of Xiphomyrma tenuierius
Emery, 2.9 mm. long, and a species of Lordomyrma, 4.8
mm long, two myrmicine ants found in the same area of rain
forest (E. O. Wilson, pers. comm.).” The colour description
given above, does not match the specimens we have seen of
either A. formicaria or “J5”, in which the cephalothorax is
only slightly darker than the yellow-amber ground colour of
the abdominal scute (see Figs 41, 42). This difference may
be due to time since preservation or could represent colour
polymorphism within the species “J5”, as is recorded in some
other myrmecomorphs (see examples in Cushing, 1997). Berland’s original description of A. formicaria (1927) gives
colouration of cephalothorax, pedicel and abdominal scuta as
“light reddish fawn” (“fauve rougeâtre clair”) but this is of
minimal help as these were dried specimens of uncertain age. Figs 1–12, 42 Anatea formicaria Berland, 1927: 55, figs 1–7. Not species figured by Reiskind & Levi, 1967: 21, figs 1–6
(misidentification, we refer this to sp. “J5”, Fig. 41). Diagnosis. Male: From other New Caledonian species
shown in Figs 38–41, 43 by pedicel and carapace
morphology and careful comparison of palp, particularly
embolus length. From Australian species, leg I longer than
leg III, pedicel with “node”, cymbium with extended apex,
ventral abdomen with ventral plates (but note male of A. elongata sp. nov. is unknown). Female: from undescribed
New Caledonian species by general morphology (as male)
and details of genitalia; from Australian species by leg I longer
than leg III, pedicel with “node”, dorsal scutum present. Type material (not examined). 1♂ type, 1♀ cotype, New
Caledonia. Found by Berland (dried) in the collection of
ants of Ernest André. Berland does not state what he did
with the specimens. p
p
g
Three of the Anatea samples we have examined each
contain a single ant, which we deduce may have been included
by collector or sorter of the specimens as a putative model. Anatea Berland, 1927: 53–55. Type species Anatea
formicaria Berland by monotypy. Diagnosis. Spiders of genus Anatea exhibit several typical
hadrotarsine characters, e.g., sperm duct entering embolus
counter-clockwise, cheliceral promargin without teeth,
shortened cheliceral base, long slender cheliceral fang,
palmate female palpal claw, grouped flat-tipped sensory
setae on tarsus I (Agnarsson, 2004). They differ from many
other hadrotarsine genera by having only one main pair of
spermathecae and carapace not elevated, and differ from all
known hadrotarsine genera by the extended and sometimes
modified pedicel. Smith et al.: The ant-mimic spider genus Anatea 5 Anatea formicaria Berland
Figs 1–12, 42
Anatea formicaria Berland, 1927: 55, figs 1–7. Not species figured by Reiskind & Levi, 1967: 21, figs 1–6
(misidentification, we refer this to sp. “J5”, Fig. 41). Anatea monteithi Smith sp. nov.
Figures 13–24, 31, 35–37 One pair of dorsal apodemes,
prominent in female, embedded in scutum of male; three
circular ventral plates/apodemes in both sexes (Figs 17,
19). Genitalia. Male: Cy blunt with two short, broad based
tooth-like modified spines (Fig. 21). Palp with Co, TTA,
MA (Fig. 35); TR extends ventrally to wrap over the ES,
Co arises from apicodorsal T and apicolateral tegular rim
(Figs 35, 37). TTA large, arising basally, extending beyond
tip of Cy. ES tip resting between TTA and Co. MA base
and basal E conjoined proventrally (Figs 35, 36). Female:
externally with ridges and grooves in lateral profile (Fig. 23),
projecting lip on posterior margin of ovoid fossa. Internally
(Fig. 22), ID exit fossa posteriorly and almost immediately
turn anterolaterally, entering spermathecae Sp at posterior
end. Fertilisation ducts FD exit adjacent to ID, with terminal
nodule (possibly degenerate second pair of Sp) before
terminal part of FD. Holotype ♀ QM S25842, Mt Formartine South, 10 km N
Kuranda, [16°43'S 145°37'E, Queensland, Australia] 700
m, 23.xi.1990, G. Monteith, G. Thompson, pyrethrum
trees & logs. Paratypes. 1♂, WAM T99420, Daintree NP,
Alexandra Range lookout point, 16°14'15"S 145°26'10"E,
6.iv.2009, K. Edward, J. Waldock, sieved litter (WT 11);
1♂, AM KS.126452, Black Mountain Rd, start of track to
Mt Formartine, 16°45'04"S 145°36'21"E, 425 m, 6.xii.2008,
G. Milledge, H. Smith, beat, sweep; [right palp on SEM stub
s/1049; right leg I on s/1050]; both Queensland, Australia. Other material examined (Queensland, Australia): 1♀
AM KS.116502, Lake Barrine, E of Yungaburra, 17°15'S
145°38'E, 31.viii.2011, M. Zabka, B. Patoleta; [abdomen
on SEM stub s/1048, legs I, IV on s/1047; epi cleared]. 1
juv. AM KS.7647, Thornton Peak, N of Daintree, 16°10'S
145°22'E, Nov. 1975, M. Gray, leaf litter sample (RF survey
site 40). QM 1 juv. (unregistered), Bellenden Ker Ra, 0.5 km
S Cable Tower 7, 17°16'S 145°51'E, 500 m, 25–31.x.1981,
Earthwatch/Qld Museum, rainforest, pyrethrum logs, stones,
tree trunks. Etymology. The species is named for Geoff Monteith, in
recognition of his enormous contributions to the study of
Australian invertebrates through his collecting activities for
the Queensland Museum. Diagnosis. From New Caledonian species by leg III longer
than legs I and II; pedicel without “node”; male without
extended tip to cymbium; from A. elongata sp. nov. by pedicel
shorter than carapace; female insemination ducts with short
conjoined section, entering spermathecae posteriorly (Fig. 22). Measurements. Male. Carapace range, 0.81 to 0.88 (n =
2). WAM T99420. Anatea formicaria Berland
Figs 1–12, 42 Female (n = 3). Carapace range, 0.83–0.92. Total length 2.40–2.55. Berland (1927) gives the total length
as 2.2 mm for the species. See Berland (1927) for full male
description. Distribution. Recorded from mountainous rainforest sites
across the main island of New Caledonia (Fig. 12). Notes. The habitus and genitalia of A. formicaria male
and female are figured for comparison with the Australian
species. Figure 1 is repeated as Fig. 42 to compare with other
New Caledonian species. Whilst Berland (1927) admitted his hypothesis that A. formicaria was a myrmecomorph could not be verified, he
pointed out that it was found in a collection of ants and that
the original collector had probably mistaken it for an ant;
unfortunately, he did not (apparently) record which species or
genera of ants the spiders were placed with in the collection. However, he comments (p. 53) that the spiders would match
species of either Pheidole or Monomorium, both genera
being well-represented in the New Caledonian ant fauna:
En étudiant les Fourmis de Nouvelle-Calédonie, on trouve
même sans trop de peine que l’Araignée se rapprocherait
beaucoup d’un Pheidole ou d’un Monomorium, genres qui
y sont très bien représentés. f Reiskind & Levi (1967) suggest different candidates as
a model for the species they identify as Anatea formicaria Figure 12. Recorded localities of Anatea formicaria Berland in
New Caledonia. Records of the Australian Museum (2017) Vol. 69 6 Description Colour (in alcohol) (Figs 13–19). Cephalothorax, mouthparts,
sternum, pedicel and male scutum dark chestnut brown, glossy,
except pedicel rugose, female abdomen and parts of male
abdomen not covered by scutum dark charcoal grey, slightly
paler ventrally, cuticle glossy between sparse but prominent
setal bases; dorsal and ventral abdominal apodemes dark
brown, obvious. Legs: (female) coxae and trochanters white
on legs III, IV, suffused with brownish black on legs I, II;
femora pale brown laterally, dark dorsally and ventrally
(legs I, II) or vis. v. (legs III, IV), femora legs III, IV with
distal white band extending onto proximal patella, traces of
same legs I, II; tibiae-tarsi mid-brown proximally to amber
brown distally, darkest coloration ventral on anterior legs to
retrolateral on leg IV; male legs similar but darker, stronger
coloration. Female. Carapace range, 0.86 (n = 2). Holotype QM S25842. Total length, 2.88; carapace length, 0.86; width, 0.69; height,
0.33; abdomen length, 1.68; width, 1.32; pedicel length, 0.56;
clypeus height, 0.16. Eyes: AME, 0.10; PME, 0.06; ALE,
0.07; PLE, 0.06; Limbs (femur + (patella-tibia) + metatarsus
+ tarsus = total): leg I, 0.49 + 0.60 + 0.31 + 0.26 = 1.66; leg
II, 0.48 + 0.58 + 0.26 + 0.30 = 1.61; leg III, 0.50 + 0.61 +
0.26 + 0.34 = 1.71; leg IV, missing (but longest in female
AM KS.116502). Distribution. Northeast Queensland. Recorded from
rainforests, mainly at higher altitudes, between Thornton
Peak and Bellenden Ker, and on the Atherton Tableland at
Yungaburra (Fig. 31). Carapace (Figs 13–15, 18) longer than wide, widest at
coxae II, smooth transition to caput; fovea absent; in profile
gently domed, highest at coxae I (male WAM T99420 rather
flatter than female holotype, other male similar to female);
eye group 3/5 carapace width; AME largest (Figs 14, 15)
and prominent above slightly concave clypeus, clypeus
> 2 × AME diameter in male, slightly lower in female. Labium bluntly triangular (Fig. 16), cheliceral bases shorter
than maxillae, flexible with slender, curved fangs. Female
palpal claw weakly palmate with three or four large teeth. Notes. Without observations of behaviour, it is unclear
exactly if, or how, ant mimicry is achieved in the Australian
species, but as in New Caledonian species, the long pedicel
and overall appearance are ant-like. Relative leg length,
with both III and IV longer than I or II is unusual and may
indicate behavioural modifications. Anatea monteithi Smith sp. nov.
Figures 13–24, 31, 35–37 Total length, 2.35; carapace length, 0.88;
width, 0.62; height, 0.37; abdomen length, 1.25; width, 0.91;
pedicel length, 0.50; clypeus height, 0.22. Eyes: AME, 0.10;
PME, 0.05; ALE, 0.06; PLE, 0.06; Limbs (femur + (patella-
tibia) + metatarsus + tarsus = total): leg I, 0.49 + 0.51 + 0.28
+ 0.24 = 1.51; leg II, 0.44 + 0.54 + 0.25 + 0.29 = 1.51; leg
III, 0.50 + 0.56 + 0.24 + 0.33 = 1.63; leg IV, 0.55 + 0.84 +
0.33 + 0.36 = 2.08. Anatea monteithi Smith sp. nov.
Figures 13–24, 31, 35–37 Legs: 4312 (holotype female missing both fourth legs but
confirmed in female from other material). Legs with sparse
dorsal macrosetae on patellae and tibiae (a few visible in Figs
13, 14), hadrotarsine flat-topped setae (see Agnarsson, 2004)
present on distal tarsus I, few on II. Pedicel sclerotized, open
“S” in lateral view, slightly expanded anterior to apex and
constriction at abdominal articulation (Fig. 18). Abdomen
egg-shaped, widest anterior to mid-point (Figs 17, 19),
constricted by sclerotized band around spinnerets; male with
dorsal scutum covering central abdomen but leaving bare
small surround of cuticle in dorsal view (Fig. 13), sclerotized
around pedicel and genital plate (Fig. 17); female without
scutum (Fig. 18) and only small sclerotized band around
pedicel insertion (Fig. 19). One pair of dorsal apodemes,
prominent in female, embedded in scutum of male; three
circular ventral plates/apodemes in both sexes (Figs 17,
19). Genitalia. Male: Cy blunt with two short, broad based
tooth-like modified spines (Fig. 21). Palp with Co, TTA,
MA (Fig. 35); TR extends ventrally to wrap over the ES,
Co arises from apicodorsal T and apicolateral tegular rim
(Figs 35, 37). TTA large, arising basally, extending beyond
tip of Cy. ES tip resting between TTA and Co. MA base
and basal E conjoined proventrally (Figs 35, 36). Female:
externally with ridges and grooves in lateral profile (Fig. 23),
projecting lip on posterior margin of ovoid fossa. Internally
(Fig. 22), ID exit fossa posteriorly and almost immediately
turn anterolaterally, entering spermathecae Sp at posterior
end. Fertilisation ducts FD exit adjacent to ID, with terminal
nodule (possibly degenerate second pair of Sp) before
terminal part of FD. Legs: 4312 (holotype female missing both fourth legs but
confirmed in female from other material). Legs with sparse
dorsal macrosetae on patellae and tibiae (a few visible in Figs
13, 14), hadrotarsine flat-topped setae (see Agnarsson, 2004)
present on distal tarsus I, few on II. Pedicel sclerotized, open
“S” in lateral view, slightly expanded anterior to apex and
constriction at abdominal articulation (Fig. 18). Abdomen
egg-shaped, widest anterior to mid-point (Figs 17, 19),
constricted by sclerotized band around spinnerets; male with
dorsal scutum covering central abdomen but leaving bare
small surround of cuticle in dorsal view (Fig. 13), sclerotized
around pedicel and genital plate (Fig. 17); female without
scutum (Fig. 18) and only small sclerotized band around
pedicel insertion (Fig. 19). Description by pedicel longer than
carapace; insemination ducts with long conjoined section,
crossing ventral spermathecae to entry point towards anterior
(Fig. 30). Description The tropical rainforests of northeastern Queensland
occupied by A. monteithi hold an extremely high diversity
of ants, with over 66 genera recorded in these areas (Antwiki, Smith et al.: The ant-mimic spider genus Anatea Smith et al.: The ant-mimic spider genus Anatea 7 Smith et al.: The ant-mimic spider genus Anatea Figures 13–19. Anatea monteithi sp. nov. (13–17) male, habitus and detail (WAM T99420): (13) dorsal; (14) lateral; (15) frontal;
(16) prosoma, ventral; (17) opisthosoma, ventral. (18, 19) Holotype female (QM S25842): (18) habitus, lateral; (19) opisthosoma,
ventral. Scale bars (13, 18) 0.5 mm. Figures 13–19. Anatea monteithi sp. nov. (13–17) male, habitus and detail (WAM T99420): (13) dorsal; (14) lateral; (15) frontal;
(16) prosoma, ventral; (17) opisthosoma, ventral. (18, 19) Holotype female (QM S25842): (18) habitus, lateral; (19) opisthosoma,
ventral. Scale bars (13, 18) 0.5 mm. Figures 13–19. Anatea monteithi sp. nov. (13–17) male, habitus and detail (WAM T99420): (13) dorsal; (14) lateral; (15) frontal;
(16) prosoma, ventral; (17) opisthosoma, ventral. (18, 19) Holotype female (QM S25842): (18) habitus, lateral; (19) opisthosoma,
ventral. Scale bars (13, 18) 0.5 mm. Records of the Australian Museum (2017) Vol. 69 8 Figures 20–24. Anatea monteithi sp. nov. genitalia, (20, 21) male palpus (AM KS.126452): (20) prolateral; (21) ventral. (22–24) Female:
(22) internal, dorsal (AM KS.116502); (23, 24) external views (holotype QM S25842): (23) lateral; (24) ventral. Scale bars 0.1 mm. FD,
fertilization duct; ID, insemination duct; Sp, spermatheca. Figures 20–24. Anatea monteithi sp. nov. genitalia, (20, 21) male palpus (AM KS.126452): (20) prolateral; (21) ventral. (22–24) Female:
(22) internal, dorsal (AM KS.116502); (23, 24) external views (holotype QM S25842): (23) lateral; (24) ventral. Scale bars 0.1 mm. FD,
fertilization duct; ID, insemination duct; Sp, spermatheca. 2017). If A. monteithi is indeed an ant mimic, candidate
models are found in several myrmecine genera. Examples
of genera with species in an appropriate size range for adult
spiders include Monomorium, Orectognathus, Pheidole,
Pristomyrmex and Vombisidris. Diagnosis. From New Caledonian species by leg III longer
than legs I and II; pedicel longer than carapace, without
“node”; from A. monteithi sp. nov. by pedicel longer than
carapace; insemination ducts with long conjoined section,
crossing ventral spermathecae to entry point towards anterior
(Fig. 30). Diagnosis. From New Caledonian species by leg III longer
than legs I and II; pedicel longer than carapace, without
“node”; from A. monteithi sp. nov. Description Anatea elongata Smith sp. nov. Figures 25–31
Holotype ♀ ANIC 42 002257, 12 km SSE Heathlands,
11°51'S 142°38'E [Queensland, Australia], 25.iv–7.vi.1992,
T. McLeod, FIT#2, F.I.T., ANIC 1248, closed forest. Colour (in alcohol). Probably similar to A. monteithi but
specimen extremely faded, pedicel appears rather reddish-
brown; coxae brown, trochanters white on legs III and IV;
otherwise apparently similar to A. monteithi except extent
of abdominal colour cannot be established as only 1 patch
remains. Figures 25–31 Holotype ♀ ANIC 42 002257, 12 km SSE Heathlands,
11°51'S 142°38'E [Queensland, Australia], 25.iv–7.vi.1992,
T. McLeod, FIT#2, F.I.T., ANIC 1248, closed forest. Etymology. The species is named in reference to the pedicel,
the longest compared to carapace length so far observed in
genus Anatea. Carapace (Figs 25, 26, 29) generally similar to female of
A. monteithi; eye group slightly over half carapace width Smith et al.: The ant-mimic spider genus Anatea
9
Figures 25–30. Anatea elongata sp. nov. holotype female (ANIC 42 002257). (25–27) Habitus: (25) dorsal; (26) lateral; (27) ventral. (28)
Opisthosoma, ventral. (29) Prosoma, frontal. (30) Epigynum and internal structure, ventral (uncleared). Scale bar (25) 0.5 mm, (30) 0.1 mm. Smith et al.: The ant-mimic spider genus Anatea 9 Figures 25–30. Anatea elongata sp. nov. holotype female (ANIC 42 002257). (25–27) Habitus: (25) dorsal; (26) lateral; (27) ventral. (28)
Opisthosoma, ventral. (29) Prosoma, frontal. (30) Epigynum and internal structure, ventral (uncleared). Scale bar (25) 0.5 mm, (30) 0.1 mm. Figures 25–30. Anatea elongata sp. nov. holotype female (ANIC 42 002257). (25–27) Habitus: (25) dorsal; (26) lateral; (27) ventral. (28)
Opisthosoma, ventral. (29) Prosoma, frontal. (30) Epigynum and internal structure, ventral (uncleared). Scale bar (25) 0.5 mm, (30) 0.1 mm. Records of the Australian Museum (2017) Vol. 69 10 Figure 31. Recorded localities of Anatea monteithi sp. nov. () and
Anatea elongata sp. nov. () in North East Queensland. damaging the specimen. Due to the lack of pigment, the
external view is sufficient to see the route of the ducts and
this is clearly different from A. monteithi. The habitat around the type locality is generally savannah
woodland, or “closed forest” on the label, a contrast to
the rainforest habitat of A. monteithi. The condition of the
specimen is not good enough to determine gloss (except
carapace) or be sure of colour. Discussion () in North East Queensland. (Fig. 29); eyes and mouthparts similar to A. monteithi. Female palpal claw strongly palmate with at least 8 teeth. Legs: 43(12). Pedicel sclerotized, almost straight ventrally
in anterior half then arching posteriorly, narrowest anteriorly
and thickest, slightly ridged at mid-point, much longer than
carapace (Figs 25–27). Abdomen as in A. monteithi. Legs
missing any visible setae but setal bases suggest macrosetae
probably present as in A. monteithi. Genitalia. Externally with fine edge curving anterior to
ovoid fossa, but otherwise apparently smooth in profile. Internal genitalia not dissected but visible through cuticle
(Fig. 30), ID exit fossa to posterior, running together for 2/3
of distance to genital groove, then turning away from each
other and simultaneously turning dorsally, crossing ventral
Sp and entering anteroventrally. Sp lying to either side of
fossa in ventral view. FD not visible. Measurements. Female.Total length, 3.04; carapace length,
0.93; width, 0.72; height, 0.38; abdomen length, 1.32; width,
0.90; pedicel length, 1.06; clypeus height, 0.17. Eyes: AME,
0.09; PME, 0.06; ALE, 0.06; PLE, 0.06; Limbs (femur +
(patella-tibia) + metatarsus + tarsus = total): leg I, 0.52 +
0.60 + 0.30 + 0.26 = 1.68; leg II, 0.50 + 0.60 + 0.28 + 0.30
= 1.68; leg III, 0.58 + 0.68 + 0.32 + 0.38 = 1.96; leg IV, 0.70
+ 0.98 + 0.42 + 0.42 = 2.52. There are several differences between the New Caledonian
and Australian species that make us question whether Anatea
is the correct placement for these new species. First, the
unusually long rear legs of the Australian species give a leg
length pattern of 4312. The typical hadrotarsine leg pattern
is 4123, which is also found in the New Caledonian Anatea
species. The long leg III is therefore highly unusual and Discussion We here describe two new hadrotarsine species (Theridiidae)
in the previously monotypic genus Anatea. Species of
this genus are remarkable in showing what appears to be
detailed ant mimicry that, unlike in most other ant mimics,
is achieved by the modification of the pedicel that connects
the cephalothorax to the abdomen. Similarities of Anatea
fang morphology to Hadrotarsinae that are known to prey on
ants may indicate another example of myrmecophagy in the
subfamily, and lends support to a potential coevolutionary
history of hadrotarsine and ant lineages (Liu et al., 2016).i y
g
(
)
The New Caledonian specimens of A. formicaria figured
here have been extracted from mixed Anatea material
deposited in the Queensland Museum (see Appendix for
details). Many of these samples were collected by Geoff
Monteith, as was at least one of the Australian specimens. These few samples alone suggest that many more Anatea
species, or relatives, await discovery in New Caledonia,
including species without elongate pedicels and sometimes
with other pedicular modifications that could mimic ants
in subfamilies other than Myrmicinae (Figs 38–41, 43). It
has also become apparent that the male of Reiskind & Levi
(1967) was not in fact A. formicaria, but a rather similar
species with a slightly shorter embolus and longer, more
stepped pedicel—both species were found among these
samples (Fig. 41). New Caledonia is not the focus of the
present Australian study, so we merely flag the potential
of this fascinating genus for further work there. However,
it is noteworthy how abundant and diverse Anatea would
appear to be in New Caledonia in comparison to Australia. One mixed New Caledonia sample in the QM collection
was found to contain three different species; another
included 8 males of one species. Our Australian material,
by contrast, is all singletons and it has proven difficult to
find more. As well as the unique female of A. elongata,
A. monteithi is represented by only four adults and two
juveniles. Considering the collecting effort put in by Geoff
Monteith over many years in Queensland compared to a
few visits to New Caledonia and use of similar collecting
techniques, the difference is astounding. Perhaps the species
we are describing tentatively as Anatea in Australia might
be utilising a quite different niche and / or behaviour to their
New Caledonian relatives.f Figure 31. Recorded localities of Anatea monteithi sp. nov. () and
Anatea elongata sp. nov. Description However, if the species is
an ant mimic the extreme extension of the pedicel, should
facilitate the identification of the model in ant samples taken
from the area. Male. Unknown Distribution. Northeast Queensland. Recorded only from
the type locality on the Cape York Peninsula (Fig. 31). Notes. The single female has lost most of its colour and may
not be robust enough to excise the genitalia without severely Smith et al.: The ant-mimic spider genus Anatea 11 Figures 32–37. Anatea spp. SEM male palpus. (32–34) undescribed species “J1” related to A. formicaria (QM S88025; see Fig. 38): (32)
ventral (unexpanded right palp, image reversed); (33) left palp expanded, prolateral; (34) ditto, retrolateral. (35–37) Anatea monteithi
sp. nov. right palp, image reversed (AM KS.126452): (35) ventral; (36) prolateral; (37) retrolateral. Scale bars 20 μm. Co conductor; Cy
cymbium; E embolus; ES embolus spire; MA median apophysis; ST subtegulum; T tegulum; TR retrolateral tegulum rim; TTA theridiid
tegular apophysis. Figures 32–37. Anatea spp. SEM male palpus. (32–34) undescribed species “J1” related to A. formicaria (QM S88025; see Fig. 38): (32)
ventral (unexpanded right palp, image reversed); (33) left palp expanded, prolateral; (34) ditto, retrolateral. (35–37) Anatea monteithi
sp. nov. right palp, image reversed (AM KS.126452): (35) ventral; (36) prolateral; (37) retrolateral. Scale bars 20 μm. Co conductor; Cy
cymbium; E embolus; ES embolus spire; MA median apophysis; ST subtegulum; T tegulum; TR retrolateral tegulum rim; TTA theridiid
tegular apophysis. Records of the Australian Museum (2017) Vol. 69 12 12
Records of the Australian Museum (2017) Vol. 69
Figures 38–43. New Caledonian Anatea spp. and relatives, diversity (males, except (39) is female). (38) “J1” (QM S88025; the species
n Figs 32–34). (39) “J2” (QM S34185; note spines on pedicel). (40) “J4” (QM S37719; with “node” but not greatly elongated pedicel). 41) “J5” (QM S107231; Reiskind & Levi’s species). (42) Anatea formicaria (AM KS.64090). (43) “J8” (QM S88024; another short-
pedicel species with only a slight step, no “node”). “J3” and “J6” missing from sequence are the Queensland species. Scale bars 0.5 mm. s evident in specimens of both known Australian species
and Hickman (1951: figs 27 33) illustrates the considerable Figures 38–43. New Caledonian Anatea spp. and relatives, diversity (males, except (39) is female). (38) “J1” (QM S88025; the species
in Figs 32–34). (39) “J2” (QM S34185; note spines on pedicel). (40) “J4” (QM S37719; with “node” but not greatly elongated pedicel). (41) “J5” (QM S107231; Reiskind & Levi’s species). (42) Anatea formicaria (AM KS.64090). (43) “J8” (QM S88024; another short-
pedicel species with only a slight step, no “node”). Male. Unknown “J3” and “J6” missing from sequence are the Queensland species. Scale bars 0.5 mm. and Hickman (1951: figs 27, 33) illustrates the considerable
differences in superficial morphology of the Australian
Anatea species, but also shows significant similarities. With
only two male specimens of A. monteithi in our possession,
it was not deemed desirable to further investigate palpal
structure. The usual theridiid sclerites of A. monteithi are
all identifiable in the definitions used by Agnarsson (2004),
whilst those of A. formicaria, like “Euryopis” petricola,
have one of either theridiid tegular apophysis or conductor
missing, or the two are fused (see Figs 32, 35). Despite the
differences, A. monteithi is currently the sister taxon of the
representative New Caledonian species “J1” in our draft
phylogeny and, although as yet incomplete, this guides our
tentative placement here. is evident in specimens of both known Australian species. The pedicel, whilst elongate, is simple, lacking the “node”
seen in most New Caledonian species; the sclerites of
the attachment zone between cephalothorax and pedicel
are also subtly different. Finally, the male palp of all the
examined New Caledonian Anatea species is apparently
rather conservative and similar to that of Australian relatives
of the New Zealand species “Euryopis” nana (O. Pickard-
Cambridge, 1880), such as “Euryopis” petricola (Hickman,
1951). Both of these species were described in the genus
Atkinsonia O. Pickard-Cambridge, 1879 (replacement name
Atkinia Strand, 1929). Indeed, Anatea, as circumscribed
here, is the sister group to these “Atkinia” taxa in a draft
phylogeny (unpublished notes). A comparison of Figs 8, 9, 21 is evident in specimens of both known Australian species. The pedicel, whilst elongate, is simple, lacking the “node”
seen in most New Caledonian species; the sclerites of
the attachment zone between cephalothorax and pedicel
are also subtly different. Finally, the male palp of all the
examined New Caledonian Anatea species is apparently
rather conservative and similar to that of Australian relatives
of the New Zealand species “Euryopis” nana (O. Pickard-
Cambridge, 1880), such as “Euryopis” petricola (Hickman,
1951). Both of these species were described in the genus
Atkinsonia O. Pickard-Cambridge, 1879 (replacement name
Atkinia Strand, 1929). Indeed, Anatea, as circumscribed
here, is the sister group to these “Atkinia” taxa in a draft
phylogeny (unpublished notes). Manuscript submitted 16 January 2017, revised 4 April 2017, and
accepted 5 April 2017. The authors are:
ORCID Smith https://orcid.org/0000-0002-0960-4367
ORCID Harvey https://orcid.org/0000-0003-1482-0109
ORCID Agnarsson https://orcid.org/0000-0003-4548-7927
ORCID Anderson https://orcid.org/0000-0002-8814-5866 Male. Unknown A comparison of Figs 8, 9, 21 and Hickman (1951: figs 27, 33) illustrates the considerable
differences in superficial morphology of the Australian
Anatea species, but also shows significant similarities. With
only two male specimens of A. monteithi in our possession,
it was not deemed desirable to further investigate palpal
structure. The usual theridiid sclerites of A. monteithi are
all identifiable in the definitions used by Agnarsson (2004),
whilst those of A. formicaria, like “Euryopis” petricola,
have one of either theridiid tegular apophysis or conductor
missing, or the two are fused (see Figs 32, 35). Despite the
differences, A. monteithi is currently the sister taxon of the
representative New Caledonian species “J1” in our draft
phylogeny and, although as yet incomplete, this guides our
tentative placement here. Smith et al.: The ant-mimic spider genus Anatea 13 Acknowledgments. We are grateful to collection managers and
curators who were most helpful in providing loans and advice
on specimens and collections: Graham Milledge (AM); Bruce
Halliday, Cate Lemann, Federica Turco (ANIC); Robert Raven,
Owen Seeman (QM); Julianne Waldock (WAM). Sue Lindsay
took SEM images. Stano Pekar assisted with a reference. Two
anonymous reviewers made helpful suggestions. Derek Smith
identified the genus of ants in the New Caledonian samples. This
work was funded under an Australian Biological Resources Study
grant 207-26 and further supported by the Australian Museum. Hickman, V. V. 1951. New Phoroncidiinae and the affinities of
the New Zealand spider Atkinsonia nana Cambridge. Papers
and Proceedings of the Royal Society of Tasmania 1950: 3–24. Liu, J., L. J. May-Collado, S. Pekár, and I. Agnarsson. 2016. A
revised and dated phylogeny of cobweb spiders (Araneae,
Araneoidea, Theridiidae): A predatory Cretaceous lineage
diversifying in the era of the ants (Hymenoptera, Formicidae). Molecular Phylogenetics and Evolution 94: 658–675. https://doi.org/10.1016/j.ympev.2015.09.023 McIver, J. D., and G. Stonedahl. 1993. Myrmecomorphy:
morphological and behavioral mimicry of ants. Annual Review
of Entomology 38: 351–379. https://doi.org/10.1146/annurev.en.38.010193.002031 References Reiskind, J. 1972. Morphological adaptation for ant-mimicry
in spiders. Arachnologorum Congressus Internationalis V,
Proceedings of the Fifth International Congress on Arachnology,
ed. C. Folk C, pp. 221–226, Brno, Czech Republic. Agnarsson, I. 2004. Morphological phylogeny of cobweb spiders
and their relatives (Araneae, Araneoidea, Theridiidae). Zoological Journal of the Linnean Society 141: 447–626. https://doi.org/10.1111/j.1096-3642.2004.00120.x Reiskind, J., and H. W. Levi. 1967. Anatea, an ant-mimicking
theridiid spider from New Caledonia (Araneae, Theridiidae). Psyche, Cambridge 74: 20–23. https://doi.org/10.1155/1967/46979 Antwiki, 2017. Australian Ant Distribution Patterns. [Accessed
28 March 2017]
http://www.antwiki.org/wiki/Australian_Ant_Distribution_Patterns Berland, L. 1927. Sur une araignée myrmécomorphe de Nouvelle
Calédonie. Bulletin de la Société Entomologique de France
1927: 52–55. World Spider Catalog. 2017. World Spider Catalog, version 17.5. Natural History Museum Bern. [Accessed 8 January 2017]. http://wsc.nmbe.ch Cushing, P. E. 1997. Myrmecomorphy and myrmecophily in
spiders: a review. Florida Entomologist 80: 165–193. https://doi.org/10.2307/3495552 Cushing, P. E. 2012. Spider-ant associations: an updated review of
myrmecomorphy, myrmecophily, and myrmecophagy in spiders. Psyche 2012: 1–23. https://doi.org/10.1155/2012/151989 Appendix. List of specimens examined representing species shown in Figs 38–41, 43 (all localities in
New Caledonia). “J1”: QM S37930, 1♀, Mt Aoupinié, 21°01'S 165°18'E, May–Oct. 1992, R. Raven, E. Guilbert, pitfalls. “J1”: QM S37930, 1♀, Mt Aoupinié, 21°01'S 165°18'E, May–Oct. 1992, R. Raven, E. Guilbert, pitfalls. p
“J1”: QM S88025, 8♂♂, 1♀, Pic d’Amoa, N slopes, 20°58'S 165°17'E, 500 m, 27.xi.2003–
30.1.2004, G. Monteith, flight intercept trap, SC11482. (1 ♂ on SEM stub). “J1”: QM S88025, 8♂♂, 1♀, Pic d’Amoa, N slopes, 20°58'S 165°17'E, 500 m, 27.xi.2003–
30.1.2004, G. Monteith, flight intercept trap, SC11482. (1 ♂ on SEM stub). ♂ “J1”: QM S88026, 1 juv. ♂, 2♀♀, Mt Aoupinié, top camp, 21°11'S 165°18'E, 850 m,
23.xi.2001, G.B. Monteith, sieved litter, QM Berlesate 1045. Sample
includes a Pheidole sp. ant, and a juv. ♂ A. formicaria. (Abdomen except
epigyne, and some legs of 1 ♀ on SEM stub). “J1”: QM S88026, 1 juv. ♂, 2♀♀, Mt Aoupinié, top camp, 21°11'S 165°18'E, 850 m,
23.xi.2001, G.B. Monteith, sieved litter, QM Berlesate 1045. Sample
includes a Pheidole sp. ant, and a juv. ♂ A. formicaria. (Abdomen except
epigyne, and some legs of 1 ♀ on SEM stub). “J2”: QM S34185, 1♀, Rivière Bleu, Parc.5, 150 m, 25.xi–8.xii.1986, L. Bonnet de Larbogne
J. Chazeau & A. & S. Tillier, Malaise trap, rainforest. ♂ “J4”: QM S37719, 1♂, Mandjélia, 20°24'S 164°32'E, 13 May–Oct. 1992, R. Raven, E. Guilbert, G. Ingram, pitfalls. References g
p
“J4”: QM S60485, 1♀, Ningua Res. camp, 21°45'S 166°09'E, 1100 m, 12–13.xi.2001, G.B. Monteith, rainforest, sieved litter, QM Berlesate 1039. “J4”: QM S55109, 1♀, Mt Do summit, 21°45'S 166°00'E, 1000 m, 21.xi.2000, G.B. Monteith,
pyrethrum, trunks and logs, SC9919. “J5”: QM S107231, 1♂, Mandjelia summit, 20°24'S 164°32'E, 750 m, 29.xi.2003–31.1.2004,
G. Monteith, flight int. trap. SC11486. “J5”: QM S107232, 1♀, 1 juv., Pic d’Amoa, N slopes, 20°58'S 165°17'E, 500 m, 27.xi.2003–
30.1.2004, G. Monteith, with a juvenile A. formicaria and a Pheidole sp. ant, flight intercept trap, SC11482. ,l
g
p
p,
“J8”: QM S88024, 3♂♂, Mandjelia summit, 20°24'S 164°32'E, 750 m, 29.xi.2003–31.1.2004,
G. Monteith, flight int. trap. SC11486. 14 Records of the Australian Museum (2017) Vol. 69 14
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Autochthonous Infections with Hepatitis E Virus Genotype 4, France
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Emerging infectious diseases
| 2,012
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cc-by
| 3,067
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Autochthonous
Infections with
Hepatitis E Virus
Genotype 4, France IgM Rapid Test (MP Biomedicals, Illkirch, France) and
the recomLine HEV IgG/IgM test (Mikrogen Diagnostik,
Neuried, Germany). HEV RNA was detected by using a
real-time reverse transcription PCR targeting open reading
frame (ORF) 2 (2). The mean age of the case-patients was 57 years (±11
years). Of the 11 case-patients, 10 were male and 3 were
kidney transplant recipients (Tables 1, 2). HEV infection
was clinically asymptomatic in all transplant recipients;
the infection was diagnosed after routine posttransplant
laboratory tests showed elevated levels of liver enzymes. Longitudinal testing indicated chronic HEV infection in 1
case-patient (no. 5), and an 80-year-old case-patient died 9
weeks after disease onset. During January–March 2011, diagnoses of hepatitis
E virus (HEV) infection increased in Marseille University
hospitals in southeastern France. HEV genotype 4, which
is described almost exclusively in Asia, was recovered from
2 persons who ate uncooked pork liver sausage. Genetic
sequences were 96.7% identical to those recently described
in swine in Europe. HEV 5′-ORF2 RNA was recovered from the serum
of 8 case-patients. Phylogenetic analysis (2) showed
that 4 patients each had HEV genotype 3c or 3f (online
Technical Appendix Figure 2). Sequences from 2
unrelated case-patients (nos. 7, 8) showed 99.8% identity;
for other pairwise comparisons, maximal identity was
93.4% (mean 83.6%). I
n industrialized countries, most cases of hepatitis E virus
(HEV) infection in humans are autochthonous (1). Pigs
are a major reservoir of HEV, and transmission of virus to
humans who ate raw or undercooked pork has been reported
(1–3). In France, >300 cases of HEV infection are reported
annually (3); most infections are autochthonous and occur
in southern France, where the prevalence of anti-HEV IgG
is higher than in northern France (3–5). Almost all cases
of autochthonous HEV infection reported in Europe have
involved genotype 3 strains (1–6). I We could not recover HEV 5′-ORF2 RNA from the
serum of 3 case-patients (nos. 1, 2, 6). For case-patient
6, this could be explained by a low viral load. When
targeting ORF1 and 3′-ORF2, we recovered HEV genotype
4 (HEV-4) from serum samples from case-patients 1 and
2 (Figure; online Technical Appendix Figure 3) (5,10). Molecular testing results for these samples were checked in
duplicate and by testing different serum samples from the
same case-patients. HEV-4 RNA from the 2 case-patients
showed 100% identity for ORF1 and 99.8% identity for
ORF2. Autochthonous
Infections with
Hepatitis E Virus
Genotype 4, France The next closest match (96.7% identity) was the
fi rst HEV-4 RNA in swine reported in Europe (GenBank
accession no. HQ857384) (Figure) (8). The next 2 closest
matches (91.0%–91.3% identity) were recovered from
swine in China (GenBank accession nos. EU676172 and
DQ279091) (Figure). However, the sequences showed only
86.0% identity with a genotype 4f HEV RNA recovered
from a patient in Germany (9). Beginning February 21, 2011, a biosurveillance
program (EPIMIC) (7) detected an increase in the number
of HEV infections diagnosed at Marseille University
hospitals in southeastern France. During February 21–
March 28, the weekly number of serum samples that were
tested and found positive for HEV was above the elected
critical threshold (online Technical Appendix Figure 1,
wwwnc.cdc.gov/EID/pdfs/11-1827-Techapp.pdf). Author affi liations: Institut Hospitalo-Universitaire Méditerranée
Infection, Marseille, France (P. Colson, P. Romanet, A. Motte); Aix-
Marseille Université, Marseille (P. Colson); Hôpital Conception,
Marseille (V. Moal, R. Purgus); and Centre Hospitalo-Universitaire
Conception, Marseille (P. Borentain, A. Benezech, R. Gérolami)
DOI: http://dx.doi.org/10.3201/eid1808.111827 The Cases None of the 11 case-patients had traveled abroad
during the 2–9 weeks before hepatitis onset. Of the 11
case-patients, 6 (55%), including the 2 with HEV-4, ate
uncooked pig liver sausage (fi gatelli [2]) (Table 2). The 2
patients with HEV-4 were not related. Because 4 contacts
of the 2 HEV-4–infected case-patients also ate some of
the fi gatelli eaten by the case-patients (Tables 1, 2), we
investigated whether these contacts had markers for HEV
infection. Test results for anti-HEV IgM were positive for
the wife of case-patient 1 and a friend of case-patient 2;
the HEV PCR result was barely positive for contact 1a,
who had an elevated level of alanine aminotransferase (165
IU/L; reference <45 IU/L). These 2 persons were clinically
asymptomatic. In Marseille during January–March 2011, a total of
11 cases of HEV infection were confi rmed by anti-HEV
IgM testing and detection of HEV RNA in serum samples. EIAgen assays (Adaltis Italia, Casalecchio di Reno, Italy)
were used to detect anti-HEV IgM and IgG. Additional anti-
HEV IgM testing was performed by using the Assure HEV Author affi liations: Institut Hospitalo-Universitaire Méditerranée
Infection, Marseille, France (P. Colson, P. Romanet, A. Motte); Aix-
Marseille Université, Marseille (P. Colson); Hôpital Conception,
Marseille (V. Moal, R. Purgus); and Centre Hospitalo-Universitaire
Conception, Marseille (P. Borentain, A. Benezech, R. Gérolami) DOI: http://dx.doi.org/10.3201/eid1808.111827 1361 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 18, No. 8, August 2012 DISPATCHES Table 1. Epidemiologic and virological findings for case-patients and contacts with HEV infection, Marseille, France, January–March
2011*
Case-patients
and contacts,
by ID no. The Cases Day obtained, serum
sample (retrospectively
tested sample)
Anti-HEV IgG
EIA result†
Anti-HEV IgM result, by testing method
HEV RNA in
serum (Ct)
HEV
genotype
EIA†
Rapid test‡
Immunoblot
1
Feb18
10.3
14.2
Positive
Positive
Positive (29)
4
1a§
Mar 2
11.4
0.2
Negative
Not tested
Positive (38)
NA
1a§
Mar 16
>10.0
2.0
Not tested
Borderline
Negative
NA
1b¶
Mar 2
Negative
Negative
Negative
Negative
Negative
NA
2
Mar 21 (Feb 7)
11.2
15.6
Positive
Positive
Positive (28)
4
2a#
Mar 21
6.3
15.6
Positive
Positive
Negative
NA
2b**
Mar 21
0.1
Negative
Negative
Negative
Negative
NA
3
Jan 4
10.7
16.6
Positive
Not tested
Positive (27)
3f
4
Feb 3 (Jan 6)
5.2
6.3
Weakly reactive††
Positive
Positive (26)
3c
5‡‡
Feb 3
0.1
1.6
Weakly reactive
Negative
Positive (31)
3f
6
Mar 1
11
14
Positive
Positive
Positive (38)
NA
7
Mar 2
10.6
15.1
Positive
Negative
Positive (27)
3c
8‡‡
Mar 9
11.1
14.9
Positive
Not tested
Positive (28)
3c
9
Mar 18
10.5
16.4
Positive
Not tested
Positive (28)
3f
10‡‡
Mar 21
0.3
15.6
Positive
Not tested
Positive (24)
3c
11
Mar 23
1.2
13.8
Positive
Positive
Positive (27)
3f
*Italics indicate results for contacts of case-patients. HEV, hepatitis E virus; ID, identification; EIA, enzyme immunoassay; Ct, cycle threshold; NA, not
applicable. †EIA score represents test/cutoff optical density ratios. ‡An immunochromatographic rapid test. §Contact (wife) of case-patient no. 1. ¶Contact (friend) of case-patient no. 1. #Contact (friend) of case patient no 2 Table 1. Epidemiologic and virological findings for case-patients and contacts with HEV infection, Marseille, France, January–March
2011* rological findings for case-patients and contacts with HEV infection, Marseille, France, January–March pp
†EIA score represents test/cutoff optical density ratios. †
p
p
‡An immunochromatographic rapid test. ¶Contact (friend) of case-patient no. 1. #Contact (friend) of case-patient no. 2. *Italics indicate results for contacts of case-patients. HEV, hepatitis E virus; ID, identification; EIA, enzyme immunoassay; Ct
applicable. Conclusions diverse clinical settings, including in solid-organ
transplant recipients. In our study, 3 (27%) of the case-
patients were solid-organ transplant recipients. This
number suggests a high incidence of HEV infection
among transplant recipients in the geographic area we
studied, and it is consistent with the incidence (≈3.2/100
person-years) of HEV infection among organ transplant
recipients in Toulouse in southwestern France (4). Liver We report a concurrent rise in testing for and diagnoses
of HEV infections in Marseille during January–March 2011. This rise may refl ect increased clinician awareness of HEV
or an increased incidence of HEV infection that may have
resulted from the use of improved tools for HEV diagnosis. In industrialized countries, HEV has increasingly
been recognized as a possible cause of hepatitis in Table 2. Epidemiologic and clinical findings for case-patients and contacts with HEV infection, Marseille, France, January–March
2011*
Case-patients and contacts,
by ID no. Age, y/sex
Clinical signs
ALT, IU/L†
Consumed uncooked
pig liver sausage
1
62/M
Asthenia, jaundice, nausea
2,124
Yes
1a‡
57/F
None
165
Yes
1b§
60/M
None
NA
Yes
2
49/M
Asthenia, jaundice
138
Yes
2a¶
46/F
None
40
Yes
2b#
51/F
None
40
Yes
3
45/F
Jaundice
1,696
Yes
4
80/M
Asthenia, jaundice
1,885
No
5**
63/M
None
340
No
6
68/M
Asthenia, fever, jaundice
1,135
Yes
7
54/M
Asthenia, jaundice
88
No
8**
58/M
None
21
No
9
38/M
Asthenia, fever, dark urine
1,554
Yes
10**
52/M
None
71
Yes
11
58/M
Asthenia, jaundice
2,500
No
*Italics indicate features for contacts of case-patients. HEV, hepatitis E virus; ID, identification; ALT, alanine aminotransferase; NA, not available. †Upper limit of normal, 40 IU/L. ‡Contact (wife) of case-patient no.1. §Contact (friend) of case-patient no.1. ¶Contact (friend) of case-patient no.2. #Contact (sister) of case-patient no.2. **Kidney transplant recipient. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 18, No. 8, August 2012 1362 Hepatitis E Virus Genotype 4 in France reported here. Subclinical HEV infection can also occur
in immunocompetent persons (1,11,12); thus, we question
whether the incidence of HEV infection among transplant
recipients in southern France might refl ect that in the
general population. Moreover, 52% of adult blood donors
sampled in southwestern France in 2003–2004 were
retrospectively shown to be HEV seropositive by using a
newly available assay (4), and HEV was retrospectively
shown to be an underdiagnosed cause of liver injury in the
United States and United Kingdom (13). Conclusions ncbi.nlm.nih.gov/Blast.cgi); italics indicates sequences obtained
from a swine in Belgium with HEV genotype 4 (8) and a human
in Germany with autochthonous HEV infection (9); underlining
indicates sequences obtained from humans in Marseille, France,
during January–March 2011; black dots indicate patients who
ate uncooked pig liver sausage. Reference sequences (rf) with
known genotypes and subtypes are indicated (6). Numbers on
right indicate genotype. For sequences from this study, nucleotide
alignments were performed by using ClustalX version 2.0 (www. clustal.org/download/current). The tree was constructed by using
MEGA4 (www.megasoftware.net) and the neighbor-joining method
as described (2). Branches were obtained from 1,000 resamplings
of the data; those with bootstrap values >50% are labeled on
the tree. The avian HEV sequence AM943646 was used as an
outgroup. Scale bar indicates nucleotide substitutions per site. HEV sequences are labeled with GenBank accession number,
host, country where isolated, and collection or submission date. Hu, human; VNM, Vietnam; BBH, best BLAST hit; Sw, swine; CHN,
China; BEL, Belgium; Pt, patient; JPN, Japan; DEU, Germany;
IND, India; Rb, rabbit; ESP, Spain; NLD, the Netherlands; USA,
United States; GBR, United Kingdom; Av, avian. Figure. Phylogenetic tree based on partial (186 nt) sequence of the
5′ end of open reading frame 1 of the hepatitis E virus (HEV) genome
(nt 133–318; GenBank accession no. AB291961). Boldface
indicates sequence with the highest BLAST scores (http://blast. ncbi.nlm.nih.gov/Blast.cgi); italics indicates sequences obtained
from a swine in Belgium with HEV genotype 4 (8) and a human
in Germany with autochthonous HEV infection (9); underlining
indicates sequences obtained from humans in Marseille, France,
during January–March 2011; black dots indicate patients who
ate uncooked pig liver sausage. Reference sequences (rf) with
known genotypes and subtypes are indicated (6). Numbers on
right indicate genotype. For sequences from this study, nucleotide
alignments were performed by using ClustalX version 2.0 (www. clustal.org/download/current). The tree was constructed by using
MEGA4 (www.megasoftware.net) and the neighbor-joining method
as described (2). Branches were obtained from 1,000 resamplings
of the data; those with bootstrap values >50% are labeled on
the tree. The avian HEV sequence AM943646 was used as an
outgroup. Scale bar indicates nucleotide substitutions per site. HEV sequences are labeled with GenBank accession number,
host, country where isolated, and collection or submission date. Conclusions biochemical testing is routinely performed after organ
transplantation; such testing enabled the detection of
subclinical HEV infection in the 3 transplant recipients A considerable proportion of HEV infections in
southern France may be linked to the consumption of
uncooked fi gatelli, which has been identifi ed as a source of
zoonotic foodborne HEV transmission (2,4). In our study,
55% of the HEV-positive patients reported eating uncooked
fi gatelli, and in another study in France, 2 HEV-positive
patients reported eating uncooked fi gatelli (11). These
cases indicate insuffi cient prevention of HEV transmission
to humans through uncooked fi gatelli consumption (2). The major fi nding in our study was the recovery
of HEV-4 sequences from 2 of 6 case-patients who ate
uncooked fi gatelli purchased in southern France. These
sequences were closely related to the sequence for the fi rst-
reported HEV-4 RNA in swine in Europe (8), which was
recovered in Belgium and classifi ed as genotype 4b. HEV-
4 is indigenous to Asia, where it has been recovered from
pigs and humans (1,6,14). For the 2 HEV-4 RNA sequences
obtained in our study, the best matches among sequences
from Asia were from HEV strains from swine in China. Since its fi rst report in China in 1999, HEV-4 has been
increasingly described as endemic in pigs and involved
in most sporadic cases of hepatitis E (1,14). Several HEV
subtypes have been described in China, including subtype
4b (1,6,14). In Europe, autochthonous infection with a
genotype 4f HEV in a person in Germany was reported
in 2008 (9), and a case of HEV-4 infection acquired in
India was reported for a person in England in 2010 (15). Studies in China have shown relatedness between HEV-4
sequences recovered from humans and swine in the same
geographic area, which suggests zoonotic transmission (1). Moreover, sequences for HEV-4 RNA recovered from a
father and son in Japan who ate barbecued pig meat were
almost identical, and sequences were identical for HEV-4
RNA from packaged pig liver sold in a grocery store and
from a patient in whom HEV infection developed after the
person ate grilled pig liver (1,2). Figure. Phylogenetic tree based on partial (186 nt) sequence of the
5′ end of open reading frame 1 of the hepatitis E virus (HEV) genome
(nt 133–318; GenBank accession no. AB291961). Boldface
indicates sequence with the highest BLAST scores (http://blast. References 11. Renou C, Pariente A, Cadranel JF, Nicand E, Pavio N. Clinically
silent forms may partly explain the rarity of acute cases of autoch-
thonous genotype 3c hepatitis E infection in France. J Clin Virol. 2011;51:139–41. http://dx.doi.org/10.1016/j.jcv.2011.02.013 1. Purdy MA, Khudyakov YE. The molecular epidemiology of hepa-
titis E virus infection. Virus Res. 2011;161:31–9. http://dx.doi. org/10.1016/j.virusres.2011.04.030 1. Purdy MA, Khudyakov YE. The molecular epidemiology of hepa-
titis E virus infection. Virus Res. 2011;161:31–9. http://dx.doi. org/10.1016/j.virusres.2011.04.030 2011;51:139–41. http://dx.doi.org/10.1016/j.jcv.2011.02.013 12. Shata MT, Navaneethan U. The mystery of hepatitis E seropreva-
lence in developed countries: is there subclinical infection due to
hepatitis E virus? Clin Infect Dis. 2008;47:1032–4. http://dx.doi. org/10.1086/591971 2. Colson P, Borentain P, Queyriaux B, Kaba M, Moal V, Gallian P,
et al. Pig liver sausage as a source of hepatitis E virus transmis-
sion to humans. J Infect Dis. 2010;202:825–34. http://dx.doi. org/10.1086/655898 13. Davern TJ, Chalasani N, Fontana RJ, Hayashi PH, Protiva P, Kleiner
DE, et al. Acute hepatitis E infection accounts for some cases of sus-
pected drug-induced liver injury. Gastroenterology. 2011;141:1665–
72. http://dx.doi.org/10.1053/j.gastro.2011.07.051 3. Bouquet J, Tessé S, Lunazzi A, Eloit M, Rose N, Nicand E, et al. Close similarity between sequences of hepatitis E virus recovered
from humans and swine, France, 2008–2009. Emerg Infect Dis. 2011;17:2018–25. http://dx.doi.org/10.3201/eid1711.110616 3. Bouquet J, Tessé S, Lunazzi A, Eloit M, Rose N, Nicand E, et al. Close similarity between sequences of hepatitis E virus recovered
from humans and swine, France, 2008–2009. Emerg Infect Dis. 2011;17:2018–25. http://dx.doi.org/10.3201/eid1711.110616 2011;17:2018–25. http://dx.doi.org/10.3201/eid1711.110616 14. Fu H, Wang L, Zhu Y, Geng J, Li L, Wang X, et al. Analysing com-
plete genome sequence of swine hepatitis E virus (HEV), strain
CHN-XJ-SW13 isolated from Xinjiang, China: putative host range,
and disease severity determinants in HEV. Infect Genet Evol. 2011;11:618–23. http://dx.doi.org/10.1016/j.meegid.2011.01.018 4. Mansuy JM, Bendall R, Legrand-Abravanel F, Sauné K, Miédouge
M, Ellis V, et al. Hepatitis E virus antibodies in blood donors, France. Emerg Infect Dis. 2011;17:2309–12. http://dx.doi.org/10.3201/
eid1712.110371 5. Legrand-Abravanel F, Mansuy JM, Dubois M, Kamar N, Peron JM,
Rostaing L, et al. Hepatitis E virus genotype 3 diversity, France. Emerg Infect Dis. 2009;15:110–4. http://dx.doi.org/10.3201/
eid1501.080296 15. Rolfe KJ, Curran MD, Mangrolia N, Gelson W, Alexander GJ,
L’Estrange M, et al. First case of genotype 4 human hepatitis E vi-
rus infection acquired in India. J Clin Virol. 2010;48:58–61. http://
dx.doi.org/10.1016/j.jcv.2010.02.004 6. Lu L, Li C, Hagedorn CH. Conclusions Hu, human; VNM, Vietnam; BBH, best BLAST hit; Sw, swine; CHN,
China; BEL, Belgium; Pt, patient; JPN, Japan; DEU, Germany;
IND, India; Rb, rabbit; ESP, Spain; NLD, the Netherlands; USA,
United States; GBR, United Kingdom; Av, avian. Together with previous fi ndings, our results bring up
the question as to whether HEV-4 was introduced into
domestic pigs or whether pig meat of Asian origin was
introduced into France. Moreover, it prompts a study of
whether HEV-4 strains circulate and spread in Europe as
an effect of globalization of HEV zoonosis. 1363 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 18, No. 8, August 2012 DISPATCHES Acknowledgments 8. Hakze-van der Honing RW, van Coillie E, Antonis AF, van der Poel
WH. First isolation of hepatitis E virus genotype 4 in Europe through
swine surveillance in the Netherlands and Belgium. PLoS ONE. 2011;6:e22673. http://dx.doi.org/10.1371/journal.pone.0022673 We are grateful to Didier Raoult for his encouragement
regarding this work. We thank Audrey Ferretti for technical
assistance. 9. Wichmann O, Schimanski S, Koch J, Kohler M, Rothe C, Plentz
A, et al. Phylogenetic and case–control study on hepatitis E virus
infection in Germany. J Infect Dis. 2008;198:1732–41. http://dx.doi. org/10.1086/593211 Dr Colson is a virologist at Aix-Marseille University and
at Marseille University Hospital. His research interests are viral
hepatitis and emerging viruses. 10. Erker JC, Desai SM, Mushahwar IK. Rapid detection of hepatitis E
virus RNA by reverse transcription–polymerase chain reaction using
universal oligonucleotide primers. J Virol Methods. 1999;81:109–
13. http://dx.doi.org/10.1016/S0166-0934(99)00052-X References Phylogenetic analysis of global hepati-
tis E virus sequences: genetic diversity, subtypes and zoonosis. Rev
Med Virol. 2006;16:5–36. http://dx.doi.org/10.1002/rmv.482 Address for correspondence: Philippe Colson, Pôle des Maladies
Infectieuses et Tropicales Clinique et Biologique, Fédération de
Bactériologie-Hygiène-Virologie,
Centre
Hospitalo-Universitaire
Timone, 264 Rue Saint-Pierre 13385, Marseille CEDEX 05, France;
email: philippe.colson@univmed.fr 7. Parola P, Colson P, Dubourg G, Million M, Charrel R, Minodier P, et
al. Letter to the Editor. Group A streptococcal infections during the
seasonal infl uenza outbreak 2010/11 in South East England. Euro
Surveill. 2011;16:pii=19816. 1364
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 18, No. 8, August 2012 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 18, No. 8, August 2012 1364
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Genetics of early-life head circumference and genetic correlations with neurological, psychiatric and cognitive outcomes
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BMC medical genomics
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University of Groningen University of Groningen Genetics of early-life head circumference and genetic correlations with neurological,
psychiatric and cognitive outcomes
Early Growth Genetics Consortium; Vogelezang, Suzanne; Bradfield, Jonathan P.; Grant,
Struan F. A.; Felix, Janine F.; Jaddoe, Vincent W. V. Published in:
BMC Medical Genomics
DOI:
10.1186/s12920-022-01281-1
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
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2022
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Early Growth Genetics Consortium, Vogelezang, S., Bradfield, J. P., Grant, S. F. A., Felix, J. F., & Jaddoe,
V. W. V. (2022). Genetics of early-life head circumference and genetic correlations with neurological,
psychiatric and cognitive outcomes. BMC Medical Genomics, 15, Article 124. https://doi.org/10.1186/s12920-022-01281-1 Genetics of early-life head circumference and genetic correlations with neurolo
psychiatric and cognitive outcomes Publication date:
2022 Link to publication in University of Groningen/UMCG research database Citation for published version (APA):
Early Growth Genetics Consortium, Vogelezang, S., Bradfield, J. P., Grant, S. F. A., Felix, J. F., & Jaddoe,
V. W. V. (2022). Genetics of early-life head circumference and genetic correlations with neurological,
psychiatric and cognitive outcomes. BMC Medical Genomics, 15, Article 124. https://doi.org/10.1186/s12920-022-01281-1 Citation for published version (APA):
Early Growth Genetics Consortium, Vogelezang, S., Bradfield, J. P., Grant, S. F. A., Felix, J. F., & Jaddoe,
V. W. V. (2022). Genetics of early-life head circumference and genetic correlations with neurological,
psychiatric and cognitive outcomes. BMC Medical Genomics, 15, Article 124. https://doi.org/10.1186/s12920-022-01281-1 Copyright
Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the
author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-
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and investigate your claim. © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract nd: Head circumference is associated with intelligence and tracks from childhood into adulthood. Background: Head circumference is associated with intelligence and tracks from childhood into adulthood. Methods: We performed a genome-wide association study meta-analysis and follow-up of head circumference in a
total of 29,192 participants between 6 and 30 months of age. Methods: We performed a genome-wide association study meta-analysis and follow-up of head circumference in a
total of 29,192 participants between 6 and 30 months of age. Results: Seven loci reached genome-wide significance in the combined discovery and replication analysis of which
three loci near ARFGEF2, MYCL1, and TOP1, were novel. We observed positive genetic correlations for early-life head
circumference with adult intracranial volume, years of schooling, childhood and adult intelligence, but not with adult
psychiatric, neurological, or personality-related phenotypes. Conclusions: The results of this study indicate that the biological processes underlying early-life head circumference
overlap largely with those of adult head circumference. The associations of early-life head circumference with cogni-
tive outcomes across the life course are partly explained by genetics. Keywords: Head circumference, Genome-wide association study, Genetic correlations, Infancy Suzanne Vogelezang1,2,3†, Jonathan P. Bradfield4,5†, the Early Growth Genetics Consortium,
Struan F. A. Grant4,6,7,8†, Janine F. Felix1,2† and Vincent W. V. Jaddoe1,2*† Suzanne Vogelezang1,2,3†, Jonathan P. Bradfield4,5†, the Early Growth Genetics Consortium,
Struan F. A. Grant4,6,7,8†, Janine F. Felix1,2† and Vincent W. V. Jaddoe1,2*† Background loci associated with child and adult head circumference,
intracranial volume and brain volume [7–10]. Heritability
estimates from GWAS range from 10 to 31% [8]. How-
ever, only two genetic loci associated with head circum-
ference between 6 and 30 months have been identified
so far [11]. Identifying additional genetic loci related to
early-life head circumference may contribute towards
our understanding of early brain development. This is
important since observational studies have associated
early brain development with several neurological and
psychiatric diseases, such as Alzheimer’s disease, schizo-
phrenia and autism [12–17]. The underlying mechanisms
are poorly understood. Both genetics and environmental
factors play a role [18]. Additionally, the shared genetic
contribution between early-life head circumference
and later-life outcomes is yet unknown. Unravelling
this shared genetic contribution may help us to better Head circumference is a complex trait, commonly used
as an indicator of brain volume during development and
associated with child and adult intelligence [1–3]. It is
also used a s measure of skeletal growth in fetal life, at
birth and in early childhood [4, 5]. Twin studies show
heritability estimates ranging from 75 to 90%, which are
consistent across the life course [6]. Large genome-wide
association studies (GWAS) have identified multiple © The Author(s) 2022. Open Access This arti
permits use, sharing, adaptation, distribution
original author(s) and the source, provide a li
other third party material in this article are inc
to the material. If material is not included in t
regulation or exceeds the permitted use, you
licence, visit http://creativecommons.org/lice
mmons.org/publicdomain/zero/1.0/) applies
†Suzanne Vogelezang, Jonathan P. Bradfield, Struan F. A. Grant, Janine F. Felix
and Vincent W. V. Jaddoe have contributed equally to this work
A list of authors and their affiliations appears at the end of the paper
*Correspondence: v.jaddoe@erasmusmc.nl
1 The Generation R Study Group, Erasmus MC, University Medical Center
Rotterdam, Rotterdam, The Netherlands
Full list of author information is available at the end of the article †Suzanne Vogelezang, Jonathan P. Bradfield, Struan F. A. Grant, Janine F. Felix
and Vincent W. V. Jaddoe have contributed equally to this work
A list of authors and their affiliations appears at the end of the paper
*Correspondence: v.jaddoe@erasmusmc.nl
1 The Generation R Study Group, Erasmus MC, University Medical Center
Rotterdam, Rotterdam, The Netherlands
Full list of author information is available at the end of the article †Suzanne Vogelezang, Jonathan P. Bradfield, Struan F. A. Grant, Janine F. University of Groningen Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the
number of authors shown on this cover page is limited to 10 maximum. Download date: 24-10-2024 Download date: 24-10-2024 Vogelezang et al. BMC Medical Genomics (2022) 15:124
https://doi.org/10.1186/s12920-022-01281-1 Open Access Background Felix
and Vincent W. V. Jaddoe have contributed equally to this work ist of authors and their affiliations appears at the end of the paper 1 The Generation R Study Group, Erasmus MC, University Medical Center
Rotterdam, Rotterdam, The Netherlands
Full list of author information is available at the end of the article Study‑level analyses
G
d Genome-wide association analyses were first run in all
discovery cohorts for birth and early-life head circum-
ference separately. Studies used high-density Illumina
or Affymetrix Single Nucleotide Polymorphism (SNP)
arrays, followed by imputation to the 1000 Genomes Pro-
ject or Haplotype Reference Consortium (HRC). Before
imputation, studies applied study specific quality filters
on sample and SNP call rate, minor allele frequency and
Hardy–Weinberg disequilibrium (see Additional file 2:
Table S1 for details). Linear regression models assum-
ing an additive genetic model were run in each study, to
assess the association of each SNP with SDS head cir-
cumference, adjusting for principal components if this
was deemed needed in the individual studies. As SDS
head circumference is age and sex specific, no further
adjustments were made. Before the meta-analysis, we
applied quality filters to each study, filtering out SNPs
with a minor allele frequency (MAF) below 1% and
SNPs with poor imputation quality (MACH r2_hat ≤ 0.3,
IMPUTE proper_info ≤ 0.4 or info ≤ 0.4). Study design We conducted a two-stage meta-analysis in children of
European ancestry to identify genetic loci associated with
birth and early-life head circumference. Sex- and age-
adjusted standard deviation scores (SDS) were created
for head circumference between 6 to 30 months (closest
to 18 months, if multiple measurements were available)
using Growth Analyzer 3.0 across all studies [19]. In the
case of twin pairs and siblings, only one of each twin or
sibling pair was included, either randomly or based on
genotyping or imputation quality. Study‑level analyses
G
d g
yp
g
p
q
y
In the discovery stage, we performed a meta-analysis of
early-life head circumference in 21 studies (N = 22,279),
including the Amsterdam Born Children and their Devel-
opment-Genetic Enrichment Study (ABCD, N = 1018),
the Avon Longitudinal Study of Parents and Children
(ALSPAC, N = 3960), Children’s Hospital of Philadel-
phia (CHOP, N = 856), the Copenhagen Prospective
Studies on Asthma in Childhood 2000 (COPSAC2000,
N = 325) and 2010 (COPSAC2010, N = 603), the Dan-
ish National Birth Cohort- preterm birth study (DNBC-
PTB, N = 508), the Generation R Study (GenerationR,
N = 2299), the Danish National Birth Cohort—the
Genetics of Overweight Young Adults offspring study
(DNBC GOYA-offspring, N = 230), the INfancia y Medio
Ambiente [Environment and Childhood] Project, with
two subcohorts that were entered into the meta-analysis
separately (INMA-Sabadell and Valencia subcohort and
INMA Menorca subcohort, N = 550), the German Infant
Study on the influence of Nutrition Intervention PLUS
environmental and genetic influences on allergy devel-
opment & Influence of life-style factors on the develop-
ment of the immune system and allergies in East and
West Germany (GINIplus&LISA, N = 1455), the Leipzig
Research Center for Civilization Diseases—Child study
(LIFE-Child, N = 1365), the Norwegian Mother Child
Cohort (MoBa, N = 836), the Northern Finland Birth
Cohort 1966 (NFBC 1966, N = 4603), the Northern Fin-
land Birth Cohort 1986 (NFBC 1986, N = 826), the Physi-
cal Activity and Nutrition in Children Study (PANIC,
N = 372), the Raine Study (Raine Study, N = 1373), the © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the
original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or
other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this
licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco
mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Vogelezang et al. BMC Medical Genomics (2022) 15:124 Page 2 of 16 understand the etiology of later-life outcomes related to
early-life head circumference. Småbørns Kost Og Trivsel study, including two subco-
horts (SKOT 1, N = 170 and SKOT 2, N = 98), the Special
Turku Coronary Risk factor Intervention Project (STRIP,
N = 505), and the TEENs of Attica: Genes and Environ-
ment (TEENAGE, N = 327). We examined the genetic background of early-life head
circumference by performing a two-stage GWAS meta-
analysis comprising 25 studies with a combined sample
size of 29,192 European-ancestry participants between
6 and 30 months of age. We also examined genetic cor-
relations of early-life head circumference with anthropo-
metrics, brain volume-related, neurological, psychiatric,
cognitive, and personality related traits. In the replication stage of early-life head cirvumference
analysis, we included 4 studies (N = 6913): 319 additional
children from the INfancia y Medio Ambiente [Environ-
ment and Childhood] (INMA) Project (INMA-Gipuzkoa
subcohort), 5644 additional children from the Norwe-
gian Mother and Child Cohort (MoBa), the European
Childhood Obesity Project (CHOP Study, N = 366), and
the Exeter Family Study of Childhood Health (EFSOCH,
N = 584). Characteristics of discovery and replication
studies can be found in Additional file 2: Table S1. The
study design of birth head circumference can be found in
the Additional file 1. The Database for Annotation, Visualization and Integrated
Discovery (DAVID) To explore biological processes, we used DAVID, with the
seven nearest genes as input, using the Kyoto Encyclope-
dia of Genes and Genomes (KEGG) database [27, 28]. Linkage‑disequilibrium score regressionh g
q
g
The use of LD score regression to estimate genetic cor-
relations between two phenotypes has been described
in detail previously [29]. Briefly, LD score is a measure
of how much a genetic variation is tagged by each vari-
ant. A high LD score indicates that a variant is in high
LD with many nearby polymorphisms. Variants with high
LD scores are more likely to contain true signals and have
a higher chance of overlap with genuine signals between
GWAS. To estimate LD scores, summary statistics from
GWAS meta-analysis are used to calculate the cross-
product of test statistics of per SNP, which is regressed
on the LD score. The slope of the regression is a function
of the genetic covariance between traits [29]: Meta‑analysis
W
f We performed fixed-effects inverse-variance weighted
meta-analysis of all discovery samples using Metal [20]. Genomic control was applied to every study before the
meta-analysis. Individual study lambdas before genomic
control ranged from 0.99 to 1.03 (Additional file 2:
Table S1). The lambda of the discovery meta-analysis was
1.02. Linkage Disequilibrium (LD) score regression anal-
ysis showed an intercept of 1.0, indicating that the slight
inflation was mainly caused by polygenicity of early-life
head circumference and not by population stratifica-
tion, cryptic relatedness or other confounders [21, 22]. After the meta-analysis, we excluded SNPs for which Vogelezang et al. BMC Medical Genomics (2022) 15:124 Page 3 of 16 information was available in less than 50% of the studies
or in less than 50% of the total sample size. Genome-wide Complex Trait Analysis (GCTA) was
used to select the independent SNPs for each locus [23]. We performed conditional analyses based on summary-
level statistics and LD estimation between SNPs from the
Generation R Study as a reference sample to select inde-
pendently associated SNPs on the basis of conditional P
values [23]. For early-life head circumference, 27 genome-
wide significant or suggestive loci (P values < 5 × 10–8 and
P values < 5 × 10−6, respectively) were taken forward for
replication in the 4 replication cohorts. Fixed-effects
inverse variance weighted meta-analysis was performed
for these 27 SNPs combining the discovery samples and
all replication samples, giving a combined analysis beta,
standard error and P value (Table 1). SNPs that reached
genome-wide significance (P value < 5 × 10–8) in the
combined analysis were considered to be significantly
associated with SDS-head circumference. For birth head
circumference, SNPs were taken forward for replication,
using the same methodology. Support for each hypothesis was quantified in terms of
posterior probabilities, defined at SNP level and indi-
cated by PP0, PP1, PP2, PP3 or PP4, corresponding to the
five hypotheses and measuring how likely these hypoth-
eses are. In most pairs, no evidence for association was
found with either trait. In case association was observed,
it was mostly with a single trait. To define colocalization
we used restriction to pairs of early-life head circumfer-
ence and eQTL signals with a high posterior probability
for colocalization, indicated by a PP4/(PP3 + PP4) > 0.9. Functional mapping and annotation of genetic
associations (FUMA) To obtain predicted functional consequences for the
SNPs that reached genome-wide significance in the com-
bined meta-analysis, we used SNP2FUNC in FUMA, a
web-based platform to facilitate and visualize functional
annotation of GWAS results [24]. To annotate the near-
est genes of the seven SNPs in biological context, we
used the GENE2FUNC option in FUMA, which provides
hypergeometric tests of enrichment of the list of near-
est genes in 53 GTEx tissue-specific gene expression sets
[24, 25]. We used GENE2FUNC for two sets of genes: 1. Nearest genes of seven SNPs; 2. Genes located in a region
of 500 kb to either side of the 7 SNPs [24]. Colocalization analysis We used Bayesian colocalization analysis to examine
evidence for colocalization between early-life head cir-
cumference and eQTL signals (GTEx v7). Colocali-
zation analyses were conducted using the R package
coloc, https://cran.r-project.org/web/packages/coloc, as
described previously [26]. Briefly, in each of the GTEx
v7 tissues, all cis-eQTLs at FDR < 5% were identified. For
each eQTL, GWAS summary statistics were extracted
for all SNPs that were present in > 50% of the studies
and > 50% of the total sample size and that were in com-
mon to both GWAS and eQTL studies, within 1 MB of
the transcription start site of the gene. For each such
locus, colocalization analyses were done with default
parameters, testing the following hypotheses [26]: E(z1jz2j) =
√N1N2ρg
M
lj +
ρNs
√N1N2 where Ni is the sample size of study i, ρg is the genetic
covariance, M is the number of SNPs in the reference
panel with a MAF between 5 and 50%, lj is the LD score
for SNP j, Ns quantifies the number of individuals that
overlap both studies, and ρ is the phenotypic correlation
amongst the Ns of overlapping samples. A sample over-
lap or cryptic relatedness between samples only affects
the intercept from the regression but not the slope. Thus,
estimates are robust even in presence of sample overlap Page 4 of 16 Vogelezang et al. Colocalization analysis BMC Medical Genomics (2022) 15:124 when comparing traits across distinct GWAS popula-
tions. Estimates of genetic covariance are therefore not
biased by overlapping samples. Similarly, in case of pop-
ulation stratification, the intercept is affected but it has
only minimal impact on the slope since population strati-
fication does not correlate with LD between variants. alleles and rounded to the nearest integer. Linear regres-
sion analysis was used to examine the associations of the
risk score with head circumference and intracranial vol-
ume at different ages. For these analyses data from the
Generation R Study and UK Biobank were used. When
calculating the risk score for the Generation R study,
effect estimates from the combined meta-analysis were
used after excluding Generation R from the meta-analy-
sis. The variance explained was estimated by the adjusted
R2 of the models. i
Because of the correlation between the imputa-
tion quality and LD score, imputation quality is a con-
founder for LD score regression. Therefore, SNPs were
excluded according to the following criteria: MAF < 0.01
or INFO ≤ 0.9. The filtered GWAS results were uploaded
on the online webtool, a web service with many GWAS
meta-analyses available on which LD score regression
has been implemented by the developers of the LD score
regression method. In case multiple GWAS meta-anal-
yses were available for the same phenotype, the genetic
correlation with early-life head circumference was esti-
mated using the most recent meta-analysis. Genetic
correlations are shown in Fig. 3 and Additional file 1:
Table S7. Colocalization analysis BMC Medical Genomics (2022) 15:124 Table 1 Results of the 27 SNPs with P values < 5 × 10 6 in the discovery phase
Bolded P values indicate genome-wide significance in the combined analysis
SNP
CHR
Position
Nearest gene
EA/non_EA
P value discovery
P value replication
EAFa
Beta (SE) combined
P value combined
Heterogeneity I2
combined analysis
rs8756b, c, d
12
66,359,752
HMGA2
C/A
5.18 × 10–12
4.62 × 10–3
0.50
0.065 (0.009)
1.84 × 10–13
0
rs9795522b
12
123,730,935
C12orf65
C/A
3.20 × 10–9
3.86 × 10–5
0.23
0.072 (0.010)
7.37 × 10–12
0
rs10883848d
10
104,973,061
NT5C2
A/G
3.06 × 10–6
0.03
0.35
0.058 (0.010)
9.99 × 10–10
54.8
rs6095360
20
47,532,536
ARFGEF2
G/A
3.53 × 10–8
6.19 × 10–4
0.32
0.055 (0.009)
4.39 × 10–9
36.6
rs3134614
1
40,363,054
MYCL1
C/G
4.31 × 10–6
7.75 × 10–4
0.88
0.075 (0.013)
1.43 × 10–8
0
rs6016511
20
39,709,255
TOP1
T/C
4.55 × 10–7
0.02
0.68
0.052 (0.010)
4.02 × 10–8
0
rs7792211d
7
50,707,141
GRB10
G/A
7.62 × 10–7
0.01
0.42
0.051 (0.009)
4.43 × 10–8
11.3
rs1490384c, d
6
126,851,160
CENPW
T/C
5.46 × 10–8
0.14
0.51
0.049 (0.009)
5.70 × 10–8*
43.9
rs116536930
17
43,855,156
CRHR1
G/T
4.19 × 10–8
0.53
0.51
0.055 (0.011)
2.27 × 10–7*
40.1
rs72952297
2
180,379,814
ZNF385B
T/G
2.07 × 10–6
0.11
0.93
0.087 (0.018)
9.19 × 10–7*
0
rs12350281
9
20,637,773
MLLT3
G/A
4.22 × 10–6
0.03
0.05
0.152 (0.031)
9.98 × 10–7*
43.1
rs7658192
4
140,023,559
ELF2
T/G
2.29 × 10–7
0.46
0.33
0.046 (0.010)
1.70 × 10–6*
62.9
rs143094271e
17
7,463,102
TNFSF12-TNFSF13
A/G
5.96 × 10–7
0.44
0.02
0.133 (0.028)
2.02 × 10–6*
33.7
rs1014098
1
119,458,215
TBX15
C/T
2.67 × 10–6
0.17
0.79
0.051 (0.011)
2.05 × 10–6*
30.3
rs77690628
8
26,189,223
PPP2R2A
A/T
1.02 × 10–6
0.31
0.07
0.080 (0.017)
3.26 × 10–6*
24.6
rs9853018
3
141,101,961
ZBTB38
T/C
5.41 × 10–7
0.62
0.45
0.040 (0.009)
5.30 × 10–6
38.4
rs4674101
2
217,487,622
RPL37A
A/G
8.66 × 10–7
0.57
0.31
0.043 (0.010)
6.56 × 10–6
65.3
rs150266910
1
23,442,265
LUZP1
C/T
1.42 × 10–6
0.65
0.83
0.052 (0.012)
1.04 × 10–5
58.2
rs13067734
3
141,963,015
GK5
A/C
2.83 × 10–6
0.55
0.52
0.037 (0.009)
1.22 × 10–5
26.5
rs17223831
14
90,601,920
KCNK13
A/G
1.24 × 10–6
0.18
0.14
0.067 (0.015)
1.30 × 10–5
67.6
rs62580922
9
125,631,594
RC3H2
C/T
1.13 × 10–8
0.35
0.07
0.072 (0.017)
1.52 × 10–5
77.8
rs2448415
1
93,469,310
FAM69A
A/G
1.97 × 10–6
0.81
0.47
0.037 (0.009)
2.56 × 10–5
60.7
rs261752
5
39,328,506
C9
A/G
1.39 × 10–6
0.70
0.55
0.035 (0.009)
7.63 × 10–5
63.7
rs72979897
6
140,881,181
MIR3668
T/A
4.13 × 10–6
0.88
0.04
0.100 (0.025)
8.26 × 10–5
50.6
rs503783
6
161,959,649
PARK2
T/C
2.98 × 10–6
0.66
0.27
0.037 (0.010)
1.59 × 10–4
74.3
rs113397574
5
31,968,320
PDZD2
T/C
4.56 × 10–6
0.61
0.02
0.118 (0.032)
2.40 × 10–4
58.5
rs77535478
9
77,533,287
TRPM6
A/G
1.28 × 10–6
0.39
0.04
0.081 (0.026)
2.05 × 10–3
75.4 Page 5 of 16 Vogelezang et al. Identification of genetic loci associated with early‑life head
circumference Individual study characteristics are shown in Additional
file 2: Table S1. In the discovery stage, we performed a
fixed-effects inverse variance-weighted meta-analysis
including data imputed to the 1000 Genomes or the Hap-
lotype Reference Consortium (HRC) reference panels
from 21 studies (N = 22,279). Using data from the discov-
ery cohorts, single nucleotide polymorphisms (SNPs) at
five independent loci reached genome-wide significance
(P values < 5 × 10–8) and SNPs at another 22 loci showed
suggestive associations with early-life head circumfer-
ence (5 × 10–8 < P values < 5 × 10−6). A Manhattan plot
of the discovery meta-analysis is shown in Fig. 1. No evi-
dence of inflation by population stratification or cryptic
relatedness was found (genomic inflation factor (λ) = 1.02
and LD-score regression intercept = 1.0) (Additional Functional characterization To gain insight into the function of the seven SNPs asso-
ciated with early-life head circumference, we used several
strategies. First, using Bayesian colocalization analysis,
we examined evidence of colocalization between GWAS
and eQTL signals for the seven index SNPs (GTEx v7),
but did not find a signal at any of the seven loci. Second,
to explore biological processes, we used the Kyoto Ency-
clopedia of Genes and Genomes (KEGG) database in the
Database for Annotation, Visualization and Integrated
Discovery (DAVID) with the seven SNPs and their near-
est genes as input [27, 28], but no enriched biological
processes were identified. Third, we did a look-up of the
seven nearest genes in mouse-knockout data but there
was no phenotypic information available for any of these
gene knockouts [31]. Fourth, we examined gene expres-
sion profiles for the nearest genes to the seven SNPs
with GTEx v7 in 53 tissues, using the tool for Functional
Mapping and Annotation of Genome-Wide Association
Studies (FUMA) [24, 25]. We did not find significant dif-
ferential expression for these seven nearest genes. Going
a step further, we included all genes within a range of
500 kb upstream to 500 kb downstream of the seven
index SNPs and found significant differential expression
in several brain structures, including the putamen, amyg-
dala, hippocampus, caudate, nucleus accumbens, sub-
stantia nigra, and anterior cingulate cortex, and in other
tissues such as the heart, pancreas and liver [25]. i
Of the 27 SNPs identified in the discovery meta-anal-
ysis, seven reached genome-wide significance in the
combined meta-analysis, in which we used data from the
discovery and replication stage. An identified locus was
defined to be a known locus if the index SNP was within a
range of 500 kb upstream to 500 kb downstream of and in
LD (r2 ≥ 0.2) with a previously reported SNP for head cir-
cumference, intracranial volume, or brain volume at any
age [7–11]. Of the seven genome-wide significant SNPs,
three were novel: rs6095360 near ARFGEF2, rs3134614
near MYCL1, and rs6016511 near TOP1 (Table 1 and
Additional file 1: Tables S2 and S3). Regional plots of
these three loci are shown in Fig. 2. The remaining four
SNPs mapped to loci previously identified from GWAS
on infant head circumference, adult intracranial vol-
ume, and/or adult brain volume (nearest genes: HMGA2,
C12orf65, NT5C2, and GRB10) [7, 10, 11]. (See figure on next page.)
Fig. 2 a–c Locus zoom plots of the 3 novel loci. Shown are the results of the meta-analysis. Regional association plot of the 3 novel loci. SNPs are
plotted with their P values from the discovery stage (as − log10; left y-axis) as a function of genomic position (x-axis). Estimated recombination rates
(right y-axis) taken from 1000 Genomes, March 2012 release are plotted to reflect the local LD-structure around the top associated SNP (indicated
with purple color) and the correlated proxies (indicated in colors) Functional characterization Six SNPs located within 500 kb (upstream or down-
stream)
from
rs6095360
(ARFGEF2),
rs3134614
(MYCL1), and rs6016511 (TOP1) have been previously
reported in relation to adult height [30]. The linkage dis-
equilibrium (LD) of the three novel SNPs near ARFGEF2,
MYCL1, and TOP1 with these six adult height SNPs was
weak to moderate. We found suggestive evidence of asso-
ciation for rs6095360 (ARFGEF2) with early-life length in
28,949 participants between 6 and 30 months of age in an
unpublished GWAS meta-analysis of 24 cohorts (P value
4.58 × 10–7), but the other two novel SNPs did not show
evidence of association (Additional file 1: Table S5). Genetic risk score and percentage of variance explainedi Genetic risk score and percentage of variance explained
We combined the seven genome-wide significant SNPs
from the combined meta-analysis into a Genetic Risk
Score (GRS) by summing up the number of alleles that
increase the SDS head circumference, weighted by the
effect sizes from the combined meta-analysis. The GRS
was rescaled to a range from 0 to 14, which is the maxi-
mum number of head circumference SDS increasing Fig. 1 Manhattan plot of results of the discovery meta-analysis of 21 GWAS. On the x-axis the chromosomes are shown. On the y-axis the − log
10 of the P value is shown. Novel SNPs are shown in green. Known SNPs are shown in black. The genome wide significance cutoff of 5 × 10–8 is
represented by the grey dotted line Fig. 1 Manhattan plot of results of the discovery meta-analysis of 21 GWAS. On the x-axis the chromosomes are shown. On the y-axis the − log
10 of the P value is shown. Novel SNPs are shown in green. Known SNPs are shown in black. The genome wide significance cutoff of 5 × 10–8 is
represented by the grey dotted line Fig. 1 Manhattan plot of results of the discovery meta-analysis of 21 GWAS. On the x-axis the chromosomes are shown. On the y-axis the − log
10 of the P value is shown. Novel SNPs are shown in green. Known SNPs are shown in black. The genome wide significance cutoff of 5 × 10–8 is
represented by the grey dotted line Vogelezang et al. BMC Medical Genomics (2022) 15:124 Page 6 of 16 shown in Additional file 1: Figs. S2 and S3. Results of the
discovery analysis for SNPs with P values < 5 × 10−6 are
shown in Additional file 4: Table S6. file 1: Fig. S1) [21]. The index SNPs from each of the 27
genome-wide and suggestive loci were followed up in
four replication cohorts (N = 6913). The results of the
discovery, replication and combined analyses are shown
in Table 1 and Additional file 1: Tables S2 and S3. Results
of the discovery analysis for SNPs with P values < 5 × 10−6
are shown in Additional file 3: Table S4. Shared genetic background of early‑life head
circumference with childhood and adult outcomes First, to examine whether associations between early-
life head circumference and phenotypes in later life from
observational studies were at least partly genetically
explained, we estimated genetic correlations between
early-life head circumference and other traits, using LD
score regression, which is based on the genome-wide
meta-analysis results [29]. We found positive genetic
correlation coefficients for early-life head circumference
with birth length (Rg = 0.57, P value = 3.61 × 10–12), birth
weight (Rg = 0.48, P value = 2.51 × 10–8), adult height
(Rg = 0.34, P value = 9.77 × 10–11), adult body mass index i
We also performed a meta-analysis of birth head cir-
cumference in a total of 32,084 participants. None of the
SNPs reached genome-wide significance in this analysis. A total of 11 SNPs with P values between 5 × 10–8 and
5 × 10–6 were taken forward for replication (N = 3750)
and combined analyses, but none were genome-wide sig-
nificant in the combined analysis. Therefore, no follow-up
analyses were performed for birth head circumference. A Manhattan plot and a Quantile–Quantile plot of the
discovery meta-analysis of birth head circumference are (i
g
p g )
Fig. 2 a–c Locus zoom plots of the 3 novel loci. Shown are the results of the meta-analysis. Regional association plot of the 3 novel loci. SNPs are
plotted with their P values from the discovery stage (as − log10; left y-axis) as a function of genomic position (x-axis). Estimated recombination rates
(right y-axis) taken from 1000 Genomes, March 2012 release are plotted to reflect the local LD-structure around the top associated SNP (indicated
with purple color) and the correlated proxies (indicated in colors) Vogelezang et al. BMC Medical Genomics (2022) 15:124 Page 7 of 16 Fig. 2 (See legend on previous page.) Fig. 2 (See legend on previous page.) Fig. 2 (See legend on previous page.) Page 8 of 16 Vogelezang et al. BMC Medical Genomics (2022) 15:124 (Additional file 1: Table S8). Additionally, rs6095360
(ARFGEF2) was associated with adult intelligence at a
genome-wide level (P value = 2.31 × 10–16). We did not
observe evidence of genome-wide significant associa-
tions with Alzheimer’s disease, neuroticism, depression
or educational attainment. Shared genetic background of early‑life head
circumference with childhood and adult outcomes Suggestive evidence for asso-
ciation was observed for rs8756 (HMGA2) with adult
intracranial volume (P value = 6.32 × 10–8), for rs9795522
(C12orf65) with adult intelligence (P value = 1.41 × 10–6)
and for rs8756 (HMGA2) with educational attainment
(P value = 1.41 × 10–6) (Additional file 1: Tables S9–13). None of the SNPs showed evidence of association with
birth head circumference in the GWAS meta-analysis
(Additional file 1: Table S14).h (BMI) (Rg = 0.13, P value = 0.03), adult intracranial vol-
ume (Rg = 0.71, P value = 1.44 × 10–5) and several cog-
nitive outcomes, including childhood intelligence (age
range 6–18 years) (Rg = 0.27, P value = 0.04), years of
schooling (Rg = 0.18, P value = 6.0 × 10–4), and adult
intelligence (Rg = 0.25, P value = 1.0 × 10–4), (Fig. 3 and
Additional file 1: Table S7). We did not find genetic corre-
lations with any psychiatric (for instance bipolar disorder
and autism spectrum disorder), neurological (Parkinson’s
disease, Alzheimer’s disease) or personality-related out-
comes (for instance neuroticism) (P values > 0.05) (Addi-
tional file 1: Table S7). i
Second, we performed a look-up of the seven identi-
fied SNPs in GWAS meta-analyses data on potentially
related phenotypes, including adult intracranial volume,
intelligence, Alzheimer’s disease, neuroticism, depres-
sion and educational attainment [7, 32–35]. Rs10883848
(NT5C2) was genome-wide significantly associated with
adult intracranial volume (P value = 1.48 × 10–9). All
effect estimates for intracranial volume were in the same
direction as those for head circumference in early-life i
Third, we calculated a combined genetic risk score
(GRS) using the seven index SNPs identified in the cur-
rent study. We summed the number of head circumfer-
ence-increasing alleles weighted by the effect sizes from
the combined meta-analysis after excluding the Genera-
tion R Study, in which we tested the GRS longitudinally. The GRS was associated with fetal head circumference Fig. 3 Genome-wide genetic correlations between early-life head circumference and adult traits and diseases. On the x axis the traits and diseases
are shown. The y-axis shows the genetic correlations (Rg) and corresponding standard errors, indicated by error bars, between early-life head
circumference and each trait, estimated by LD score regression. The genetic correlation estimates (Rg) are colored according to their intensity and
direction. Red indicates a positive correlation, blue indicates a negative correlation. References can be found in Additional file 1: Table S7 Fig. Discussion In a GWAS meta-analysis including 29,192 participants
of European ancestry aged 6 to 30 months of age, we
identified seven genome-wide significant SNPs associ-
ated with early-life head circumference, of which three
were novel and had not been related with head circum-
ference, intracranial volume or brain volume before. We
observed positive genetic correlations between early-life
head circumference and adult intracranial volume as well
as cognitive outcomes. The potential roles of the nearest genes to the novel
loci are still poorly understood. MYCL1, (MYCL proto-
oncogene, BHLH transcription factor), and TOP1 (DNA
topoisomerase 1) have been suggested to play a role in
various types of cancer [40–44]. The role of these genes
in the development of head circumference in early life is
currently unknown. A mutation in ARFGEF2 has been
previously associated with several phenotypes related
to brain development, including microcephaly [45, 46]. The three novel SNPs are located near regions that have
been previously reported for adult height, indicating that
they might represent loci involved in growth [30]. How-
ever, the strong association of rs6095360 (nearest gene:
ARFGEF2) with adult intelligence (P value = 2.31 × 10–16) We used multiple approaches to identify potential
underlying mechanisms. As there is no strong evidence
linking the nearest genes to the seven SNPs as causal
genes, we included all genes within 500 kb to either side
of the genome-wide significant SNPs in a GTEx analy-
sis. We found differential expression of these genes in
different brain structures that are related to cognitive Fig. 4 Associations of early-life head circumference genetic risk score with head circumference at different time points in the Generation R Study
and from UK Biobank data. On the x axis the different ages are shown at which the genetic risk score of the seven early-life head circumference SNPs
is tested. On the y axis the beta’s and 95% confidence intervals from linear regression analyses are shown. Detailed data can be found in Additional
file 1: Table S15 Fig. 4 Associations of early-life head circumference genetic risk score with head circumference at different time points in the Generation R Study
and from UK Biobank data. On the x axis the different ages are shown at which the genetic risk score of the seven early-life head circumference SNPs
is tested. On the y axis the beta’s and 95% confidence intervals from linear regression analyses are shown. Shared genetic background of early‑life head
circumference with childhood and adult outcomes 3 Genome-wide genetic correlations between early-life head circumference and adult traits and diseases. On the x axis the traits and diseases
are shown. The y-axis shows the genetic correlations (Rg) and corresponding standard errors, indicated by error bars, between early-life head
circumference and each trait, estimated by LD score regression. The genetic correlation estimates (Rg) are colored according to their intensity and
direction. Red indicates a positive correlation, blue indicates a negative correlation. References can be found in Additional file 1: Table S7 Page 9 of 16 Vogelezang et al. BMC Medical Genomics (2022) 15:124 functions and emotional control, indicating a potential
functional role of these genes in the brain [36–39]. How-
ever, as donors aged 20–79 years were included in the
GTEx data source, we were not able to look at expression
of the genes in brain structures in early life. Using colo-
calization analysis, no potentially causal genes were iden-
tified [26]. Future studies should also determine whether
the nearest genes, identified in this study, are indeed the
causal genes and assess their expression in child brain
structures. in the third trimester of pregnancy (N = 1984), at post-
natal ages 1 month (N = 1501), 6 months (N = 1662),
11 months (N = 1528), and 6 years (N = 4010) and at
the mean (SD) age of 64 (7.5) years (N = 22,152) in UK
Biobank data (P values < 0.05) (Fig. 4 and Additional
file 1: Table S15). in the third trimester of pregnancy (N = 1984), at post-
natal ages 1 month (N = 1501), 6 months (N = 1662),
11 months (N = 1528), and 6 years (N = 4010) and at
the mean (SD) age of 64 (7.5) years (N = 22,152) in UK
Biobank data (P values < 0.05) (Fig. 4 and Additional
file 1: Table S15). Abbreviations
GWAS G GWAS: Genome-wide association studies; SDS: Standard deviation scores;
MAF: Minor allele frequency; KEGG: Kyoto Encyclopedia of Genes and
Genomes; HRC: Haplotype Reference Consortium; SNPs: Single nucleotide
polymorphisms; LD: Linkage disequilibrium; DAVID: Database for Annota-
tion, Visualization and Integrated Discovery; FUMA: Functional Mapping and
Annotation of Genome-Wide Association Studies; BMI: Body mass index; GRS:
Genetic risk score. It has been suggested that heritability estimates are
consistent from childhood onwards [6]. Whether this
genetic stability starts from early life onwards, is cur-
rently not well studied. We observed evidence for asso-
ciation of two of the seven SNPs with adult intracranial
volume [7]. We combined the seven index SNPs into a
weighted GRS. Although we have used effect sizes of the
meta-analysis after excluding the Generation R Study, the
discovery of the seven SNPs was based on the meta-anal-
ysis including the Generation R Study, potentially result-
ing in overfitting of the GRS. We found an association of
the GRS with fetal head circumference in third trimester,
head circumference in infancy and childhood and intrac-
ranial volume in adulthood. The effect estimates were
largely similar for the different time points. We did not
observe an association of the GRS with birth head cir-
cumference. This may be explained by the larger vari-
ance in birth head circumference that might be present Supplementary Information The online version contains supplementary material available at https://doi.
org/10.1186/s12920-022-01281-1. Additional file 1. Additional data includes 15 tables, 3 figures and addi-
tional cohort information and methods. Additional file 1. Additional data includes 15 tables, 3 figures and addi-
tional cohort information and methods. Additional file 2. Characteristics of discovery and replication studies. Additional file 3. Results of the discovery analysis for SNPs with P-values
<5 × 10−6. Additional file 4. Results of the discovery analysis for SNPs with P-values
<5 × 10−6 for birth head circumference. le 2. Characteristics of discovery and replication studies. Additional file 3. Results of the discovery analysis for SNPs with P-values
<5 × 10−6. Additional file 4. Results of the discovery analysis for SNPs with P-values
<5 × 10−6 for birth head circumference. Additional file 4. Results of the discovery analysis for SNPs with P-values
<5 × 10−6 for birth head circumference. Discussion Detailed data can be found in Additional
file 1: Table S15 Vogelezang et al. BMC Medical Genomics (2022) 15:124 Page 10 of 16 Page 10 of 16 might indicate a role in brain development as well [32]. Future functional studies should investigate the role of
these genes and should determine whether these genes
are indeed causal. due to the deformation of the head during birth. Also, in
the GWAS meta-analysis of head circumference at birth,
no SNPs were genome-wide significantly associated with
birth head circumference. Thus, the genetic background
of early-life head circumference seems to partially over-
lap with the genetic background of related measures in
later life. The SNPs identified in infancy seem to repre-
sent effects across multiple ages. However, as not all
SNPs identified for early-life head circumference were
associated with adult intracranial volume, it has been
suggested that some of the underlying mechanisms are
age-specific. Observational studies suggest that early life head cir-
cumference is not only related to intracranial volume
in adults, but also to adult intelligence, Alzheimer’s dis-
ease, schizophrenia and autism [1–3, 12–17]. In observa-
tional studies of such associations, effect estimates may
be influenced by confounding factors and reverse causa-
tion, potentially evoking spurious associations [47, 48]. Genetic studies such as ours can provide more insight
into the etiology of complex diseases. We found a strong
genetic correlation of head circumference in early life
with intracranial volume in adults, underlining the idea
that early-life head circumference is a valid measure for
brain growth during early development [3, 7]. Abnormal
growth trajectories of head circumference are related to
adverse neurological outcomes [49]. Additionally, varia-
tion within the normal range of head circumference has
been reported to be associated with cognitive and behav-
ioral traits [2, 50, 51]. In the current study, we observed
positive genetic correlations for head circumference in
early life with childhood intelligence, years of school-
ing and adult intelligence. These findings indicate that
the association of early-life head circumference with
cognitive function from observational studies is at least
partly explained by a shared genetic background, which
is in line with the observed positive genetic correlations
between intracranial volume and cognitive function in
the literature [7, 8, 52]. Altogether, the findings from the
current study and from previous literature suggest that
the associations between measures of early-life brain vol-
ume and cognition decades later are partly genetically
explained. Conclusions
W id
ifi d We identified seven SNPs associated with early-life head
circumference. Three of these are novel and four mapped
to loci that are known for head circumference, intracra-
nial volume, or brain volume. We observed a strong posi-
tive genetic correlation of early-life head circumference
with adult intracranial volume and cognitive outcomes in
childhood and adulthood. The well-known associations
of head circumference with later cognitive phenotypes
are partly explained by genetics. Our findings may con-
tribute to the understanding of the early-life brain devel-
opment, which may lay the foundation for diseases later
in life. The Early Growth Genetics Consortiumi Suzanne Vogelezang1–3, Jonathan P Bradfield4,5, Stefan Johansson6,7,
Evie Stergiakouli8–10, Elisabeth Thiering11,12, Craig E Pennell13, Tarunveer S
Ahluwalia14–16, Ville Karhunen17,18, Markus Scholz19,20, Xueping Liu21, Carmen
Iñiguez22,23, Olli T Raitakari24–26, Jonas Bacelis27,28, Theresia M Schnurr29, Timo
A Lakka30–32, Ioanna Ntalla33, Mads V Lind34, Lotte Lauritzen34, Sauli Herrala18,
Frederick TJ Lin35, Christine Frithioff-Bøjsøe29,36, Robin N Beaumont37, Moham-
med H Zafarmand38,39, Peter Rzehak40, Jose R Bilbao41–43, Nella Junna44,
Judith M Vonk45, Sheryl L Rifas-Shiman46, Vimala D Janjanam47, Maria J Knol3,
Shweta Ramdas48, Lawrie Beilin49, Klaus Bønnelykke14, Maribel Casas23,50,51,
Johan G Eriksson52–54, Joaquin Escribano55, Tavia E Evans3,56, Ulrike Gehring57,
Frank Geller21, Veit Grote40, Darek Gruszfeld58, Hakon Hakonarson4,59, Andrew
T Hattersley37,60, Christian T Have29, M Geoffrey Hayes35,61,62, Joachim
Heinrich11,63, Øvind Helgeland6,64, Jhon Holloway65, Raimo Joro30, Julius
Juodakis27, Bridget A Knight60, Bert Koletzko40, Antje Körner20,66, Jean-Paul
Langhendries67, Jaakko T Leinonen44, Virpi Lindi68, Morten AV Lund36,69,
Stephen Lye70, Mads Melbye21,71,72, Kim F Michaelsen34, Camilla S Morgen73,74,
Harri Niinikoski26,75, Katja Pahkala24,26,76, Kalliope Panoutsopoulou77,78, Oluf
Pedersen29, Fernando Rivadeneira3,79, Loreto Santa-Marina80–82, Barbera DC
Schaik39, Denise Scholtens83, Sylvain Sebert18,84, Ibon Tamayo85,86, Nicholas
J Timpson8,9, Maties Torrent87,88, André G Uitterlinden3,79,89, Marc Vaudel6,
Elvira Verduci90, Rebecca Vinding14, Mandy Vogel20,66, Eleftheria Zeggini77,91,
Christopher Brown92, Hieab HH Adams3,56,93, Wilfried Karmaus47, Marie-France
Hivert46,94,95, Gerard H Koppelman96, Elisabeth Widén44, Nora Fernandez-
Jimenez41,42, Melanie Waldenberger97, Tanja GM Vrijkotte38, Rachel M Freathy37,
Jens-Christian Holm29,36, William L Lowe, Jr.35, Niels Grarup29, Torben Hansen29,
George V Dedoussis98, Mustafa Atalay30, Ellen A Nohr99,100, Bo Jacobsson101–103,
Niina Pitkänen24,26, Martine Vrijheid23,50,51, Bjarke Feenstra21, Wieland Kiess20,66,
Marjo-Riita Jarvelin17,18,84, Hans Bisgaard14, Carol Wang C13, Marie Standl11,
Mark I McCarthy104–106, Beate St Pourcain8,107,108, Pål R Njølstad6,109, Struan FA
Grant4,59,110–111, Janine F Felix1,2, Vincent WV Jaddoe1,2 These authors contributed equally: Suzanne Vogelezang and Jonathan P
Bradfield. These authors contributed equally: Struan FA Grant, Janine F Felix, Vincent WV
Jaddoe. 1. The Generation R Study Group, Erasmus MC, University Medical Center
Rotterdam, The Netherlands. 2. Department of Pediatrics, Erasmus MC,
University Medical Center Rotterdam, The Netherlands. 3. Department of
Epidemiology, Erasmus MC, University Medical Center Rotterdam, The
Netherlands. 4. Center for Applied Genomics, Division of Human Genetics,
Children’s Hospital of Philadelphia, Philadelphia, PA, USA. 5. Quantinuum
Research LLC, San Diego, CA, USA. 6. KG Jebsen Center for Diabetes Research,
Department of Clinical Science, University of Bergen, Bergen, Norway. 7. Department of Medical Genetics, Haukeland University Hospital, Bergen,
Norway. 8. MRC Integrative Epidemiology Unit, University of Bristol, Bristol, UK. 9. Population Health Sciences, Bristol Medical School, University of Bristol,
Bristol, UK. Acknowledgements Division of
Endocrinology, Metabolism, and Molecular Medicine, Department of
Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL. 36. The Children’s Obesity Clinic, Department of Pediatrics, Copenhagen
University Hospital Holbæk, Holbæk, Denmark. 37. Institute of Biomedical and
Clinical Science, University of Exeter Medical School, University of Exeter, Royal
Devon and Exeter Hospital, Exeter, EX2 5DW, UK. 38. Department of Public
Health, Amsterdam Public Health Research Institute, Amsterdam UMC,
location Academic Medical Center, University of Amsterdam, 1105 AZ
Amsterdam, The Netherlands. 39. Department of Clinical Epidemiology,
Biostatistics and Bioinformatics, Amsterdam Public Health Research Institute,
Amsterdam UMC, location Academic Medical Center, University of Amster-
dam, 1105 AZ Amsterdam, The Netherlands. 40. Division of Metabolic and
Nutritional Medicine, Dr. von Hauner Children’s Hospital, Ludwig-Maximilians
Universität München (LMU), Munich, Germany. 41. Dpt. of Genetics, Physical
Anthropology and Animal Physiology, University of the Basque Country (UPV/
EHU) Leioa, Spain. 42. Biocruces-Bizkaia Health Research Institute, Barakaldo,
Spain. 43. Spanish Biomedical Research Center in Diabetes and Associated
Metabolic Disorders (CIBERDEM), Spain. 44. Institute for Molecular Medicine
Finland, FIMM, University of Helsinki, Finland. 45. Department of Epidemiology,
GRIAC (Groningen Research Institute for Asthma and COPD), University of
Groningen, University Medical Center Groningen, Groningen, The Netherlands. 46. Department of Population Medicine, Harvard Medical School, Harvard
Pilgrim Health Care Institute, Boston, MA, USA. 47. Division of Epidemiology,
Biostatistics, and Environmental Health, University of Memphis, USA. 48. Department of Genetics, University of Pennsylvania. 49. University of Western
Australia, Faculty of Health and Medical Sciences. 50. ISGlobal, Barcelona
(Spain). 51. Pompeu Fabra University (UPF). 52. Department of General Practice
and Primary Health Care, University of Helsinki and Helsinki University Hospital,
Helsinki, Finland. 53. Folkhälsan Research Center, Helsinki Finland. 54. Chronic
Disease Prevention Unit, National Institute for Health and Welfare, Helsinki,
Finland. 55. Pediatrics, Nutrition and Development Research Unit, Universitat
Rovira i Virgili, IISPV, Reus, Spain. 56. Department of Radiology and Nuclear
Medicine, Erasmus MC University Medical Center, Rotterdam, The Netherlands. 57. Institute for Risk Assessment Sciences, Utrecht University, Utrecht, The
Netherlands. 58. Neonatal Department, Children’s Memorial Health Institute,
Warsaw, Poland. 59. Department of Pediatrics, Perelman School of Medicine,
University of Pennsylvania, Philadelphia, PA, USA. 60. NIHR Exeter Clinical
Research Facility, University of Exeter Medical School and Royal Devon and
Exeter NHS Foundation Trust, Exeter, EX2 5DW, UK. 61. Acknowledgements Center for Genetic
M di i
N
th
t
U i
it F i b
S h
l f M di i
Chi
IL and MFH were responsible for the management of an individual contributing
study. SV, IN, GVD, JGE, RV, KB, HB, CSM, EAN, PRN, SJ, ØH, MV, CFB, MAVL, JCH,
NJT, GDS, PR, VG, BK, JPL, EV, JE, DG, TAL, VL, MA, MV, AK, WK, HH, SFAG, BAK,
ATH, LSM, MC, CEP, SL, LB, TEE, OTR, KP, HN, TGMV, FG, and BF were involved in
sample collection and phenotyping of an individual contributing study. FR, EZ,
JGE, TSA, HB, TMS, CTH, CSM, TH, NG, PRN, SJ, ØH, MV, CFB, MAVL, OP, JCH, ES,
NJT, GDS, BP, MW, TAL, MA, MS, HH, SFAG, JMV, RNB, NFJ, JRB, CEP, SL, LW, OTR,
MHZ, AGU, MMcC, and TGMV genotyped the data of an individual contribut-
ing study. SV, FR, JFF, IN, KP, NJ, JTL, TSA, TMS, CTH, NG, ØH, MV, BP, PR, VL, RJ,
MS, JPB, RNB, IT, NFJ, JRB, CW, MJK, NP, MHZ, XL, FG, BF, VDJ, and SLRS analyzed
data of an individual contributing study. All authors provided comments and
consultation regarding the analyses, interpretation of the results and manu-
script. SV, JFF and VWVJ had primary responsibility for final content. All authors
gave final approval of the version to be published. Acknowledgements SV, JFF and VWVJ designed the research and wrote the paper. SV, JFF and JPB
analyzed the meta-analysis data. IN, JFF, VWVJ, GVD, KB, HB, KFM, LL, JCH, PR,
VG, BK, JPL, EV, JE, DG, TAL, VL, MA, AK, WK, UG, CEP, SL, LW, OTR, KP, HN, TGMV,
MM, BF, WK, and JH were responsible for the study design of an individual
contributing study. JFF, VWVJ, IN, GVD, JTL, EW, TSA, KB, HB, MVL, KFM, LL, CFB,
MAVL, JCH, NJT, GDS, PR, VG, BK, JPL, EV, JE, DE, TAL, MV, AK, WK, HH, SFAG,
BAK, ATH, RMF, LSM, CI, MT, MV, CEP, SL, LW, HHHA, OTR, KP, HN, MM, BF, WK, JH, Page 11 of 16 Page 11 of 16 Vogelezang et al. BMC Medical Genomics (2022) 15:124 Vogelezang et al. BMC Medical Genomics (2022) 15:124 Vogelezang et al. BMC Medical Genomics (2022) 15:124 Copenhagen, Copenhagen, Denmark. 17. Department of Epidemiology and
Biostatistics, Imperial College London, London, UK. 18. Center for Life-Course
Health Research, Faculty of Medicine, University of Oulu, Oulu, Finland. 19. Institute for Medical Informatics, Statistics and Epidemiology, University of
Leipzig, Leipzig, Germany. 20. LIFE Research Center for Civilization Diseases,
University of Leipzig, Leipzig, Germany. 21. Department of Epidemiology
Research, Statens Serum Institut, Copenhagen, Denmark. 22. Department of
Statistics & Computational research, Universitat de València, València (Spain). 23. CIBER of Epidemiology and Public Health (CIBERESP). 24. Research Centre
of Applied and Preventive Cardiovascular Medicine, University of Turku,
Finland. 25. Department of Clinical Physiology and Nuclear Medicine,
University of Turku, Turku University Hospital, Turku, Finland. 26. Centre for
Population Health Research, University of Turku and Turku University Hospital,
Turku, Finland. 27. Department of Obstetrics and Gynecology, Institute of
Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothen-
burg Sweden. 28. Region Västra Götaland, Sahlgrenska University Hospital,
Department of Obstetrics and Gynecology, Gothenburg Sweden. 29. Novo
Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health and
MedicalSciences, University ofCopenhagen, Copenhagen, Denmark. 30. Institute of Biomedicine, Physiology, University of Eastern Finland, Kuopio,
Finland. 31. Foundation for Research in Health Exercise and Nutrition, Kuopio
Research Institute of Exercise Medicine, Kuopio, Finland. 32. Department of
Clinical Physiology and Nuclear Medicine, Kuopio University Hospital, Kuopio,
Finland. 33. Clinical Pharmacology, William Harvey Research Institute, Barts and
The London School of Medicine and Dentistry, Queen Mary University of
London, London, United Kingdom. 34. Department of Nutrition, Exercise and
Sports, Faculty of Science, University of Copenhagen. 35. The Early Growth Genetics Consortiumi 10. School of Oral and Dental Sciences, University of Bristol, Bristol,
UK. 11. Institute of Epidemiology, Helmholtz Zentrum München- German
Research Center for Environmental Health, Neuherberg, Germany. 12. Division
of Metabolic and Nutritional Medicine, Dr. von Hauner Children’s Hospital,
University of Munich Medical Center, Munich, Germany. 13. University of
Newcastle, School of Medicine and Public Health, Faculty of Medicine and
Health. 14. COPSAC, Copenhagen Prospective Studies on Asthma in
Childhood, Herlev and Gentofte Hospital, University of Copenhagen,
Copenhagen, Denmark. 15. Steno Diabetes Center Copenhagen, Herley,
Denmark. 16. The Bioinformatics Center, Department of Biology, University of Page 12 of 16 Page 12 of 16 Vogelezang et al. BMC Medical Genomics (2022) 15:124 Vogelezang et al. BMC Medical Genomics (2022) 15:124 Vogelezang et al. BMC Medical Genomics (2022) 15:124 Bioinformatics, Health Data and Digitalization, Norwegian Institute of Public
Health, Oslo, Norway. 65. Faculty of Medicine, University of Southampton,
Southampton, UK. 66. Center of Pediatric Research, University Hospital for
Children and Adolescents, University of Leipzig, Leipzig, Germany. 67. CHC, St
Vincent, Liège-Rocourt, Belgium. 68. Library, University of Eastern Finland,
Kuopio, Finland. 69. Department of Biomedical Sciences, University of
Copenhagen, Copenhagen N, Denmark. 70. Lunenfeld-Tanenbaum Research
Institute Mount Sinai Hospital Joseph & Wolf Lebovic Health Complex. 71. Department of Clinical Medicine, Faculty of Health and Medical Sciences,
University of Copenhagen, Copenhagen, Denmark. 72. Department of
Medicine, Stanford University School of Medicine, Stanford, California, USA. 73. National Institute of Public Health, University of Southern Denmark,
Copenhagen, Denmark. 74. Department of Public Health, Section of
Epidemiology, University of Copenhagen, Copenhagen, Denmark. 75. Department of Physiology and Department of Pediatrics, University of Turku,
Turku, Finland. 76. Paavo Nurmi Centre, Sports and Exercise Medicine Unit,
Department of Health and Physical Activity, University of Turku, Finland. 77. Institute of Translational Genomics, Helmholtz Zentrum München – German
Research Center for Environmental Health, Neuherberg, Germany. 78. Wellcome Trust Sanger Institute, Hinxton, CB10 1SA, UK. 79. Department of
internal medicine, Erasmus MC, University Medical Center Rotterdam, The
Netherlands. 80. Basque Government Public Health Department-Gipuzkoa,
San Sebastian, Spain. 81. Biodonostia Health Research Institute, San Sebastian,
Spain. 82. Spanish Biomedical Research Center in Epidemiology and Public
Health (CIBERESP), Spain. 83. Department of Preventive Medicine, Division of
Biostatistics, Northwestern University Feinberg School of Medicine, Chicago,
Illinois, USA. 84. Biocenter Oulu, Oulu, Finland. 85. Division of Immunology and
Immunotherapy, Cima Universidad de Navarra. 86. Instituto de Investigación
Sanitaria de Navarra (IdISNA). 87. The Early Growth Genetics Consortiumi Area de Salut de Menorca ib-salut. 88. Institut
d’Investigacio Sanitaria Illes Balears (IdISBa). 89. Netherlands Genomics
Initiative (NGI)-sponsored Netherlands Consortium for Healthy Aging NCHA),
Leiden, The Netherlands. 90. Department of Pediatrics, San Paolo Hospital,
University of Milan, Milan, Italy. 91. Technical University of Munich (TUM) and
Klinikum Rechts der Isar, TUM School of Medicine, Munich, Germany. 92. Department of Genetics, Perelman School of Medicine, University of
Pennsylvania. 93. Department of Clinical Genetics, Erasmus MC University
Medical Center, Rotterdam, The Netherlands. 94. Diabetes Unit, Massachusetts
General Hospital, Boston, MA, USA. 95. Department of Medicine, Universite de
Sherbrooke, QC, Canada. 96. Department of Pediatric Pulmonology and
Pediatric Allergology, Beatrix Children’s Hospital, GRIAC (Groningen Research
Institute for Asthma and COPD), University of Groningen, University Medical
Center Groningen, Groningen, The Netherlands. 97. Research Unit of Molecular
Epidemiology, Institute of Epidemiology, Helmholtz Zentrum Muenchen,
Munich, Germany. 98. Harokopi University of Athens, Athens, Greece. 99. Institute of Clinical Research, University of Southern Denmark, Odense,
Denmark. 100. Centre of Women’s, Family and Child Health, University of
South-Eastern Norway, Kongsberg, Norway. 101. Department of Obstetrics
and Gynecology, Institute of Clinical Science, Sahlgrenska Academy, University
of Gothenburg, Gothenburg Sweden (SA). 102. Region Västra Götaland,
Sahlgrenska University Hospital, Department of Obstetrics and Gynecology,
Gothenburg Sweden (SU). 103. Department of Genetics and Bioinformatics,
Domain of Health Data and Digitalization, Institute of Public Health, Oslo,
Norway (FHI). 104. Oxford National Institute for Health Research (NIHR)
Biomedical Research Centre, Churchill Hospital, Oxford, UK. 105. Wellcome
Centre for Human Genetics, University of Oxford, Oxford, UK. 106. Oxford
Centre for Diabetes, Endocrinology and Metabolism, University of Oxford,
Oxford, UK. 107. Max Planck Institute for Psycholinguistics, The Netherlands. 108. Donders Institute for Brain, Cognition and Behaviour, Radboud University,
The Netherlands. 109. Department of Pediatrics and Adolescents, Haukeland
University Hospital, Bergen, Norway. 110. Division of Endocrinology and
Diabetes, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA. 111. Center for Spatial and Functional Genomics, Division of Human Genetics,
Children’s Hospital of Philadelphia, Philadelphia, PA, USA. Author contributions
SV, JFF and VWVJ designed the research and wrote the paper. SV, JFF and JPB
analyzed the meta-analysis data. JFF and VWVJ were responsible for the study
design of an individual contributing study. JFF, VWVJ and SFAG were respon-
ibl f
th
t f
i di id
l
t ib ti
t d SV
d SFAG SV, JFF and JPB analyzed data of an individual contributing study. CHOP Europe The research of the CHOP study reported herein was partially supported by
the Commission of the European Community, specific RTD Programme “Qual-
ity of Life and Management of Living Resources,” within the 5th Framework
Programme (research grant nos. QLRT-2001-00389 and QLK1-CT-2002-30582);
the 6th Framework Programme contract no. 007036 (FP6-007036); the
European Union’s Seventh Framework Programme Project EarlyNutrition
under grant agreement no. 289346 (FP7-289346), the Horizon 2020 research
and innovation programme DYNAHEALTH (no. 633595) and the European
Research Council Advanced Grant META-GROWTH (ERC-2012-AdG—no. 322605). Additional support from the German Ministry of Education and
Research, Berlin (Grant Nr. 01 GI 0825) and the University of Munich Innovative
Research Priority Project MC-Health is gratefully acknowledged. This manu-
script does not necessarily reflect the views of the Commission and in no way
anticipates the future policy in this area. The funders of this study had no role
in study design, data collection, data analysis, data interpretation, decision to
publish, or preparation of the manuscript. p
,
p p
p
Copenhagen Prospective Studies on Asthma in Childhood (COPSAC)
Cohorts (COPSAC 2000, COPSAC 2010 and COPSAC registry):
All funding received by COPSAC is listed on www.copsac.com. The Lundbeck
Foundation (Grant no R16-A1694); The Ministry of Health (Grant no 903516);
Danish Council for Strategic Research (Grant no 0603-00280B) and The Capital
Region Research Foundation have provided core support to the COPSAC
research center. p
,
p p
p
Copenhagen Prospective Studies on Asthma in Childhood (COPSAC)
Cohorts (COPSAC 2000, COPSAC 2010 and COPSAC registry):
All funding received by COPSAC is listed on www.copsac.com. The Lundbeck
Foundation (Grant no R16-A1694); The Ministry of Health (Grant no 903516);
Danish Council for Strategic Research (Grant no 0603-00280B) and The Capital
Region Research Foundation have provided core support to the COPSAC
research center Copenhagen Prospective Studies on Asthma in Childhood (COPSAC)
Cohorts (COPSAC 2000, COPSAC 2010 and COPSAC registry): DNBC GOYA-offspring The Danish National Research Foundation has established the Danish
Epidemiology Science Centre that initiated and created the Danish National
Birth Cohort (DNBC). The cohort is furthermore a result of a major grant from
this foundation. Additional support for the Danish National Birth Cohort is
obtained from the Pharmacy Foundation, the Egmont Foundation, the March
of Dimes Birth Defects Foundation, the Augustinus Foundation, and the
Health Foundation. The DNBC biobank is a part of the Danish National Biobank
resource, which is supported by the Novo Nordisk Foundation. The Danish National Birth Cohort-Preterm Birth Study (DNBC-PTB) The Danish National Birth Cohort-Preterm Birth Study (DNBC-PTB)
DNBC: The Danish National Research Foundation has established the Danish
Epidemiology Science Centre that initiated and created the Danish National
Birth Cohort (DNBC). The cohort is furthermore a result of a major grant from
this foundation. Additional support for the Danish National Birth Cohort
is obtained from the Pharmacy Foundation, the Egmont Foundation, the
March of Dimes Birth Defects Foundation, the Augustinus Foundation, and
the Health Foundation. The DNBC biobank is a part of the Danish National
Biobank resource, which is supported by the Novo Nordisk Foundation. The
DNBC preterm birth (DNBC-PTB) study is a nested study within the DNBC. The
generation of GWAS genotype data for the DNBC-PTB sample was carried out
within the Gene Environment Association Studies (GENEVA) consortium with
funding provided through the National Institutes of Health’s Genes, Environ-
ment, and Health Initiative (U01HG004423; U01HG004446; U01HG004438). EFSOCH The Early Growth Genetics Consortiumi All authors
provided comments and consultation regarding the analyses, interpretation
of the results and manuscript. SV, JFF and VWVJ had primary responsibility for
final content. All authors gave final approval of the version to be published. All
authors read and approved the final manuscript. Funding Funding
Amsterdam Born Children and their Development
The ABCD study has been supported by grants from The Netherlands
Organisation for Health Research and Development (ZonMW) and Sarphati
Amsterdam. Genotyping was funded by the BBMRI-NL grant CP2013-50. Dr
M.H. Zafarmand was supported by BBMRI-NL (CP2013-50). Dr. T.G.M. Vrijkotte
was supported by ZonMW (TOP 40–00812–98–11010). Avon Longitudinal Study of Parents and Children (ALSPAC)
The UK Medical Research Council and Wellcome (Grant ref: 102215/2/13/2)
and the University of Bristol provide core support for ALSPAC. A comprehen-
sive list of grants funding for ALSPAC is available online (http://www.bristol.ac.
uk/alspac/external/documents/grant-acknowledgements.pdf). ES, NJT and
GDS work in a unit that receives funding from the University of Bristol and the
UK Medical Research Council (MC_UU_00011/1). Author contributions SV, JFF and VWVJ designed the research and wrote the paper. SV, JFF and JPB
analyzed the meta-analysis data. JFF and VWVJ were responsible for the study
design of an individual contributing study. JFF, VWVJ and SFAG were respon-
sible for the management of an individual contributing study. SV and SFAG
were involved in sample collection and phenotyping of an individual con-
tributing study. SFAG genotyped the data of an individual contributing study. Lifestyle—Immune System—Allergy Study and German Infant Study
on the influence of Nutrition Intervention (LISA + GINI)i Lifestyle—Immune System—Allergy Study and German Infant Study
on the influence of Nutrition Intervention (LISA + GINI)i on the influence of Nutrition Intervention (LISA + GINI)
The GINIplus study was mainly supported for the first 3 years of the Federal
Ministry for Education, Science, Research and Technology (interventional arm)
and Helmholtz Zentrum Munich (former GSF) (observational arm). The 4 year,
6 year, 10 year and 15 year follow-up examinations of the GINIplus study
were covered from the respective budgets of the 5 study centres (Helmholtz
Zentrum Munich (former GSF), Research Institute at Marien-Hospital Wesel,
LMU Munich, TU Munich and from 6 years onwards also from IUF—Leibniz
Research-Institute for Environmental Medicine at the University of Düssel-
dorf) and a grant from the Federal Ministry for Environment (IUF Düsseldorf, INfancia y Medio Ambiente [Environment and Childhood] (INMA)
Project j
The INMA-GIP cohort was funded by the Spanish Biomedical Research Center
in Epidemiology and Public Health (CIBERESP) and by grants from the Instituto
de Salud Carlos III (PI06/0867, PI09/00090, PI13/02187 and PI18/01142)
cofunded by ERDF, “A way to make Europe”, the Basque Department of
Health (Projects 2005111093, 2009111069, 2013111089, 2015111065 and
2018111086), the Provincial Government of Gipuzkoa (Projects DFG06/002,
DFG08/001 and DFG15/221) and annual agreements with the municipali-
ties of the study area (Zumarraga, Urretxu, Legazpi, Azkoitia y Azpeitia and
Beasain). Northern Finnish Birth Cohort Study 1966 (NFBC66) and 1968
(NFBC86) The Northern Finland Birth Cohort (NFBC) Research program, including
the study of the NFBC1966 and 1986 studies, received financial support
from Academy of Finland (1114194, 24300796), NHLBI grant 5R01HL087679
through the STAMPEED program (1RL1MH083268-01), ENGAGE project and
grant agreement HEALTH-F4-2007-201413, the Medical Research Council
(grant G0500539, centre grant G0600705, PrevMetSyn), and the Wellcome
Trust (project grant GR069224), UK. The program is currently being funded by
the H2020-633595 DynaHEALTH action and Academy of Finland EGEA-project. h
h
i
l
i i
d
i i
i
hild
(
)
d The INMA Menorca (MEN), Valencia (VAL) and Sabadell (SAB) cohorts were
funded by grants from the Instituto de Salud Carlos III (CB06/02/0041,
G03/176, FIS PI041436, PI081151, PI041705, and PS09/00432, FIS-FEDER
03/1615, 04/1509, 04/1112, 04/1931, 05/1079, 05/1052, 06/1213, 07/0314, and
09/02647), Fundació La Marató de TV3 (090430), Generalitat de Catalunya-
CIRIT (1999SGR 00241), Conselleria de Sanitat Generalitat Valenciana, and
Fundación Roger Torné. ISGlobal is a member of the CERCA Programme,
Generalitat de Catalunya. The Physical Activity and Nutrition in Children (PANIC) Study
The PANIC Study has financially been supported by grants from Ministry
of Education and Culture of Finland, Ministry of Social Affairs and Health of
Finland, Research Committee of the Kuopio University Hospital Catchment
Area (State Research Funding), Finnish Innovation Fund Sitra, Social Insurance
Institution of Finland, Finnish Cultural Foundation, Foundation for Paediatric
Research, Diabetes Research Foundation in Finland, Finnish Foundation for
Cardiovascular Research, Juho Vainio Foundation, Paavo Nurmi Foundation,
Yrjö Jahnsson Foundation, and the city of Kuopio. The PhD students and post-
doctoral researchers of The PANIC Study have financially been supported by
personal grants from Program for Clinical Research and Program for Health Sci-
ences of Doctoral School of University of Eastern Finland, Finnish Doctoral Pro-
grams in Public Health, Finnish Doctoral Program in Oral Sciences, European
Commission Marie Curie Fellowship Programme, North Savo Regional Fund
of Finnish Cultural Foundation, Juho Vainio Foundation, Päivikki and Sakari
Sohlberg Foundation, Foundation for Paediatric Research, Diabetes Research
Foundation in Finland, Finnish Foundation for Cardiovascular Research, Emil
Aaltonen Foundation, Paulo Foundation, Jalmari and Rauha Ahokas Founda-
tion, Aarne and Aili Turunen Foundation, Finnish Medical Foundation, Jenny
and Antti Wihuri Foundation, Orion Foundation, Urho Känkänen Foundation,
Finnish Association for the Study of Pain, Finnish Dental Society Apollonia,
Northern Savo Dental Association, Health Centre Dentist Association, Finnish
Female Dentists Association, Kuopio Naturalists’ Society, Olvi Foundation, and
the city of Kuopio. LIFE-Child LIFE-Child is funded by the Leipzig Research Center for Civilization Diseases
(LIFE). LIFE is an organizational unit affiliated to the Medical Faculty of the
University of Leipzig. LIFE is funded by means of the European Union, by the
European Regional Development Fund (ERDF) and by funds of the Free State
of Saxony within the framework of the excellence initiative. The study was
further supported by the German Research Foundation (DFG) for the CRC1052
ObesityMechanisms, project C05. IOW National Institutes of Health, USA, award numbers R01 AI091905 and R01
HL132321 National Institutes of Health, USA, award numbers R01 AI091905 and R01
HL132321. ational Institutes of Health, USA, award numbers R01 AI091905 and National Institutes of Health, USA, award numbers R01 AI091905 and R01
HL132321. EFSOCH The Exeter Family Study of Childhood Health (EFSOCH) was supported by
South West NHS Research and Development, Exeter NHS Research and
Development, the Darlington Trust and the Peninsula National Institute of Page 13 of 16 Page 13 of 16 Vogelezang et al. BMC Medical Genomics (2022) 15:124 Vogelezang et al. BMC Medical Genomics (2022) 15:124 Vogelezang et al. BMC Medical Genomics (2022) 15:124 Health Research (NIHR) Clinical Research Facility at the University of Exeter. The
opinions given in this paper do not necessarily represent those of NIHR, the
NHS or the Department of Health. Genotyping of the EFSOCH study samples
was funded by the Welcome Trust and Royal Society grant 104150/Z/14/Z. Generation R FKZ20462296). Further, the 15 year follow-up examination of the GINIplus
study was supported by the Commission of the European Communities, the
7th Framework Program: MeDALL project, and as well by the companies Mead
Johnson and Nestlé. The LISA study was mainly supported by grants from
the Federal Ministry for Education, Science, Research and Technology and in
addition from Helmholtz Zentrum Munich (former GSF), Helmholtz Centre for
Environmental Research—UFZ, Leipzig, Research Institute at Marien-Hospital
Wesel, Pediatric Practice, Bad Honnef for the first 2 years. The 4 year, 6 year,
10 year and 15 year follow-up examinations of the LISA study were covered
from the respective budgets of the involved partners (Helmholtz Zentrum
Munich (former GSF), Helmholtz Centre for Environmental Research—UFZ,
Leipzig, Research Institute at Marien-Hospital Wesel, Pediatric Practice, Bad
Honnef, IUF—Leibniz-Research Institute for Environmental Medicine at the
University of Düsseldorf) and in addition by a grant from the Federal Ministry
for Environment (IUF Düsseldorf, FKZ 20462296). Further, the 15-year follow-up
examination of the LISA study was supported by the Commission of the Euro-
pean Communities, the 7th Framework Program: MeDALL project. Health Research (NIHR) Clinical Research Facility at the University of Exeter. The
opinions given in this paper do not necessarily represent those of NIHR, the
NHS or the Department of Health. Genotyping of the EFSOCH study samples
was funded by the Welcome Trust and Royal Society grant 104150/Z/14/Z. Generation R and the
HBCS Helsinki Birth Cohort Study has been supported by grants from the Academy
of Finland (JGE grant no. 129369, 129907, 135072, 129255 and 126775), the
Finnish Diabetes Research Society, Samfundet Folkhälsan, Novo Nordisk
Foundation, Finska Läkaresällskapet, Juho Vainio Foundation, Signe and Ane
Gyllenberg Foundation, Liv och Hälsa, University of Helsinki and Ministry of
Education. JTL has been supported by the Finnish Cultural Foundation. INfancia y Medio Ambiente [Environment and Childhood] (INMA) y
Norwegian Mother and Child (MoBa) Cohort Study (Bo Jacobsson)
The analyses were supported by the Research Council of Norway (grant
FRIMEDBIO ES547711 to Bo Jacobsson), the March of Dimes Foundation Pre-
maturity Research Initiative Program (grant 21-FY16-121 to Bo Jacobsson). y
Generation R The general design of Generation RStudy is made possible by financial sup-
port from the Erasmus Medical Center, Rotterdam, the Erasmus University
Rotterdam, the Netherlands Organization for Health Research and Develop-
ment (ZonMw), the Netherlands Organisation for Scientific Research (NWO),
the Ministry of Health, Welfare and Sport and the Ministry of Youth and
Families. The project has received funding from the European Union’s Horizon
2020 research and innovation programme under grant agreement No 633595
(DynaHEALTH) and under grant agreements No 733206 (LifeCycle). HAPO HAPO is supported by grants from the Eunice Kennedy Shriver National
Institute of Child Health and Human Development and the National
Institute of Diabetes and Digestive and Kidney Diseases (R01-HD34242 and
R01-HD34243); the National Center for Research Resources (M01-RR00048 and
M01-RR00080); and the American Diabetes Association; and grants to local
field centers from Diabetes UK (RD04/0002756), Kaiser Permanente Medical
Center, KK Women’s and Children’s Hospital, Mater Mother’s Hospital, Novo
Nordisk, the Myre Sim Fund of the Royal College of Physicians of Edinburgh,
and the Howard and Carol Bernick Family Foundation. Norwegian Mother and Child (MoBa) Cohort Study (Pål R. Njølstad)
MOBA: The Norwegian Mother and Child Cohort Study was supported by
grants from the European Research Council (AdG #293574), the Bergen
Research Foundation (“Utilizing the Mother and Child Cohort and the Medical
Birth Registry for Better Health”), Stiftelsen Kristian Gerhard Jebsen (Trans-
lational Medical Center), the University of Bergen, the Research Council of
Norway (FRIPRO grant #240413), the Western Norway Regional Health Author-
ity (Strategic Fund “Personalized Medicine for Children and Adults”, and Open
Grants («Understanding infant weight biology through genomics and deep
phenotyping» grant #912250), and the Norwegian Diabetes Foundation; the
Research Council of Norway through its Centres of Excellence funding scheme
(#262700), Better Health by Harvesting Biobanks (#229624). SKOT (1 & 2) The SKOT 1 and 2 study have their base at the Department of Human Nutri-
tion, Faculty of Life Sciences, University of Copenhagen and was supported
by grants from The Danish Directorate for Food, Fisheries and Agri Business
as part of the ‘Complementary and young child feeding (CYCF)—impact on
short- and long-term development and health’ project and partly by contribu-
tions from the research program ‘Governing Obesity’ by the University of
Copenhagen Excellence Program for Interdisciplinary Research (www.go.ku.
dk Project Viva This study was supported by the National Institutes of Health (UG3OD23286,
R01 HD034568, and R01 AI102960). This study was supported by the National Institutes of Health (UG3OD23286,
R01 HD034568, and R01 AI102960). Prevention and incidence of asthma and mite allergy birth cohort
study—(PIAMA) Netherlands Organization for Health Research and Development (grant 912-
03-031), the Stichting Astmabestrijding and the Ministry of the Environment. Genome-wide genotyping was funded by the European Commission as part
of GABRIEL (A multidisciplinary study to identify the genetic and environmen-
tal causes of asthma in the European Community) contract number 018996
under the Integrated Program LSH-2004-1.2.5-1 Post genomic approaches to
understand the molecular basis of asthma aiming at a preventive or therapeu-
tic control and a Grant from BBMRI-NL (CP 29). The Raine Study We are grateful to the Raine Study participants and their families and we thank
the Raine Study and Lions Eye Institute research staff for cohort coordina-
tion and data collection. The Raine Study acknowledges the National Health
and Medical Research Council (NHMRC) for their long term contribution
to funding the study over the last 29 years. The core management of the
Raine Study has been funded by the University of Western Australia, Curtin
University, Telethon Kids Institute, Women and Infants Research Foundation,
Edith Cowan University, Murdoch University, The University of Notre Dame
Australia, and the Raine Medical Research Foundation. The collection of the
GWAS data for Gen2 of the Raine Study participants were supported by the
Canadian Institutes of Health Research—CIHR (MOP-82893) and the National
Health and Medical Research Council of Australia [grant numbers 634445,
634509, and 1021105]. The authors gratefully acknowledge the assistance
of the Western Australian DNA Bank (National Health and Medical Research
Council of Australia National Enabling Facility). This work was supported by
resources provided by the Pawsey Supercomputing Centre with funding from
the Australian Government and Government of Western Australia. S O ( & ) Author details y (
)
TEENAGE study has been co-financed by the European Union (European
Social Fund—ESF) and Greek national funds through the Operational Pro-
gram “Education and Lifelong Learning” of the National Strategic Reference
Framework (NSRF)—Research Funding Program: Heracleitus II. Investing in
knowledge society through the European Social Fund. This work was funded
by the Wellcome Trust (098051). 1 The Generation R Study Group, Erasmus MC, University Medical Center Rot-
terdam, Rotterdam, The Netherlands. 2 Department of Pediatrics, Erasmus MC,
University Medical Center Rotterdam, Rotterdam, The Netherlands. 3 Depart-
ment of Epidemiology, Erasmus MC, University Medical Center Rotterdam,
Rotterdam, The Netherlands. 4 Division of Human Genetics, Center for Applied
Genomics, Children’s Hospital of Philadelphia, Philadelphia, PA, USA. 5 Quantin-
uum Research LLC, San Diego, CA, USA. 6 Department of Pediatrics, Perelman
School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. 7 Division
of Endocrinology and Diabetes, The Children’s Hospital of Philadelphia, Phila-
delphia, PA, USA. 8 Division of Human Genetics, Center for Spatial and Func-
tional Genomics, Children’s Hospital of Philadelphia, Philadelphia, PA, USA. Availability of data and materials y
GWAS summary data will be deposited at the EGG website (https://egg-conso
rtium.org/) at publication. Individual study data are available from the cor-
responding author on reasonable request. Ethics approval and consent to participate All studies were approved by the national research ethics committee. Study-
specific details are shown in the acknowledgements and supplemental cohort
information. Informed consent to participate was obtained for all participants. Detailed and study-specific information can be found in the acknowledge-
ments and supplemental cohort information. Prevention and incidence of asthma and mite allergy birth cohort
study—(PIAMA) The PIAMA study was funded by grants from the Dutch Asthma Founda-
tion (grant 3.4.01.26, 3.2.06.022, 3.4.09.081 and 3.2.10.085CO), the ZON-MW Page 14 of 16 Vogelezang et al. BMC Medical Genomics (2022) 15:124 Vogelezang et al. BMC Medical Genomics (2022) 15:124 Vogelezang et al. BMC Medical Genomics (2022) 15:124 Vogelezang et al. BMC Medical Genomics (2022) 15:124 (BRC-1215-20011), the MRC Integrative Epidemiology Unit (MC_UU_12013/3)
and works within the CRUK Integrative Cancer Epidemiology Programme
(C18281/A19169). BSTP is funded by Max Planck Society core funds and the
Simons Foundation (514787). BK received personal funding by the European
Research Council Advanced Grant META-GROWTH (ERC-2012-AdG—no. 322605). RNB and RMF are funded by a Wellcome Trust and Royal Society Sir
Henry Dale Fellowship, grant 104150/Z/14/Z. VK was funded by Marie Curie
CAPICE EU project (European Union’s Horizon 2020 research and innova-
tion programme under grant agreement no. 721567). SFAG was financially
supported by an Institute Development Award from the Children’s Hos-
pital of Philadelphia, a Research Development Award from the Cotswold
Foundation, the Daniel B. Burke Endowed Chair for Diabetes Research, the
Children’s Hospital of Philadelphia Endowed Chair in Genomic Research and
NIH grant R01 HD056465. NFJ received funding from Basque Dpt. of Health
(SAN2018111086). HHHA was supported by ZonMW grant number 916.19.151. MHZ was supported by BBMRI-NL (CP2013-50). TGMV was supported by
ZonMW (TOP 40–00812–98–11010). XL was supported by the Nordic Center
of Excellence in Health-Related e-Sciences. BF was supported by the Oak
Foundation and the Novo Nordisk Foundation (12955). MFH is supported by
ADA Pathways to Stop Diabetes award 1-15-ACE-26. VWVJ received funding
from the Netherlands Organization for Health Research and Develop-
ment (VIDI 016.136.361) and the European Research Council (ERC-2014-
CoG-648916). MMcC is a Wellcome Senior Investigator and an NIHR Senior
Investigator. Relevant funding support for this work comes from Wellcome
(090532, 106130, 098381, 203141, 212259), NIDDK (U01-DK105535), and NIHR
(NF-SI-0617-10090). Netherlands Organization for Health Research and Development (grant 912-
03-031), the Stichting Astmabestrijding and the Ministry of the Environment. Genome-wide genotyping was funded by the European Commission as part
of GABRIEL (A multidisciplinary study to identify the genetic and environmen-
tal causes of asthma in the European Community) contract number 018996
under the Integrated Program LSH-2004-1.2.5-1 Post genomic approaches to
understand the molecular basis of asthma aiming at a preventive or therapeu-
tic control and a Grant from BBMRI-NL (CP 29). )
Special Turku Coronary Risk Factor Intervention Project (STRIP) Special Turku Coronary Risk Factor Intervention Project (STRIP)
STRIP was supported by the Academy of Finland (grants 206374, 294834,
251360, and 275595), the Juho Vainio Foundation, the Finnish Foundation for
Cardiovascular Research, the Finnish Ministry of Education and Culture, the
Finnish Cultural Foundation, the Sigrid Juselius Foundation, Special Govern-
mental grants for Health Sciences Research (Turku University Hospital), the
Yrjö Jahnsson Foundation, and the Turku University Foundation. Received: 20 August 2021 Accepted: 30 May 2022 j
TDCOB MMcC: The views expressed in this article are those of the author(s) and not
necessarily those of the NHS, the NIHR, or the Department of Health. He serves
on advisory panels for Pfizer, NovoNordisk, Zoe Global; has received honoraria
from Merck, Pfizer, NovoNordisk and Eli Lilly; has stock options in Zoe Global;
has received research funding from Abbvie, Astra Zeneca, Boehringer Ingel-
heim, Eli Lilly, Janssen, Merck, NovoNordisk, Pfizer, Roche, Sanofi Aventis, Ser-
vier & Takeda. Ms Receives funding from Pfizer Inc. for a project not related to
this research. The other authors declare that they have no competing interests. TDCOB received support from the Danish Innovation Foundation and The
Region Zealand Health and Medical Research Foundation. This study was
partly supported by grants from the Program Committee for Individuals,
Disease and Society of the Danish Innovation Foundation (grant numbers:
0603-00484B [TARGET] and 0603-00457B [BIOCHILD]). The Novo Nordisk
Foundation Center for Basic Metabolic Research, an independent Research
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J Epidemiol. 2006;163(9):829–37. 52. Hagenaars SP, Harris SE, Davies G, Hill WD, Liewald DC, Ritchie SJ, et al. Publisher’s Note
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English
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An Inverse Switch in DNA Base Excision and Strand Break Repair Contributes to Melphalan Resistance in Multiple Myeloma Cells
|
PloS one
| 2,013
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| 13,757
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Abstract Alterations in checkpoint and DNA repair pathways may provide adaptive mechanisms contributing to acquired drug
resistance. Here, we investigated the levels of proteins mediating DNA damage signaling and -repair in RPMI8226 multiple
myeloma cells and its Melphalan-resistant derivative 8226-LR5. We observed markedly reduced steady-state levels of DNA
glycosylases UNG2, NEIL1 and MPG in the resistant cells and cross-resistance to agents inducing their respective DNA base
lesions. Conversely, repair of alkali-labile sites was apparently enhanced in the resistant cells, as substantiated by alkaline
comet assay, autoribosylation of PARP-1, and increased sensitivity to PARP-1 inhibition by 4-AN or KU58684. Reduced base-
excision and enhanced single-strand break repair would both contribute to the observed reduction in genomic alkali-labile
sites, which could jeopardize productive processing of the more cytotoxic Melphalan-induced interstrand DNA crosslinks
(ICLs). Furthermore, we found a marked upregulation of proteins in the non-homologous end-joining (NHEJ) pathway of
double-strand break (DSB) repair, likely contributing to the observed increase in DSB repair kinetics in the resistant cells. Finally, we observed apparent upregulation of ATR-signaling and downregulation of ATM-signaling in the resistant cells. This was accompanied by markedly increased sensitivity towards Melphalan in the presence of ATR-, DNA-PK, or CHK1/2
inhibitors whereas no sensitizing effect was observed subsequent to ATM inhibition, suggesting that replication blocking
lesions are primary triggers of the DNA damage response in the Melphalan resistant cells. In conclusion, Melphalan
resistance is apparently contributed by modulation of the DNA damage response at multiple levels, including
downregulation of specific repair pathways to avoid repair intermediates that could impair efficient processing of
cytotoxic ICLs and ICL-induced DSBs. This study has revealed several novel candidate biomarkers for Melphalan sensitivity
that will be included in targeted quantitation studies in larger patient cohorts to validate their value in prognosis as well as
targets for replacement- or adjuvant therapies. Editor: Kamaleshwar Singh, Texas Tech University, United States of America Received September 13, 2012; Accepted December 23, 2012; Published February 6, 2013 Received September 13, 2012; Accepted December 23, 2012; Published February 6, 2013 Copyright: 2013 Sousa et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding was provided by the Research Council of Norway (http://www.forskningsradet.no/en/Home_page/1177315753906), grant no. 183423 and
183432; the Norwegian Cancer Society (https://kreftforeningen.no/en/), grant no. An Inverse Switch in DNA Base Excision and Strand Break
Repair Contributes to Melphalan Resistance in Multiple
Myeloma Cells Mirta M. L. Sousa1,2, Kamila Anna Zub1,3., Per Arne Aas1., Audun Hanssen-Bauer1, Aida Demirovic1,
Antonio Sarno1, Erming Tian1,4, Nina B. Liabakk1, Geir Slupphaug1,2,3* 1 Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology (NTNU), Trondheim, Norway, 2 The Proteomics and
Metabolomics Core Facility (PROMEC), Norwegian University of Science and Technology (NTNU), Trondheim, Norway, 3 The KG Jebsen Center for Myeloma Research,
Norwegian University of Science and Technology (NTNU), Trondheim, Norway, 4 Laboratory of Myeloma Genetics, Myeloma Institute for Research and Therapy, University
of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America Abstract TS-02-2008-0185; the Cancer Fund at St. Olavs Hospital, Trondheim (http://www. stolav.no/en/),grant no. 47063403; the Liaison Committee between the Central Norway Regional Health Authority (RHA) and the Norwegian University of Science
and Technology (NTNU) (http://www.ntnu.no/dmf/rad/samorg), grant no. 81133200; and Familien Blix fond til fremme av medisinsk forskning (http://www. legatsiden.no/innhold/visettlegat.php?id = 1017). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
manuscript. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: geir.slupphaug@ntnu.no * E-mail: geir.slupphaug@ntnu.no * E-mail: geir.slupphaug@ntnu.no . These authors contributed equally to this work. . These authors contributed equally to this work. Results and Discussion Melphalan Resistance in RPMI8226 Cells is Accompanied
by Increased S-phase Progression and Reduced G2/M
Arrest Compared to Melphalan Sensitive Parental Cells Melphalan is a bifunctional alkylating agent belonging to the
nitrogen mustard class of chemotherapeutic agents, and induces
both DNA monoadducts and ICLs [9,10]. Although ICLs
apparently constitute a minor fraction of the DNA lesions
introduced by Melphalan [11,12] they have been regarded the
major cytotoxic lesions since they block DNA replication and
induce the formation of DNA double-strand breaks (DSBs) [9]. Modulation of several cellular processes have been suggested to
contribute to resistance, including reduced drug uptake due to
defective drug transport [13], elevated glutathione levels [14,15],
decreased apoptosis [16,17] and modulated interaction of the
myeloma cells with extracellular matrix [18]. More recently,
modulation of DNA damage signaling- and repair pathways have
also been suggested to be major contributors to Melphalan
resistance [19–21]. This work has primarily focused upon the role
of the Fanconi Anemia (FA)/BRCA1 pathway in enhancing ICL
repair, thereby promoting cell survival [17,21–24]. However,
Melphalan may form a number of different adducts in DNA,
including (but likely not limited to) intra- and interstrand guanine
N7 crosslinks, adenine N3 alkylations and DNA-protein crosslinks
[25,26]. Moreover, a marked elevation of oxidative stress markers
has been observed in patients undergoing MP-based conditioning
regimen prior to ACST, as well as subsequent to transplant [27]
suggesting that oxidative DNA damage may also contribute to
cytotoxicity. The quantitative contribution of each type of DNA
lesion
to
Melphalan
cytotoxicity
remains,
however,
to
be
established. To mimic Melphalan concentrations observed in plasma of
patients receiving oral Melphalan treatment [35,36] and to
maintain similar growth rate of the sensitive/resistant cell lines,
the LR5 cells were added Melphalan to a final concentration of
1 mM at each passage. At this drug concentration we observed
essentially identical growth rate (Fig. 1A) and similar cell cycle
distribution (Fig. 1B, 0 hrs) of LR5 compared to the unexposed,
parental 8226 cells (referred to as steady state growth conditions in
the following). This is important when DNA repair proteins are
the subject of analysis, since the expression of many proteins
involved in DNA damage signaling and repair are strictly cell cycle
regulated [37–43]. Results and Discussion Whereas addition of Melphalan to a final
concentration of 2.5 mM to both cell lines mediated a weak
reduction
of
the
proliferation
of
the
resistant
LR5
cells,
proliferation of the sensitive cells essentially ceased 3 days after
Melphalan addition (Fig. 1A). Flow cytometry analysis indicated
that Melphalan exposure mediated a markedly delayed S-phase
progression in the sensitive- compared to the resistant cells,
followed by accumulation of cells in G2 (Fig. 1B). This delayed S-
phase progression conforms to introduction of DNA lesions that
delay or stall replication forks, as well as to potential accumulation
of unrepaired damage that activates the G2/M checkpoint and
induces apoptosis. This is supported by a markedly increased sub-
G1 fraction among the sensitive cells from 72 to 96 hours post
Melphalan addition, concomitant with a selective loss of cells in
G2 (Fig. 1B, bottom panels). Conversely, S-phase progression was
much less affected in the resistant LR5 cells, in agreement with a
more efficient removal or bypass of replication-blocking DNA
lesions, potentially accompanied with less efficient intra-S and G2/
M checkpoints. Repair of ICLs is not yet fully understood. However, proteins
belonging to several different DNA repair pathways cooperate to
resolve these complex lesions, including proteins contributing to
the FA-pathway, nucleotide excision repair (NER), mismatch
repair (MMR) and double-strand break repair (DSBR) ([28] and
references therein). More recently, the involvement of base
excision repair (BER) in development of resistance to DNA
crosslinkers has been increasingly recognized [29,30]. Interesting-
ly, while enhanced BER activities mediate resistance against
antitumor agents in some types of cancer [31–33], BER deficiency
has also been connected with development of Cisplatin resistance
[34]. To gain more insight into potential contribution to
Melphalan resistance by proteins involved in DNA damage
signaling and repair, we investigated the expression of a broad
panel of proteins involved in the DNA damage response in the
Melphalan-sensitive multiple myeloma cell line RPMI8226 and its
Melphalan
resistant
derivative
8226-LR5. Interestingly,
our
findings demonstrate that expression of several factors not
previously associated with Melphalan resistance is altered in the
resistant cells. Specifically we observed a striking downregulation
of several DNA glycosylases in the resistant cells in parallel with
apparently increased efficiency of single- (SSB) and double strand
break (DSB) repair. This suggest that resistant cells modulate the
repair pathways to avoid activation of multiple stress response
mechanisms that could interfere with processing of highly
cytotoxic ICLs and DSBs. Introduction phoma and constitute about 1% of all malignancies. It is also
showing substantial and systematic mortality (1% of total cancer
deaths) in the elderly of most areas worldwide [1,2]. Multiple
myeloma
(MM)
is
a
clonal
B-cell
malignancy
characterized by abnormal proliferation of malignant plasma cells
in the bone marrow, leading to impaired hematopoiesis as well as
osteolytic bone destruction [1]. As a consequence, MM patients
often experience bone pain, bone fractures, hypercalcemia and
fatigue. In addition, MM cells produce excessive amounts of non-
functional antibodies, which mediate increased susceptibility to
infections. MM is the second most prevalent haematological
malignancy (approximately 10%) following non-Hodgkin’s lym- Since its introduction in 1958, Melphalan (L-phenylalanine
mustard, Alkeran, CAS 148-82-3) [3] has been a common agent to
treat MM. In combination with prednisone (MP) it has been the
core treatment for patients with newly diagnosed MM who are not
eligible for autologous stem cell transplant (ASCT) and is also
central in high dose therapy (HDM) prior to ASCT [4,5]. More
recently, MP has also been combined with novel agents such as
thalidomide, lenalidomide and bortezomib in patients not eligible February 2013 | Volume 8 | Issue 2 | e55493 1 PLOS ONE | www.plosone.org February 2013 | Volume 8 | Issue 2 | e55493 DNA Repair Switch in Melphalan Resistance Melphalan-induced downregulation of specific BER proteins
may provide clues adjuvant therapies to overcome Melphalan
resistance. for ASCT [6] and this has increased survival ([7] and references
therein). Although
the
initial
response
to
Melphalan-based
treatment is generally good, treatment is limited by development
of acquired drug resistance (ADR) [8] and eventually all cases
become refractive [6]. There is thus an urgent need to develop
means for early detection of ADR to improve prognosis and
treatment. for ASCT [6] and this has increased survival ([7] and references
therein). Although
the
initial
response
to
Melphalan-based
treatment is generally good, treatment is limited by development
of acquired drug resistance (ADR) [8] and eventually all cases
become refractive [6]. There is thus an urgent need to develop
means for early detection of ADR to improve prognosis and
treatment. Melphalan-induced downregulation of specific BER proteins
may provide clues adjuvant therapies to overcome Melphalan
resistance. PLOS ONE | www.plosone.org DNA Repair Switch in Melphalan Resistance DNA Repair Switch in Melphalan Resistance proliferation of resistant cells was observed. (B) Flow cytometric analysis
of cell cycle phases shows that 2.5 mM Melphalan caused delayed S-
phase progression in sensitive cells followed by G2/M arrest. Conversely, progression throughout S-phase was less affected in the
resistant cells exposed to Melphalan. Moreover, the resistant cells were
able to overcome the G2/M arrest induced by Melphalan and re-enter
the cell cycle. Per cent cells in each of the cell cycle phases are indicated
above the histograms. The fraction of cells in subG1 is indicated, but
not illustrated in the gated histograms. doi:10.1371/journal.pone.0055493.g001 Figure 1. Melphalan resistance in RPMI8226 cells is
nied by increased S-phase progression and redu
arrest. Cell growth rate (A) and cell cycle distribution (B)
(8226) and resistant (LR5) cells exposed or non-exposed
Melphalan. (A) Exposure to 2.5 mM Melphalan greatly
proliferation of the sensitive cells, while a minor effec
PLOS ONE | www plosone org increased capability to detect and process replication-blocking
lesions in the resistant cells. Moreover, several proteins involved in
DSB repair by NHEJ were found to be upregulated in the resistant
cells, most notably LIG4 and XRCC4. Conversely, three BER
glycosylases (NEIL1, UNG2 and MPG) were found to be
downregulated, indicating a reduced base excision capacity in
the resistant cells. Somewhat surprisingly, we found no apparent
up-regulation of the FA-pathway in the LR5 cells, as determined
from the total level of FANCD2 as well as the apparent absence of
monoubiquitinylated FANCD2 (Fig. 2). Ub-FANCD2 is consid-
ered an essential link between the FA protein complex and the
BRCA1 repair machinery [44], and has previously been reported
to be modestly upregulated in LR5 cells adapted to growth in 5mM
Melphalan [24]. However, in another multiple myeloma cell line,
U266, no increase in FANCD2 was observed subsequent to
growth adaption to 6 mM Melphalan [17]. Figure 1. Melphalan resistance in RPMI8226 cells is accompa-
nied by increased S-phase progression and reduced G2/M
arrest. Cell growth rate (A) and cell cycle distribution (B) of sensitive
(8226) and resistant (LR5) cells exposed or non-exposed to 2.5 mM
Melphalan. DNA Repair Switch in Melphalan Resistance (A) Exposure to 2.5 mM Melphalan greatly reduced the
proliferation of the sensitive cells, while a minor effect upon the Among the cell-cycle and growth regulatory proteins we found
that the oncogenes c-MYC and 14-3-3b were upregulated in the
resistant cells, while Cdc25B/C and the tumor suppressor 14-3-3s
are markedly downregulated in the same cells. 14-3-3s is a known
inhibitor of G2/M-progression in many cell types subsequent to
DNA damage [45], while 14-3-3b has been shown to stimulate
proliferation [46]. The markedly shifted expression of these 14-3-3
proteins might thus contribute to the apparently reduced G2/M
checkpoint-activation in the resistant cells. Melphalan Resistance in RPMI8226 Cells is Accompanied
by Modulated Expression of Proteins Belonging to
Several DNA Damage Response Pathways To investigate the molecular mechanisms underlying Melpha-
lan resistance in the adapted LR5 cell line in more detail, and the
potential role of DNA-repair and signaling proteins, we performed
a quantitative western analysis of a panel of more than 50 proteins
directly and/or indirectly involved in the DNA damage response. We chose western analysis since many of the involved proteins
exist in the cells at levels that precluded a broad quantitative mass
spectrometry profiling by our current instrumentation unless pre-
enrichment strategies were included. Protein levels were analyzed
both during continuous growth of the sensitive/resistant cells, as
well as subsequent to a 6 h exposure to high-dose Melphalan
(50 mM), corresponding to the lag-period reported where the
conversion of mono-adducts into ICLs reaches a maximum [15]. Expression levels in the Melphalan resistant LR5 cells relative to
the sensitive parental cells under continuous growth are summa-
rized in Fig. 2, and demonstrate marked changes in the steady-
state levels of several proteins belonging to different DNA damage
signaling- and repair pathways. Most notably, we observed
upregulation of the DNA damage sensory kinase ATR in the
resistant cells with a concomitant decrease in ATM, indicating The present results contribute novel candidate factors to be
included in targeted protein profiling analyses by e.g. high-
throughput MRM-mass spectrometry of patient samples to further
evaluate
their prognostic value. In addition, the
apparent PLOS ONE | www.plosone.org February 2013 | Volume 8 | Issue 2 | e55493 February 2013 | Volume 8 | Issue 2 | e55493 2 Melphalan Resistant Cells have Reduced Expression of
Several BER-initiating Glycosylases and Display Cross-
resistance to Agents that Mediate Non-bulky DNA Base
Alterations Each
bar represent the mean expression ratio of the target protein in the LR5 (resistant) cells relative to the reference value of 1 (dotted line) in the
sensitive cells subsequent to normalization against either b-actin or tubulin (to avoid overlapping signals). Quantitative analysis enclosed an average
of 3 to 5 biological replicates with standard deviations as indicated. The P values were calculated by one sample two tailed t test against a
hypothetical expression ratio set to 1 (no change in expression). .90%, .95% and .98% confidence levels indicated by *, **, and ***, respectively. #
Only non-ubiquitinylated FANCD2 was detected in the analyses. doi:10.1371/journal.pone.0055493.g002 Figure 2. Steady state expression levels of proteins involved in DNA repair and DNA damage signaling response in 8226-LR5 cells
relative to parental cells. Protein levels were assessed by quantitative western blot analysis using specific antibodies against target proteins. Each
bar represent the mean expression ratio of the target protein in the LR5 (resistant) cells relative to the reference value of 1 (dotted line) in the
sensitive cells subsequent to normalization against either b-actin or tubulin (to avoid overlapping signals). Quantitative analysis enclosed an average
of 3 to 5 biological replicates with standard deviations as indicated. The P values were calculated by one sample two tailed t test against a
hypothetical expression ratio set to 1 (no change in expression). .90%, .95% and .98% confidence levels indicated by *, **, and ***, respectively. #
Only non-ubiquitinylated FANCD2 was detected in the analyses. doi:10.1371/journal.pone.0055493.g002 tion from other glycosylases than UNG to the total uracil-excision
activity was ,10% in both cell lines (Fig. 3B). Thus the reduced
uracil-excision capacity in the resistant cells was mediated
primarily by reduced level of UNG protein. cell-cycle regulated with the highest expression in late G1/S-phase
[40,49], the most likely explanation for the apparent upregulation
is likely accumulation in S-phase of cells at the high (50 mM)
concentration of Melphalan used in the experiment. This was also
supported by the more pronounced upregulation in the sensitive
cells
which
conforms
to
the
strong
S-phase
accumulation
subsequent to Melphalan treatment (Fig. 1B). The reduced level
of UNG2 protein was also accompanied by a reduced uracil-
excision activity in resistant cell free extracts and in both cell
extracts this activity increased subsequent to acute exposure to
50 mM melphalan (Fig. 3B). Melphalan Resistant Cells have Reduced Expression of
Several BER-initiating Glycosylases and Display Cross-
resistance to Agents that Mediate Non-bulky DNA Base
Alterations Melphalan Resistant Cells have Reduced Expression of
Several BER-initiating Glycosylases and Display Cross-
resistance to Agents that Mediate Non-bulky DNA Base
Alterations BER is the dominating cellular pathway for repair of non-bulky
DNA base damage [47]. A function of BER in the repair of
Melphalan-induced DNA damage is thus not obvious, unless
secondary lesions are induced by yet unidentified mechanisms. One exception is, however, the finding that the DNA glycosylase
NEIL1 is able to excise a bulky unhooked psoralen-induced ICL
by cleavage of the glycosidic bond between the deoxyribose and
the crosslinked base [48] Surprisingly, however, we observed a
marked downregulation at steady state of 3 out of 6 initiator
glycosylases of BER (Fig. 3 A) in the LR5 cells whereas levels of
downstream (damage- general) enzymes (APE1, POLB, FEN1,
LIG1 and LIG3) remained unaltered (Fig. 2). Interestingly, the
downregulated
glycosylases
NEIL1
(0.3060.20),
UNG2
(0.4560.06) and MPG (0.7360.09), share a common character-
istic in that they are able to excise their respective base substrates
(oxidised pyrimidines, uracil, and alkylated bases, respectively)
from both dsDNA- as well as ssDNA contexts. Conversely, the
levels of TDG, MBD4 and OGG1 that are all strictly dsDNA-
specific, were essentially unaltered in the LR5 cells compared to
the parental 8226 cells. Subsequent to a 6 h treatment with 50 mM
Melphalan, UNG2 was upregulated in both the sensitive and
resistant cells whereas little modulation of the protein levels of the
other glycosylases was observed (Fig. 3A). Since UNG2 is strictly Figure 1. Melphalan resistance in RPMI8226 cells is accompa-
nied by increased S-phase progression and reduced G2/M
arrest. Cell growth rate (A) and cell cycle distribution (B) of sensitive
(8226) and resistant (LR5) cells exposed or non-exposed to 2.5 mM
Melphalan. (A) Exposure to 2.5 mM Melphalan greatly reduced the
proliferation of the sensitive cells, while a minor effect upon the PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org February 2013 | Volume 8 | Issue 2 | e55493 3 DNA Repair Switch in Melphalan Resistance Figure 2. Steady state expression levels of proteins involved in DNA repair and DNA damage signaling response in 8226-LR5 cells
relative to parental cells. Protein levels were assessed by quantitative western blot analysis using specific antibodies against target proteins. February 2013 | Volume 8 | Issue 2 | e55493 doi:10.1371/journal.pone.0055493.g003 total level of reactive oxygen species (ROS) in the two cell lines. We observed about 20% lower ROS level in the resistant-
compared to the sensitive cells, but no significant difference in any
of the cell lines after treatment with 50 mM Melphalen. In
contrast, treatment with 50 mM of the positive control tert-butyl
hydroperoxide (TBHP) mediated about threefold increased ROS
in both cell lines (Fig. S1). The most likely interpretation of these
results is that the markedly increased accumulation of genomic 8-
oxodG in the resistant cells is not primarily caused by Melphala-
induced ROS, but more likely result from lack of removal of 8-
oxodG formed by endogenous sources of ROS. This would
conform to the apparently very low level of OGG1 in both cell
lines as well as the markedly (threefold) downregulated level of
NEIL1 in the resistant cells. Next we investigated whether the reduced levels of DNA
glycosylases were accompanied by increased sensitivity towards
agents inducing BER substrate lesions. To this end we subjected
the cells to treatment with the alkylator methyl methanesulfonate
(MMS), H2O2 and the antimetabolite 5-fluorouracil (5-FU). The
latter induces both genomic uracil and 5-FU, both of which are
substrates for UNG2 (reviewed in [51]). In addition, we monitored
potential cross-resistance towards the ICL-inducing agent mito-
mycin C (MMC) and UVB-irradiation (Fig. 4). Notably, the
Melphalan-resistant cells displayed a markedly increased resistance
to both MMS and H2O2 (Fig. 3A,B). This was rather surprising,
given the observed reduced levels of the respective glycosylases
(MPG and NEIL1) that excise the major base lesions induced by
these agents [52–53]. A similar cross-resistance was observed with
low concentrations (1 mM) of 5-FU, whereas at high concentra-
tions (10 mM) the Melphalan-sensitive cells displayed higher
resistance to 5-FU (Fig. 4 B,C). Finally, the Melphalan-resistant
cells displayed cross-resistance to the ICL-inducing agent MMC
(Fig. 4E) whereas both cell lines were equally sensitive to UVB
(Fig. 3F). The latter suggests that modulation of the expression of
NER-factors does not likely contribute to the Melphalan-resistant
phenotype in the 8226 cells. The apparently increased tolerance of the Melphalan-resistant
cell line towards agents causing structurally minor DNA-base-
lesions (BER substrates) was intriguing. Previous studies have,
however, demonstrated that overexpression of MPG mediates
increased sensitivity towards MMS [54], likely because the
number of AP-sites generated exceeds the capacity of downstream
BER-factors and results in increased levels of cytotoxic repair
intermediates. Melphalan Resistant Cells have Reduced Expression of
Several BER-initiating Glycosylases and Display Cross-
resistance to Agents that Mediate Non-bulky DNA Base
Alterations Moreover, the increased activity was
significant (P,0.05) in the sensitive cells only, in agreement with
the UNG2 western analysis. Repeating the excision experiments in
presence of the UNG-specific inhibitor Ugi encoded by the
bacteriophage PBS2 [50] demonstrated that the overall contribu- To monitor whether the markedly reduced level of NEIL1 as
well as the low level of OGG1 (barely above detection level,
Fig. 3A) was reflected in the genomic content of genomic oxidative
damage, we analyzed the genomic 8-oxodG in both cell lines at
steady state as well as 6 h subsequent to acute 50 mM Melphalan
exposure. As illustrated in Fig. 3C, the steady state 8-oxodG level
in the resistant cells was about twofold higher than that of the
sensitive cells. Remarkably, however, acute exposure to 50 mM did
not mediate any increase in 8-oxodG in any of the cell lines. To
investigate the underlying cause of genomic 8-oxodG accumula-
tion in the resistant cell line in more detail, we also monitored the February 2013 | Volume 8 | Issue 2 | e55493 PLOS ONE | www.plosone.org February 2013 | Volume 8 | Issue 2 | e55493 4 DNA Repair Switch in Melphalan Resistance observed for the dsDNA-specific glycosylases TDG, MBD4 and OGG1,
the latter barely above the detection level in the cells (A). In agreement
with UNG2 protein levels, activity assays showed that uracil excision
activity was significantly higher in sensitive cells against [3H]dUMP-
containing ss- and dsDNA substrates. Moreover, high dose Melphalan
mediated an apparent increase in UDG activity in both cell lines,
although this was only significant (P,0.05) for the resistant cells. Addition of the UNG-specific inhibitor Ugi to the reactions demon-
strated that the observed reduced uracil-excision activity in the resistant
cell line was mainly accounted for by reduction of UNG-activity. Each
bar constitutes an average from four independent experiments, each
run in triplicate, with standard deviations as demonstrated. P-values
were calculated using unpaired Student t-test (B). A twofold increased
steady state level of genomic 8-oxodG was found in the resistant
compared to the sensitive cells. Each bar constitutes and average from
three independent experiments with standard deviations as indicated
(C). Figure 3. DNA glycosylases with dual ss/dsDNA substrate
specificity are downregulated in Melphalan-resistant cells. Levels of DNA glycosylases (A), relative UDG activities (B) and genomic
8-oxodG-content in sensitive and resistant cells prior and after exposure
to 50 mM Melphalan. Melphalan Resistant Cells have Reduced Expression of
Several BER-initiating Glycosylases and Display Cross-
resistance to Agents that Mediate Non-bulky DNA Base
Alterations Western blot analysis revealed downregulation of
UNG2, MPG and NEIL1 (dual ss/dsDNA-specific glycosylases) in the
Melphalan-resistant cells compared to sensitive cells while this was not doi:10.1371/journal.pone.0055493.g003 Furthermore, it was recently demonstrated that
UNG-deficient cells displayed a Cisplatin-resistant phenotype
accompanied by enhanced repair of ICLs and ICL-induced DSBs
[34]. The authors demonstrated that (extrahelical) cytosines
flanking
the
Cisplatin-ICL
underwent
preferential
oxidative Figure 3. DNA glycosylases with dual ss/dsDNA substrate
specificity are downregulated in Melphalan-resistant cells. Levels of DNA glycosylases (A), relative UDG activities (B) and genomic
8-oxodG-content in sensitive and resistant cells prior and after exposure
to 50 mM Melphalan. Western blot analysis revealed downregulation of
UNG2, MPG and NEIL1 (dual ss/dsDNA-specific glycosylases) in the
Melphalan-resistant cells compared to sensitive cells while this was not PLOS ONE | www.plosone.org February 2013 | Volume 8 | Issue 2 | e55493 5 DNA Repair Switch in Melphalan Resistance deamination in vitro and proposed that UNG-mediated removal of
h fl
ki
il i d
AP i
d BER i
di
h
preferential deamination of cytosine also oc
ICL
i
h
b
bli h d
Figure 4. Melphalan resistance is accompanied by increased resistance to agents inducing non-bulky BER s
exposed to MMS (A), H2O2 (B), low (C) and high (D) doses of 5-FU, MMC (E) and UVB (F) assessed by the MTT assay. Viability c
Melphalan-resistant cells are cross-resistant to MMS, H2O2, low dose 5-FU and MMC. Conversely, high dose of 5-FU affects the p
cells in higher extent compared to sensitive cells. Exposure to UVB similarly affected growth of both cell lines. Increasing
directly with the number of living cells. Standard deviation bars are indicated. doi:10.1371/journal.pone.0055493.g004
DNA Repair Switch in Figure 4. Melphalan resistance is accompanied by increased resistance to agents inducing non-bulky BER substrates. Cell were
exposed to MMS (A), H2O2 (B), low (C) and high (D) doses of 5-FU, MMC (E) and UVB (F) assessed by the MTT assay. Viability curves showed that the
Melphalan-resistant cells are cross-resistant to MMS, H2O2, low dose 5-FU and MMC. Conversely, high dose of 5-FU affects the proliferation of resistant
cells in higher extent compared to sensitive cells. Exposure to UVB similarly affected growth of both cell lines. Increasing absorbance correlates
directly with the number of living cells. Standard deviation bars are indicated. doi:10.1371/journal.pone.0055493.g004 preferential deamination of cytosine also occurs at Melphalan-
ICLs remains, however, to be established. Melphalan-Resistance is Accompanied by Reduced Level
of Genomic Alkali-labile Sites (ALS) and Increased
Sensitivity to PARP-1 Inhibition In addition SSBs are formed by oxidative attack at
the sugar-phosphate backbone or as intermediates in other DNA-
processing pathways, including repair of ICLs. An alternative or
additional explanation to the reduced levels of ALS in the resistant
cells could thus be increased processing of AP sites and SSBs. Notably, we did not detect any overt changes in expression of
proteins
involved
in
damage-general
steps
of
BER/SSBR,
including APE1, POLB, PCNA, LIG1, and LIG3 (Fig. 2). One
exception was a modestly increased level of the scaffolding protein
XRCC1. However, whereas the total level of the nick-sensing
enzyme Poly(ADP-ribose) polymerase 1 (PARP-1) was similar in
the two cell lines, a high molecular weight (HMW) form
apparently constituting autoribosylated and activated PARP-1
[55] was observed exclusively in the resistant cells, even after
treatment with 50 mM Melphalan (Fig. 5B, upper and middle
panels). To further substantiate the activation of PARP-1 by
autoribosylation, we treated the cells with 4-amino-1,8-naphtha-
limide (4-AN), a potent inhibitor of PARP-1 and of SSB repair
[56]. This mediated a complete loss of the HMW (PARylated)
form
and a concomitant increase in the faster
migrating
unmodified PARP-1 (Fig. 5C), in agreement with loss of
autoribosylation. The markedly increased PARP-1 activation
was somewhat surprising given the lower level of PARP-1
trigger-lesions (ALS) in the resistant cells and suggests that the
majority of these sites are efficiently repaired in the resistant cells. Notably, the increased enzymatic activity of PARP-1 in the
resistant cells conforms to the upregulated level of c-MYC (Fig. 2). c-MYC was recently demonstrated to regulate the activity of
PARP-1 via downregulation of the PARP-1 inhibitor protein
BIN1, thereby increasing Cisplatin resistance [57]. No marked alteration in the steady-state level of most proteins
involved in HRR (RAD50, RAD51, RAD52, MRE11) was
observed in the Melphalan-resistant cells (Fig. 2). One exception
was, however, a moderate increase in BRCA1 (1.4360.10). However, subsequent to high dose Melphalan, the level of BRCA1
was reduced in both the sensitive (0.5460.32) and resistant
(0.5660.11) cells, suggesting a down-regulation of HRR during
high-dose treatment (Fig. 6B). In summary, these results indicate
that upregulation of NHEJ, including a potential involvement of
PARP-1 to facilitate synapsis of DSBs [60], may contribute to the
Melphalan-resistant phenotype. To test whether increased PARP-1 activation also mediated
altered sensitivity to PARP-1 inhibition, we next treated both cell
lines with 4-AN and subjected the cells to MTT assay. As
illustrated in Fig. Melphalan-Resistance is Accompanied by Reduced Level
of Genomic Alkali-labile Sites (ALS) and Increased
Sensitivity to PARP-1 Inhibition DNA DSBs are repaired via two major pathways; homologous
recombination repair (HRR), and non-homologous end-joining
(NHEJ) [58]. HRR takes advantage of homologous DNA
templates present in late S and G2 and is thus error-free. NHEJ
simply re-joins broken DNA ends, but is error-prone since it often
involves processing of the ends to produce ligatable ends. NHEJ
may thus result in chromosomal translocations, which are
commonly observed in multiple myeloma. NHEJ may operate
throughout the entire cell-cycle and appears to be the major route
for DSBs repair in vertebrates. The classical NHEJ pathway is
initiated by binding of the KU heterodimer to the ends of a DSB,
and subsequent recruitment of the catalytic subunit of DNA-PK
(DNA-PKcs) to form the active holoenzyme. DNA-PKCS is
autophosphorylated and also phosphorylates other NHEJ proteins
such as RPA2, WRN, XLF, LIG4, XRCC4 and Artemis to
facilitate end-joining and repair ([59] and references therein). More recently alternative NHEJ pathways have been described,
termed Alt-NHEJ, which is dominated by microhomology-
mediated end-joining [58]. Alt-NHEJ is mediated by PARP-1/2,
and involves WRN and ligation by LIG3/XRCC1. Interestingly,
the protein quantitation experiments indicate that several factors
of the classical NHEJ pathway are upregulated in the Melphalan-
resistant cells (Fig. 2 and 6A), with LIG4 (2.5160.08, p = 0.001),
XRCC4 (1.6960.19, p = 0.02), and RPA2 (1.6960.0.34, p = 0.03)
showing significant upregulation. Artemis, which is involved in
DNA end-processing prior to ligation, is present in two forms in
both parental and resistant cells. Notably, however, a marked shift
from the low-MW form conforming to unphosphorylated Artemis,
to the high-MW form conforming to phosphorylated (activated)
Artemis was observed in the resistant cells (Fig. 6A). These results
indicate that the final steps of NHEJ encompassing end-processing
and ligation are upregulated in the Melphalan-resistant cells. y
To investigate whether the reduced levels of BER glycosylases
was also accompanied modulated levels of ALS, including AP-sites
and SSBs, we performed an alkaline comet assay of both cell lines
in the absence of- and subsequent to high dose (50 mM and
100 mM) Melphalan. Notably the resistant cells displayed signif-
icantly lower values of tail DNA both in the absence- and presence
of high dose Melphalan (Fig. 5A) in agreement with the decreased
level of initiator BER glycosylases. However, ALS are also formed
by
glycosylase-independent
routes. AP-sites
are
formed
by
spontaneous base loss and are further processed to SSBs by AP-
endonucleases. Melphalan-Resistance is Accompanied by Reduced Level
of Genomic Alkali-labile Sites (ALS) and Increased
Sensitivity to PARP-1 Inhibition 5D (upper panel), the Melphalan-resistant cells
were significantly more sensitive to PARP-1 inhibition than the
parental cell line. The increased sensitivity of Melphalan-resistant
cells to PARP-1 inhibition was also observed when using another
PARP-1 inhibitor, KU58684 (Fig. 5D, lower panel). doi:10.1371/journal.pone.0055493.g003 Nevertheless, since
UNG2, NEIL1 and MPG are major BER initiators of endogenous deamination in vitro and proposed that UNG-mediated removal of
such flanking uracils induce AP-sites and BER intermediates than
compete with productive ICL DNA repair. Whether such February 2013 | Volume 8 | Issue 2 | e55493 PLOS ONE | www.plosone.org 6 DNA Repair Switch in Melphalan Resistance effect of PARP-1 inhibition via such alternative routes could thus
not be excluded. DNA base damage, decreased steady-state levels of BER repair
intermediates (AP-sites and single-strand breaks (SSBs)) could
mediate a survival benefit during the more complex processing of
Melphalan-induced DNA damage. ATM and ATR Signaling Pathways are Modulated in
Melphalan Resistant Cells (A) Comet assays demonstrated that the resistant LR
cells contained a significantly lower number of alkali-labile sites than the sensitive cells both at steady-state and subsequent to high-dose melphalan
treatment. Per cent tail DNA of the comets is presented in dot plots with average and corresponding SEM. P values were calculated using an unpaired
two-tailed t-test. R: resistant, S: sensitive. 100 comets were randomly selected and evaluated from one experiment at each treatment. (B) Western blot
analysis showed that PARP-1 is activated via poly-ADP(ribosyl)ation only in the resistant cells suggesting that SSB repair and potentially also DSB
synapsis is enhanced in the resistant cells. (C) Activation of PARP1 by PARylation in the resistant LR5 cells was completely abolished subsequent to
treatment with the PARP-1 inhibitor 4-AN. (D) Inhibition of PARP-1 by 4-AN or KU58684 obstructs the proliferation of resistant cells while having a
minor effect on the sensitive cells. doi:10 1371/journal pone 0055493 g005 Figure 5. Assessment of alkali-labile sites in Melphalan sensitive and resistant cells. (A) Comet assays demonstrated that the resistant LR
cells contained a significantly lower number of alkali-labile sites than the sensitive cells both at steady-state and subsequent to high-dose melphalan
treatment. Per cent tail DNA of the comets is presented in dot plots with average and corresponding SEM. P values were calculated using an unpaired
two-tailed t-test. R: resistant, S: sensitive. 100 comets were randomly selected and evaluated from one experiment at each treatment. (B) Western blot
analysis showed that PARP-1 is activated via poly-ADP(ribosyl)ation only in the resistant cells suggesting that SSB repair and potentially also DSB
synapsis is enhanced in the resistant cells. (C) Activation of PARP1 by PARylation in the resistant LR5 cells was completely abolished subsequent to
treatment with the PARP-1 inhibitor 4-AN. (D) Inhibition of PARP-1 by 4-AN or KU58684 obstructs the proliferation of resistant cells while having a
minor effect on the sensitive cells. doi:10.1371/journal.pone.0055493.g005 ATM and ATR in fact may directly interact and activate each
other [62]. cell cycle analyses (Fig. 1B), the lack of modulation of pro- (BAX)
and anti- (BCL2) apoptotic proteins (Fig. 2) and the low degree of
PARP-1 cleavage (Fig. 3B). Whereas no difference in the level of DNA-PKcs was observed
in the resistant and sensitive cells (Fig. 6A), marked alterations
were observed for ATM and ATR. As illustrated in Fig. ATM and ATR Signaling Pathways are Modulated in
Melphalan Resistant Cells The apparent switch in the levels of NHEJ versus HRR
prompted us to investigate the involvement the damage-signaling
kinases involved in DSB repair. DSB repair is initiated by three
kinases belonging to the family of phosphatidylinositol 3-kinase-
related kinases (PIKKs); DNA-PK, ATM (ataxia telangiectasia
mutated) and ATR (ataxia telangiectasia mutated) [61]. While
DNA-PK and ATM primarily respond to DSBs induced by e.g. ionizing radiation, ATR responds to replication blocks or other
factors that induce extended stretches of ssDNA. Recent research
has, however, demonstrated that the ATM- and ATR-initiated
pathways are not separated, but highly interconnected and that In summary, this suggests that the Melphalan-resistant cells
have a lower level of ALS that may impede productive processing
of Melphalan-ICLs. The lower level of ALS is apparently
mediated both by reduced formation of AP-sites by glycosylases
and by improved PARP-1- induced processing of ALS as
substantiated by the unique sensitivity of the resistant cells to
blockage of SSB repair. However, PARP-1 is also involved in
other cellular defense mechanisms such as repair of DSBs and an February 2013 | Volume 8 | Issue 2 | e55493 7 PLOS ONE | www.plosone.org DNA Repair Switch in Melphalan Resistance Figure 5. Assessment of alkali-labile sites in Melphalan sensitive and resistant cells. (A) Comet assays demonstrated that the resistant LR
cells contained a significantly lower number of alkali-labile sites than the sensitive cells both at steady-state and subsequent to high-dose melphalan
treatment. Per cent tail DNA of the comets is presented in dot plots with average and corresponding SEM. P values were calculated using an unpaired
two-tailed t-test. R: resistant, S: sensitive. 100 comets were randomly selected and evaluated from one experiment at each treatment. (B) Western blot
analysis showed that PARP-1 is activated via poly-ADP(ribosyl)ation only in the resistant cells suggesting that SSB repair and potentially also DSB
synapsis is enhanced in the resistant cells. (C) Activation of PARP1 by PARylation in the resistant LR5 cells was completely abolished subsequent to
treatment with the PARP-1 inhibitor 4-AN. (D) Inhibition of PARP-1 by 4-AN or KU58684 obstructs the proliferation of resistant cells while having a
minor effect on the sensitive cells. doi:10.1371/journal.pone.0055493.g005 Figure 5. Assessment of alkali-labile sites in Melphalan sensitive and resistant cells. (A) Comet assays de Figure 5. Assessment of alkali-labile sites in Melphalan sensitive and resistant cells. Phosphorylated cH2AX foci are Formed more Rapidly
and Persist for a Shorter Time in Melphalan Resistant
8226 Cells Phosphorylated cH2AX foci are Formed more Rapidly
and Persist for a Shorter Time in Melphalan Resistant
8226 Cells The level of nuclear foci of cH2AX is generally used to monitor
the number of DSBs that are marked for processing subsequent to
genotoxic stress and is also a sensitive marker of DNA damage
induced by ICL-inducing agents [67]. To monitor a potential
differential formation of- and persistence of such foci in the two
cell lines, cells were fixed at different time points subsequent to
treatment with 25 mM Melphalan, incubated with cH2AX
antibodies, and nuclear cH2AX quantified subsequent to confocal
analysis. Due to the half-life of Melphalan in cell culture medium
at 37 degrees (, 1 h), cell washing after pulse drug exposure was
not performed [68,69]. Notably, a significantly higher number of Figure 7. Effect of PI3-kinase-like kinase (PIKK)- and CHK1/2
inhibitors on the proliferation of 8226-LR5 cells. The MTT assay
was used to monitor the survival after 72h incubation with the various
inhibitors 62.5 Melphalan. CHK1/2 inhibitor AZD7762 (0.1 mM), DNA-PK
inhibitor NU7441 (0.2 mM), ATM inhibitor KU55933 (3 mM), ATR inhibitor
VE821 (1 mM). Whereas a moderate inhibition on proliferation was
observed with either inhibitor alone, strong inhibition was observed in
combination with melphalan for the CHK1/2, DNA-PK and ATR
inhibitors. Each bar represents the mean of at least 5 independent
experiments with standard deviations as indicated. P-values were
calculated using 2-sample t-test with equal variance. .95%, .99% and
.99.9% confidence levels indicated by *, **, and ***, respectively. doi:10.1371/journal.pone.0055493.g007 Figure 6. Level of DNA repair proteins (A) and damage
signaling kinases (B) involved in DSB repair in Melphalan-
sensitive and -resistant cells. (A) Quantitative western blot analysis
revealed that the NHEJ proteins DNA ligase IV, XRCC4 and RPA2 are
significantly and constitutively upregulated in Melphalan-resistant cells. In addition, KU80 and phosphorylated Artemis was consistently,
although not significantly upregulated. Exposure to high dose
Melphalan did not mediate further alteration of NHEJ proteins in the
cell lines. (B) The steady-state levels of the kinase ATR and its
phosphorylated form are increased in Melphalan resistant cells. Conversely, ATM is constitutively downregulated in the same cells. Rather than activation of CHK1, increased steady-state phosphorylation
of pCHK2 was observed in resistant cells. Notably, high dose Melphalan
strongly activates CHK2 in both sensitive and resistant cells but was
consistently higher in resistant cells. DNA Repair Switch in Melphalan Resistance Figure 6. Level of DNA repair proteins (A) a
signaling kinases (B) involved in DSB repair in
sensitive and -resistant cells. (A) Quantitative western
revealed that the NHEJ proteins DNA ligase IV, XRCC4
significantly and constitutively upregulated in Melphalan-
In addition, KU80 and phosphorylated Artemis was
although not significantly upregulated. Exposure to
Melphalan did not mediate further alteration of NHEJ p
cell lines. (B) The steady-state levels of the kinase
phosphorylated form are increased in Melphalan re
Conversely, ATM is constitutively downregulated in th
Rather than activation of CHK1, increased steady-state ph
of pCHK2 was observed in resistant cells Notably high do contribute to NHEJ activation via activation of DNA-PK. A
function of DNA-PK downstream of ATR would also conform to
the lower effect of DNA-PK- compared to ATR inhibition on
Melphalan sensitivity (Fig. 7). Notably ATR-dependent activation
of DNA-PK has previously been observed subsequent to UV-
induced replication stress in HeLa cells and this activation was
mediated by phosphorylations on DNA-PK distinct from those
observed subsequent to ionizing radiation [65]. Moreover, DNA-
PK has been shown to phosphorylate CHK2 [66]. Although we
did not observe Melphalan-induced phosphorylation of DNA-PK
by using a phospho-specific pT2609 antibody (data not shown),
potential activation of DNA-PK and NHEJ via ATR as well as the
individual roles of CHK1/2 in mediating Melphalan resistance
clearly warrants further investigation. Figure 6. Level of DNA repair proteins (A) and damage
signaling kinases (B) involved in DSB repair in Melphalan-
sensitive and -resistant cells. (A) Quantitative western blot analysis
revealed that the NHEJ proteins DNA ligase IV, XRCC4 and RPA2 are
significantly and constitutively upregulated in Melphalan-resistant cells. In addition, KU80 and phosphorylated Artemis was consistently,
although not significantly upregulated. Exposure to high dose
Melphalan did not mediate further alteration of NHEJ proteins in the
cell lines. (B) The steady-state levels of the kinase ATR and its
phosphorylated form are increased in Melphalan resistant cells. Conversely, ATM is constitutively downregulated in the same cells. Rather than activation of CHK1, increased steady-state phosphorylation
of pCHK2 was observed in resistant cells. Notably, high dose Melphalan
strongly activates CHK2 in both sensitive and resistant cells but was
consistently higher in resistant cells. Moreover, BRCA1, which is slightly
induced in resistant cells, becomes markedly reduced in both sensitive
and resistant cells after high dose Melphalan indicating temporary
downregulation of HRR. Also note the the lack of apparent
monoubiquitinylation of FANCD2 (B). DNA Repair Switch in Melphalan Resistance b-actin was used as reference
for quantitative analysis. d i 10 1371/j
l
0055493 006 ATM and ATR Signaling Pathways are Modulated in
Melphalan Resistant Cells 6B, the
steady state-levels of ATM were significantly higher in the
Melphalan-sensitive cells, whereas this was reversed for ATR. Moreover, subsequent to high-dose Melphalan, substantially more
activated ATM (pS1981) was observed in the sensitive cells,
whereas the level of activated ATR (pS428) was highest in the
resistant cells. The increased CHK2-activation in the resistant cell
line was also intriguing, since CHK2 is generally regarded as a
tumor suppressor mediating cell cycle delay/arrest, apoptosis and
DNA repair and is a canonical target of ATM [63]. Likely, an
effect of CHK2 is not mediated via phosphorylation of p53, since
p53 is mutated in RPMI8226 cells [64], as is also substantiated by
the reduced level of p21 in the resistant cells (Fig. 2). Furthermore,
this would have resulted in increased cell-cycle arrest and
apoptosis in the resistant cells, which is not substantiated by the To gain further insight in the relative roles of PI3-kinase-like
kinases (PIKKs) as well as the downstream kinases CHK1/2 in
mediating Melphalan resistance, we treated the resistant cells with
varying concentrations of inhibitors against ATR (VE821), ATM
(KU55933), DNA-PK (NU7441) and CHK1/2 (AZD7762) in the
presence- or absence of Melphalan. Whereas a modestly decreased
proliferation was observed with each of the inhibitors in the
absence of Melphalan, a marked inhibition was observed when
either CHK1/2, DNA-PK or ATR was inhibited in the presence
of Melphalan. Notably, inhibition of ATR caused the strongest
inhibition of proliferation, whereas inhibition of ATM mediated
no significant reduction in proliferation. This corroborates the
marked shift in expression and activation of ATM and ATR in the
resistant cells and strongly supports that ATR plays a significant
role in mediating Melphalan resistance in our experimental
system. Moreover, it is tempting to speculate that ATR may February 2013 | Volume 8 | Issue 2 | e55493 February 2013 | Volume 8 | Issue 2 | e55493 PLOS ONE | www.plosone.org 8 DNA Repair Switch in Melphalan Resistance Concluding Remarks The present study reveals several DNA damage response factors
as novel potential contributors to Melphalan resistance. Our
results are in agreement with recent findings that productive repair
of Cisplatin-ICLs is suppressed by base excision repair interme-
diates in the vicinity of the Cisplatin-ICL [34]. Here we
demonstrate that three DNA glycosylases, UNG2, NEIL1 and
MPG are significantly downregulated in the Melphalan-resistant
cells. This is further corroborated by a markedly reduced UNG
enzyme activity as well as doubling of the genomic 8-oxodG
content in the resistant cells. Moreover, the lowered expression of
the glycosylases was accompanied by an increased tolerance
towards agents inducing their respective DNA base substrates. This is in agreement with previous findings demonstrating that
overexpression of MPG increases sensitivity towards temozolo-
mide [70] as well as MMS [71]. The genomic accumulation of 8-
oxodG was apparently not caused by an increased level of ROS in
the resistant cells and did not change subsequent to high-dose
Melphalan. Thus a more likely explanation is that the observed
accumulation of 8-oxodG is mediated by reduced excision by
DNA glycosylases. Notably, OGG1 is regarded the prime
glycosylase for removal of genomic 8-oxoG, with NEIL1 as a
ubiquitously expressed [72]. Since the OGG1 expression was
barely above the detection level in both cell lines, the markedly
increased 8-oxodG level is most likely mediated by the threefold
reduced level of NEIL1 in the resistant cells. It is currently not
clear whether the marked downregulation of UNG2 mediates a
corresponding increase in overall genomic uracil. Furthermore, it
remains to be elucidated whether preferential deamination of
cytosine occurs in regions flanking Melphalan-ICLs similar to
what is observed at Cisplatin-ICLs. To our knowledge, the three-
dimensional structure of a DNA Melphalan-ICL has not been
resolved. Whether this ICL mediates the same degree of single-
stranded DNA and extrahelical bases flanking the lesion as seen in
the cisplatin-ICL [73], thus remain to be investigated. This could
certainly also impact the rate of oxidative base damage at
Melphalan ICLs. In conclusion, downregulation of initiator BER
glycosylases may provide a selective advantage by lowering the
levels of ALS repair intermediates that would obstruct ICL
processing. Reduced excision of base lesions such as uracil and 8-
oxoG, which both per se are regarded non-cytotoxic, would also
make biological sense from another point of view, by increasing
the overall mutation frequency in the resistant cells. Phosphorylated cH2AX foci are Formed more Rapidly
and Persist for a Shorter Time in Melphalan Resistant
8226 Cells Melphalan-resistant cells have a more rapid kinetics
of cH2AX foci formation than sensitive cells. (A) The number of c-
H2AX foci per cell nucleus (N.13) was manually quantified for each
time point and the experiment was performed in triplicate. Corre-
sponding standard deviation bars are indicated. P,0.01 at all time
points as calculated using 2-sample t-test with equal variance. (B)
Representative confocal images of the cells at different time points
subsequent to Melphalan treatment. Foci formation was monitored
using cH2AX polyclonal antibody (red) and due to the significant
fraction of NFkB protein present in the cytoplasm of these cells, NFkB
p65 mouse monoclonal antibody was used as control for cytoplasmic
staining (green). doi:10.1371/journal.pone.0055493.g008 Phosphorylated cH2AX foci are Formed more Rapidly
and Persist for a Shorter Time in Melphalan Resistant
8226 Cells Moreover, BRCA1, which is slightly
induced in resistant cells, becomes markedly reduced in both sensitive
and resistant cells after high dose Melphalan indicating temporary
downregulation of HRR. Also note the the lack of apparent
monoubiquitinylation of FANCD2 (B). b-actin was used as reference
for quantitative analysis. d i 10 1371/j
l
0055493 006 Figure 7. Effect of PI3-kinase-like kinase (PIKK)- and CHK1/2
inhibitors on the proliferation of 8226-LR5 cells. The MTT assay
was used to monitor the survival after 72h incubation with the various
inhibitors 62.5 Melphalan. CHK1/2 inhibitor AZD7762 (0.1 mM), DNA-PK
inhibitor NU7441 (0.2 mM), ATM inhibitor KU55933 (3 mM), ATR inhibitor
VE821 (1 mM). Whereas a moderate inhibition on proliferation was
observed with either inhibitor alone, strong inhibition was observed in
combination with melphalan for the CHK1/2, DNA-PK and ATR
inhibitors. Each bar represents the mean of at least 5 independent
experiments with standard deviations as indicated. P-values were
calculated using 2-sample t-test with equal variance. .95%, .99% and
.99.9% confidence levels indicated by *, **, and ***, respectively. doi:10.1371/journal.pone.0055493.g007 doi:10.1371/journal.pone.0055493.g006 PLOS ONE | www.plosone.org February 2013 | Volume 8 | Issue 2 | e55493 9 DNA Repair Switch in Melphalan Resistance Figure 8. Melphalan-resistant cells have a more rapid kinetics
of cH2AX foci formation than sensitive cells. (A) The number of c-
H2AX foci per cell nucleus (N.13) was manually quantified for each foci were observed in the non-treated, resistant cells (0 hours). Subsequent to Melphalan treatment, a markedly increased
accumulation of cH2AX foci was observed at early time points
in the resistant- compared to the sensitive cell line (Fig. 8 A,B). In
the Melphalan-resistant cells the number of foci decreased at 24 h,
whereas an increase in the number of foci was observed in the
sensitive cells. These results are entirely in agreement with the
proposed increased repair of Melphalan-induced DNA damage in
the resistant cells. foci were observed in the non-treated, resistant cells (0 hours). Subsequent to Melphalan treatment, a markedly increased
accumulation of cH2AX foci was observed at early time points
in the resistant- compared to the sensitive cell line (Fig. 8 A,B). In
the Melphalan-resistant cells the number of foci decreased at 24 h,
whereas an increase in the number of foci was observed in the
sensitive cells. These results are entirely in agreement with the
proposed increased repair of Melphalan-induced DNA damage in
the resistant cells. Figure 8. Western Blot Analysis y
Proteins were heated in LDS loading buffer (Invitrogen) prior to
separation on pre-cast 4–12% denaturing NuPAGE gels using
MOPS run buffer (Invitrogen) and electroblotting to PVDF
membranes (Immobilon, Millipore) for 1:30 h at 30 V. Western
analysis was performed using the antibodies targeted against the
following (dilutions or concentrations of antibodies are indicated):
Artemis (ab35649, 1:500), ATR (ab10312, 1:15000), Chk1
(ab2845,
1:3000
dilution),
Cdc5L
(ab51320,
1:100),
p21
(ab18209,
0.5 mg/mL),
c-H2AX
(ab2893,
1:1000),
14-3-3b
(ab16859, 1:2000), PRP19 (ab27692, 1 mg/mL), DNA polymerase
b (ab3181, 1:200), PCNA (ab29, 0.5 mg/mL), KU80 (ab3107,
0.5 mg/mL), MSH2 (ab16833-50, 1 mg/mL) DNA ligase 1 (ab615,
1:3000), DNA ligase 3 (ab587, 2 mg/mL) and XPA (ab65963,
1 mg/mL), MPG (ab55461, 1 mg/mL) from Abcam; pATM
(4526S, 1:1000), pATR (2853, 1:1000), pChk2 (2661, 1:1000),
pS317Chk1 (2344, 1:1000), pS345Chk1 (2348, 1:1000), Cdc25B
(9525, 1:1000), Cdc25C (4688, 1:1000), pCdk1 (9111, 1:1000) and
BAX (2772, 1:1000) from Cell Signaling; Cdk2 (sc-748, 1:1000), c-
Myc (sc-42, 1:500), Bcl-2 (sc-65392, 1:1000), MLH1 (sc-56159,
1:1000), XRCC1 (sc-11429, 1:500), ERCC1 (sc-10785, 1:1000),
FANCD2 (sc-20022, 1:1000), DNA-PK (sc-9051, 1:1000), MBD4
(sc-10753, 1:1000), MRE11 (sc-5859, 1:500), Rad50 (sc-20155,
1:1000), Rad51 (sc-8349, 1:1000), Rad52 (sc-8350, 1:1000), RPA2
(sc-53496, 1:1000) from Santa Cruz; ATM (A300-299A, 1:5000),
BRCA1 (A300-000A, 1:5000), FEN1 (A300-256A, 1:10000) from
Bethyl laboratories; p53 (MS-105-p1, 0.5 mg/mL) from Neo
markers; PARP-1 (04–575, 1:1000) from Millipore; poly(ADP-
ribose) from Trevigen (4335, 1:1000) DNA ligase 4 (HPA001334,
1:1000) from Sigma; XRCC4 (GTX109632, 1:1000) from Gene
Tex;
CDC4
(39–5800,
0.5 mg/mL)
from
Zymed;
14-3-3s
(H00002810, 1:1000), TDG (H00006996, 1:1000) and CDK1
(HPA003387, 1:1000) from Abnova; WEE1 (AP8106b, 0.5 mg/
mL) from Abgent. The polyclonal antibodies PAPE1 and PU059
recognizing APE1 and the catalytic domain of UNG, respectively,
were generated in our own lab (0.5 mg/mL dilution). The NEIL-1
and OGG1 polyclonal antibodies were kindly provided by Prof. Magnar Bjøra˚s at the University of Oslo. Anti-b-actin mouse
monoclonal antibody (ab8226, Abcam, 1:10000) was used to
normalize the data. After incubation with HRP-conjugated
secondary antibodies (Dako Denmark, 1:5000), membranes were
incubated with SuperSignal West Femto Maximum Sensitivity
Substrate (Thermo Scientific) and bands were detected on a digital
imaging system IS4000R Kodak (Fisher Scientific). One exception
was parallel detection of PARP-1/PARylated PARP-1, which was
performed on the Odyssey platform (Li-Cor Biosciences – GmbH)
subsequent to incubation with Goat anti-mouse IRDye800LT and
goat
anti-rabbit
IRDye680LT secondary
antibodies
(Li-Cor
Biosciences). Concluding Remarks This would
likely be beneficial under the selective pressure imposed by
Melphalan therapy. Figure 8. Melphalan-resistant cells have a more rapid k
of cH2AX foci formation than sensitive cells. (A) The num
H2AX foci per cell nucleus (N.13) was manually quantified f
time point and the experiment was performed in triplicate
sponding standard deviation bars are indicated. P,0.01 at
points as calculated using 2-sample t-test with equal varia
Representative confocal images of the cells at different time
subsequent to Melphalan treatment. Foci formation was mo
using cH2AX polyclonal antibody (red) and due to the sig
fraction of NFkB protein present in the cytoplasm of these ce
p65 mouse monoclonal antibody was used as control for cyto Figure 8. Melphalan-resistant cells have a more rapid kinetics
of cH2AX foci formation than sensitive cells. (A) The number of c-
H2AX foci per cell nucleus (N.13) was manually quantified for each
time point and the experiment was performed in triplicate. Corre-
sponding standard deviation bars are indicated. P,0.01 at all time
points as calculated using 2-sample t-test with equal variance. (B)
Representative confocal images of the cells at different time points
subsequent to Melphalan treatment. Foci formation was monitored
using cH2AX polyclonal antibody (red) and due to the significant
fraction of NFkB protein present in the cytoplasm of these cells, NFkB
p65 mouse monoclonal antibody was used as control for cytoplasmic
staining (green). d i 0 3
/j
l
00
93 008 A second factor contributing to reduced steady state levels of
ALS would be increased repair of such sites. Although we did not
observe a general upregulation of factors involved in AP-site or
SSB processing, the observation of PARP1 autoribosylation
exclusively in the resistant cells strongly support that such factors
are more efficiently recruited to ALS in the resistant cells. This was
also corroborated by the increased sensitivity to PARP-1 inhibition
of the resistant cell line. Cell Lines Multiple myeloma cell lines 8226 and its Melphalan resistant
derivative 8226-LR5 [15], were kindly provided by Prof. William
S. Dalton at the H. Lee Moffitt Cancer Center & Research
Institute at the University of South Florida. Cells were cultured in
RPMI 1640 (Sigma Aldrich) medium containing 10% (v/v) FBS
(GIBCO) heated for 30 minutes at 56uC, 1% (v/v) of 200 mM L-
glutamine (Lonza), 1% (v/v) of 10 mg/mL gentamicin (GIBCO)
and
1% (v/v) of 250 UG/mL
Amphotericin B
(GIBCO). Melphalan resistant 8226-LR5 cells were maintained under
constant selection through the addition of 1 mM Melphalan
(Sigma-Aldrich, St. Louis, MO) twice per week. Subsequent to
addition of the maintenance Melphalan, 8226-LR5 cells were
allowed to grow at least for 3 days prior to any additional
treatment or cell extract preparation. Concluding Remarks Whether this increased sensitivity is
mediated exclusively via reduced SSB-repair in the cells, however, doi:10.1371/journal.pone.0055493.g008 February 2013 | Volume 8 | Issue 2 | e55493 PLOS ONE | www.plosone.org 10 DNA Repair Switch in Melphalan Resistance containing 10 mM Tris-HCl pH 8.0, 200 mM KCl and diluted in
the same volume (cells+buffer 1) of buffer 2 (10 mM Tris-HCl
pH 8.0, 200 mM KCl, 2 mM EDTA, 40% glycerol, 0.5% NP40,
1 mM DTT, 1% phosphatase inhibitor cocktails 1 and 3 (Sigma),
and 2% Complete EDTA-free (Roche)). After incubation for 1.5 h
at
4uC,
cell
extracts
were
sonicated
for
2 min,
following
centrifugation at 14,000 rpm for 10 min. Supernatants were
collected and used for western blot analysis. Protein concentration
was determined by using the BioRad protein assay (BioRad
laboratories). remains elusive, since PARP-1 is also involved in other processes
including DSB repair by mediating synapsis of strand breaks in
NHEJ [60]. A recent study also demonstrated that PARP-1 bound
with higher affinity to Cisplatin-induced ICLs than to undamaged
DNA [74], supporting that PARP-1 may be involved in the
recruitment of factors contributing to ICL- or ICL-induced DSB
processing. If this is so, co-treatment with PARP-1 inhibitors
should certainly be investigated further as a means to overcome
sensitivity to ICL-inducing agents by inducing symbiotic lethality. Our data suggest the potential involvement of an ATR/DNA-
PK/CHK2 pathway in the stress response triggered by Melphalan
and underscores that the classical ATM-CHK2/ATR-CHK1
pathways are likely much more versatile and dynamic than
previously anticipated. This is also substantiated by studies of
cisplatin-induced cytotoxicity in renal cells, in which ATR, but not
ATM and DNA-PK is specifically activated during Cisplatin
treatment, and that this results in ATR-dependent phosphoryla-
tion of both CHK1 and CHK2 [75]. In the same study CHK1 was
degraded via the proteosomal pathway following phosphorylation,
whereas CHK2 remained activated. The authors also report that
Cisplatin-induced p53 activation and apoptosis are suppressed in
ATR-deficient fibroblasts. The lack of apparent apoptosis-induc-
tion in the RPMI8226 cells may be explained by the mutant p53
protein, and the ATR-CHK2 activation rather mediates increased
efficiency in the repair of DSBs and/or modulated checkpoint
signaling. Moreover our data also support that DNA-PK may be
involved in CHK2 activation, in accordance with previous
observations [66]. DNA-PK may thus have a dual function in
the cells in mediating CHK2 activation (together with ATR) and
in initiating NHEJ. Western Blot Analysis Quantitative analysis was performed using 3 to 5
biological replicates, each consisting of a set of sensitive and
resistant cells both at normal growth and subsequent to treatment
with high dose Melphalan for 6 hours. Band densities of target
proteins were measured for each replicate by using the Kodak In summary, we have identified several novel candidate DNA
damage response proteins that may contribute to development of
Melphalan resistance. We will now include these candidate
proteins in SID-MRM-based targeted quantitative proteomics
analyses in a larger patient cohort to determine their value in
overcoming drug resistance in multiple myeloma, either as targets
for substitution therapies or adjuvants for existing therapies. Concluding Remarks Upregulation of several proteins involved in
the classical NHEJ pathway could then contribute to the observed
increased efficiency in the repair DSBs, potentially involving
increased DSB synapsis by autoribosylated PARP-1 [60]. Viability Assays The 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium
bromide (MTT) assay was performed using Melphalan, methyl
methanesulfonate (MMS), H2O2, 5-Fluorouracil, Mitomycin C
(MMC) and 4-Amino-1,8-naphthalimide (4-AN), ATM inhibitor
(KU55933) from Santa Cruz ATR inhibitor (VE821) from Axon
Medchem, and DNA-PK inhibitor (NU7441) and CHK1/2
inhibitor (AZD7762) from Selleckchem. The PARP1-inhibitor
KU58684 (Kudos/AstraZeneca) was kindly provided by Dr. Franc¸oise Dantzer at the University of Strasbourg. Exposure to
UVB (312 nm) was performed using ultraviolet lamp from Vilber
Lourmat. Briefly, myeloma cells resuspended in RPMI 1640
medium were added to 96-well plates (16104 cells/100 ml/well),
and then exposed to varying concentrations of agents (100 ml/well). Drugs used in the cytotoxicity assays were used at concentrations in
the same range as previously reported IC50 values in RPMI8226 for
5-FU [76] and H2O2 [77], and in lymphocytes for MMS and MMC
[78]. Cells exposed to ultraviolet radiation were resuspended in PBS
instead of RPMI medium prior to addition in 96-well plates (26104
cells/50 ml/well). After irradiation with 100 J/m2 UVB, 150 ml of
RPMI medium were added to each well to allow cell proliferation. Wells containing only medium and medium with a particular agent
were also included as controls. Results were measured at 0, 48, 72
and 96 hours after treatment. After each time point, 100 ml were
removed from each well and 100 ml of 1 mg/ml MTT were added
to each well, followed by incubation at 37uC in culture hood for 4
hours. Subsequently, 130 ml were carefully removed from each well
and acidified isopropanol (100 ml) was added to the plates. Plates
were covered with tin foil and agitated in orbital shaker for 1 hour
prior to absorbance measurement in a Fluostar optima plate reader
(BMG labtech). Measurements were performed at 590 nm with a
reference filter of 620 nm. Standard deviations for each time point
are indicated. Genomic 8-oxodG Analysis y
Genomic DNA was isolated using the DNeasyH Blood and
Tissue kit (Qiagen, Hilden, Germany) according to the manufac-
turer’s instructions. The kit’s elution buffer contains EDTA, which
inhibits nucleases during sample preparation
for
8-oxo-dG
measurement, so the final elution step was performed using
150 ml water instead of the provided elution buffer. The DNA was
then enzymatically hydrolyzed to deoxynucleosides. To this end,
0.5 mg DNA was added to 40 ml of 100 mM NH4HCO3, pH 7.6,
1 mM CaCl3, 10 mM MgCl2, 1 U recombinant DNase I (Roche),
0.2 mU phosphodiesterase I from Crotalus adamanteus venom
(Sigma-Aldrich), 0.1 U alkaline phosphatase from bovine intestinal
mucosa (Sigma-Aldrich), 1.25 pmol [15N5]8-oxodG (Cambridge
Isotope Laboratories) and incubated at 37 uC for 6 h. To
precipitate contaminants that could potentially clog the HPLC
column, five volumes of ice-cold methanol were added to the
samples, mixed by vortexing, and centrifuged at 16,100 x g for
20 min at 4uC. The supernatants were transferred to new tubes
and vacuum centrifuged at room temperature until dry. The
resulting pellets were dissolved in 25 ml 5% methanol. Liquid
chromatography/tandem mass spectrometric analysis was per-
formed using an LC-20AD HPLC system (Shimadzu Corporation,
Kyoto, Japan) coupled to an API 5000 triple-quadrupole mass
spectrometer (Applied Biosystems, Carlsbad, CA, USA). The
HPLC column was a Zorbax SB-C18 reverse phase chromatog-
raphy column (2.16150 mm, i.d., 3.5mm, Agilent Technologies,
Santa Clara, CA, USA), protected with a Zorbax Reliance guard-
column
(4.6 mm612.5 mm,
Agilent
Technologies)
and
the
injection volume was 20 ml. The gradient used consisted of solvent
A (water, 0.1% formic acid) and B (methanol, 0.1% formic acid)
starting at 5% B for 0.5 min, ramping to 90% B over 6 min,
holding at 90% B for 1.5 min and re-equilibrating with 5% B for
5 min at a flow rate of 300 ml/min. Mass spectrometric detection
was performed using positive electrospray ionization in multiple
reaction monitoring mode, monitoring the mass transitions 284.1/
168.2 and 289.2/173.1 for 8-oxodG and [15N5]8-oxodG, respec-
tively. Flow Cytometry For FACS analysis, one million cells were fixed in ice-cold
100% methanol and stored at 4uC until DNA measurement. The
cells were washed with cold phosphate-buffered saline (PBS) and
incubated with 200 ml of DNase-free RNAse A in PBS (100 mg/
ml) for 30 min at 37uC before DNA staining with 200 ml of
propidium iodide (50 mg/ml) at 37uC for 30 min. Cell cycle
analyses were performed by using a BD FACSAria cell sorter (BD
Biosciences). Propidium iodide stained cells were analysed at
488nm excitation (blue laser) and 575 nm emission. Cell cycle
fractions were determined by using the BD FACSDiva software
(BD Biosciences). Comet Assay Cells (1 million cells/mL RPMI medium) were treated with
different doses of Melphalan (50 mM and 100 mM) or vehicle
control (acidified ethanol) for 6 h. The cells were harvested by
centrifugation, embedded in 1% low-melt agar (LMA) and
mounted on microscope slides. The embedded cells were lysed
overnight at 4uC in lysis solution, treated 30 min in alkaline
running buffer (pH.13.3) and subjected to electrophoresis as
described [79]. DNA was visualized by ethidium bromide, and
100 comets were randomly selected and evaluated using Komet
5.0 imaging software (Andor Technology). DNA Repair Switch in Melphalan Resistance 1 mM EDTA and 7.5 mM MgCl2) and incubated at 30uC for 10
minutes with ss- or dsDNA substrate. The reaction was quenched
by addition of 50 ml salmon DNA and 500 ml of 5% TCA
following centrifugation at 14,000 rpm for 10 minutes at 4uC. The
supernatant was analyzed by liquid scintillation counting. Exper-
iments to monitor residual UDG activity in cell extracts were
performed by incubating cell extracts with the UNG inhibitor Ugi
on ice for 15 minutes prior to addition of substrate. Molecular Imaging software version 4.0.1. After subtracting
background intensity values, densities of target proteins of each
replicate were normalized according to -b-actin or -b-tubulin
densities. After normalization, ratios of band densities of target
proteins
in resistant
untreated
cell
extract
versus
sensitive
untreated cell extract were calculated. The mean of 3 to 5
measurements of ratios Rc/Sc was calculated and the correspon-
dent standard deviations were obtained using the following
formula:
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
P
(x{x)2
(n{1)
r
, where x is the sample mean average and n
is the sample size, described as STEDVA function in Excel. Molecular Imaging software version 4.0.1. After subtracting
background intensity values, densities of target proteins of each
replicate were normalized according to -b-actin or -b-tubulin
densities. After normalization, ratios of band densities of target
proteins
in resistant
untreated
cell
extract
versus
sensitive
untreated cell extract were calculated. The mean of 3 to 5
measurements of ratios Rc/Sc was calculated and the correspon-
dent standard deviations were obtained using the following
formula:
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
P
(x{x)2
(n{1)
r
, where x is the sample mean average and n
is the sample size, described as STEDVA function in Excel. Cell Treatment with Melphalan and Preparation of Cell
Extracts Cells were treated with 50 mM Melphalan or vehicle control
(acidified ethanol) for 6 hours in a ratio 1.106/mL, harvested and
washed 3 times with PBS. Cells were resuspended in buffer 1 February 2013 | Volume 8 | Issue 2 | e55493 PLOS ONE | www.plosone.org 11 DNA Repair Switch in Melphalan Resistance References Cancer Res 63:
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resistance in an isogenic multiple myeloma cell line model. Immunofluorescence Uracil
DNA
glycosylase
activity
was
measured
against
[3H]dUMP-containing calf thymus DNA (U:A) as described
(Kavli et al, 2002). Briefly, cell extracts were diluted in assay
buffer (20 mM Tris-HCl pH 7.5, 60 mM NaCl, 1 mM DTT, Cells were treated with 25 mM Melphalan for different time
points and then fixed in freshly prepared 4% paraformaldehyde
for 10 minutes on ice prior to addition of cold methanol (220uC). After incubation for 20 minutes at 220uC, cells were washed three February 2013 | Volume 8 | Issue 2 | e55493 PLOS ONE | www.plosone.org 12 DNA Repair Switch in Melphalan Resistance times with 2% FCS in PBS and incubated with anti-c-H2AX
rabbit polyclonal (ab11174, Abcam, 1:200 dilution) and anti-NFkB
p65 mouse monoclonal antibodies (sc-80086, Santa Cruz, 1:200
dilution) following overnight incubation at 4uC. Anti-NFkB p65
mouse monoclonal antibody was chosen as control for cytoplasmic
staining, since the portion of NFkB present in the cytoplasm is
easily visualized. Cells were then incubated with Alexa Fluor
647 goat anti–rabbit and 532 goat anti-mouse secondary antibod-
ies (Invitrogen, 1:4000 dilution) for 1 h at 37uC. Immunofluores-
cence data were acquired in a laser-scanning microscope (LSM
510 Meta;
Carl
Zeiss)
equipped
with a
Plan
Apochromat
6361.4 NA oil immersion objective and images were analyzed
using LSM 510 software (Carl Zeiss). No image processing except
from contrast and intensity adjustments were performed. hydrofluorescein diacetate (carboxy-H2DCFDA, Molec-
ular Probes) according to the manufacturer’s protocol. Briefly, cells (16106 cells/mL) were washed with PBS and
incubated in PBS buffer containing 10 uM dye for 30 min at
37uC. H2DCFDA-stained cells were returned to growth medium
and treated with 50 uM Melphalan. Flow cytometry analysis was
performed after 6 hours of incubation, at 488 nm excitation and
530 nm emission. Parallel treatment of cells with the oxidizing
agent tert-butyl hydroperoxide (TBHP, Sigma) was included as a
positive control for oxidative stress in the assay. (EPS) Author Contributions Obtained permission for use of cell line: ET. Conceived and designed the
experiments: MMLS KAZ PAA GS. Performed the experiments: MMLS
KAZ PAA AHB AD AS NBL. Analyzed the data: MMLS KAZ PAA AD
AS AHB ET NBL GS. Contributed reagents/materials/analysis tools: ET
GS. Wrote the paper: MMLS GS. Figure S1
Intracellular level of ROS was detected by
flow cytometry using 5-(and-6)-carboxy-29,79-dichlorodi- Figure S1
Intracellular level of ROS was detected by
flow cytometry using 5-(and-6)-carboxy-29,79-dichlorodi- Figure S1
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The First New Zealanders? An Alternative Interpretation of Stable Isotope Data from Wairau Bar, New Zealand
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RESEARCH ARTICLE Editor: Sharon N DeWitte, University of South
Carolina, UNITED STATES Editor: Sharon N DeWitte, University of South
Carolina, UNITED STATES
Received: April 8, 2015
Accepted: June 28, 2015
Published: October 28, 2015 The First New Zealanders? An Alternative
Interpretation of Stable Isotope Data from
Wairau Bar, New Zealand Andrew A. Brown1*, Tim Thomas2
1 Institute of Archaeology, University College London, London, United Kingdom, 2 Department of
Anthropology and Archaeology, University of Otago, Dunedin, New Zealand Andrew A. Brown1*, Tim Thomas2 1 Institute of Archaeology, University College London, London, United Kingdom, 2 Department of
Anthropology and Archaeology, University of Otago, Dunedin, New Zealand * andrew.brown.12@ucl.ac.uk * andrew.brown.12@ucl.ac.uk OPEN ACCESS Citation: Brown AA, Thomas T (2015) The First New
Zealanders? An Alternative Interpretation of Stable
Isotope Data from Wairau Bar, New Zealand. PLoS
ONE 10(10): e0135214. doi:10.1371/journal. pone.0135214 Editor: Sharon N DeWitte, University of South
Carolina, UNITED STATES
Received: April 8, 2015
Accepted: June 28, 2015
Published: October 28, 2015 Abstract PLOS ONE Volume 8 includes an article “The First New Zealanders: Patterns of Diet and
Mobility Revealed through Isotope Analysis”. The paper proposes that burial groups within
the settlement phase site of Wairau Bar differ in terms of dietary stable isotopes and
87Sr/86Sr. The authors argue this difference is probably due to one group being a founding
population while the other burials are later. Here we review the work of Kinaston et al. and
present an alternative analysis and interpretation of the isotopic data. Treating the isotope
data independently from cultural and biological factors we find that sex best explains dietary
variation. Our reassessment of 87Sr/86Sr confirms the authors original finding of high mobil-
ity of early New Zealanders but suggests a larger range of individuals should be considered
‘non-local’ on current evidence. a1111 Introduction The manner of Polynesian adaptation to, and exploitation of, the New Zealand environment in
the decades following colonisation is a central focus for archaeological research in the region. Some of the most important and far-reaching questions archaeologists have asked are also the
most basic: what were people eating and how mobile were early groups? The answers to these
questions have important implications for a broader understanding of settlement, seasonality,
socio-political complexity and culture change in New Zealand. Diet and mobility reconstruc-
tions therefore remain an important component of New Zealand archaeology [1, 2, 3]. Here,
we consider a reconstruction of human diet and mobility based on stable isotopes presented in
the recent paper by Kinaston et al. [4] entitled “The First New Zealanders: Patterns of Diet and
Mobility Revealed through Isotope Analysis”. The study is based on koiwi tangata (human
remains) from Wairau Bar, a large settlement occupied early in New Zealand prehistory, ca. AD 1300 [5, 6]. Wairau Bar contains a rich array of early material culture unmatched in New
Zealand archaeology. The abundance and range of artefacts has led to the site being considered
the type-site of the East Polynesian Archaic phase in New Zealand [5, 7]. Throughout New Published: October 28, 2015 Published: October 28, 2015 Copyright: © 2015 Brown, Thomas. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited. Data Availability Statement: All relevant data are
within the paper and its Supporting Information files. Funding: The authors have no support or funding to
report. Competing Interests: The authors have declared
that no competing interests exist. Competing Interests: The authors have declared
that no competing interests exist. 1 / 16 PLOS ONE | DOI:10.1371/journal.pone.0135214
October 28, 2015 The First New Zealanders? Zealand and East Polynesia the ‘Archaic’ is associated with colonising populations and, as
such, represents the culture of the first generation of settlers in the region. Zealand and East Polynesia the ‘Archaic’ is associated with colonising populations and, as
such, represents the culture of the first generation of settlers in the region. As the first large-scale isotopic analysis of human remains in New Zealand, and one of the
few in East Polynesia, the analysis by Kinaston et al. Introduction represents a significant advance in refining
our understanding of diet and mobility in the region. The authors model diet using carbon
(δ13C) and nitrogen (δ13N) stable isotope ratios in human bone collagen and analyse mobility
through strontium isotopes (87Sr/86Sr) in tooth enamel. Traditional reconstructions of diet
using faunal remains and mobility using proxies such as lithic exchange present aggregate or
population level behaviours. However, the analysis of stable isotopes affords the opportunity to
assess diet and mobility at an individual level, and thus identify sub-groups or outliers in a pop-
ulation. Kinaston et al. ([4]: 8–9) argue that isotopic analysis of the skeletons from Wairau Bar
reveals distinctions between sub-groups within the cemetery population and that, tantalisingly,
one group (Group 1) may represent an immigrant colonising population at the site and, by
extension, New Zealand. While we strongly support the attempt to reveal patterns of diet and
mobility through isotopic analysis, we believe the authors interpretations are constrained by
the focus on a single explanatory variable and by the use of a priori divisions in the analysed
sample. Ultimately, these divisions introduce bias, which leads to conclusions that we feel are
not supported by the available data. Here we review the work of Kinaston et al. [4] and offer an
alternative interpretation of the isotopic patterns at Wairau Bar. Critical Review of Isotopic Analysis from Wairau Bar Central to the analysis of Kinaston et al. [4] is the idea that distinct groups can be identified
within the wider Wairau Bar cemetery population prior to isotopic analysis. Kinaston et al. argue that certain cultural markers differentiate Burials 1–7 from the rest of the cemetery popu-
lation ([4]: 2). Their goal is to assess whether the supposed cultural differences are apparent in
the isotopic signatures or if the cemetery represents a homogenous isotopic group. While we
recognise the obvious utility of defining sub-groups in such an analysis, we have identified two
concerns with their construction and application in this case. Below we consider each of these
in turn. PLOS ONE | DOI:10.1371/journal.pone.0135214
October 28, 2015 Group construction The basis of sub-dividing the cemetery population is ostensibly a distinction between Burials
1–7 (Group 1) and the remaining Burials 8–44 (Group 2/3) due to differences in burial posi-
tion, burial wealth and spatial location. However, in practice both burial wealth and burial
position are used only to emphasise the distinction between groups rather than as exclusive cri-
teria for membership. Reference to ‘burial position’ in S1 Table makes this clear. Here it can be
seen that whilst the majority of Group 1 individuals were interred in the prone position, two
individuals were not, and at least six individuals in Group 2/3 were also interred in the prone
position. Likewise, while the authors note that the average amount of grave goods is much
higher in Group 1, some burials with many grave goods are included in Group 2/3. Moreover,
it is not certain that the burial position and wealth data are reliable indicators of difference
since the Group 2/3 burials are known to have been much more disturbed and fragmented at
recovery than the relatively intact Group 1 [8]. Consequently some of the perceived variation
may be due to taphonomic bias. Effectively this means that Kinaston et al. [4] define groups primarily by their spatial loca-
tion, with the other attributes being used as suggestive secondary evidence indicating that the
spatial differences might be culturally meaningful. PLOS ONE | DOI:10.1371/journal.pone.0135214
October 28, 2015 2 / 16 The First New Zealanders? The use of a single defining criterion for a group in concert with supporting information is,
arguably, valid. The spatial clustering of burials in mortuary contexts has been demonstrated to
have social correlates such as kinship groups [9, 10] but the veracity of these must be statisti-
cally robust, and, moreover, independent of the subsequent explanatory framework and vari-
ables tested. In this instance we believe that the spatial criteria for developing analytical groups
adopted by Kinaston et al. is inconsistent. Duff [11] argues for the existence of three burial
groups at the site: Burials 1–7 (Group 1), Burials 8–11 (Group 2) and a ‘southern burial ground’
comprising Burials 12–44 (Group 3), which extends over roughly one-hundred metres (fifty
metres excluding burial 14) along the edge of the lagoon and contains discrete burial clusters of
comparable size to Groups 1 and 2 [12] (Fig 1). Group construction These designations are adopted by the authors;
however, for their purposes, they combine Burials 8–11 and 12–44 into a single group labelled
Group 2/3, despite the Group 2 burials being spatially closer to Group 1. The effect of this decision is that Group 1 is defined using different spatial criteria from Group
2/3. Group 1 consists of a small, coherent group of well-preserved individuals aligned with each
other in 9 x 6 m unit, while Group 2/3 (as evidenced by its name) is a large composite or ‘leftovers’
group from multiple units spread over several hundred metres of site. This is important to take
into consideration when interpreting the results of statistical analysis comparing these groups. Statistical analysis of dietary stable isotopes Kinaston et al. carry out a series of statistical tests to ascertain if Group 1 and Group 2/3 are
dietarily distinct. In the first instance their analysis involves a comparison of the Group 1 and
Group 2/3 mean δ13C and δ15N values. The authors report no statistically significant difference
in δ13C values; however, the Group 1 average δ15N value, representing the plant/protein por-
tion of diet was significantly lower (Student’s t-test p = 0.025) than Group 2/3. This result
comes with the caveat that a significant result was not found after adjusting for sex (p = 0.150);
therefore, caution must be used when interpreting the initial significant result. This is clearly a
reflection of the influence a predominance of males in Group 1 (n = 5 of six individuals) has on
the unadjusted value. At this point then, no reliable significant difference has been found
between these groups to suggest that they are distinct populations. In the second instance, a significant difference in the variance of δ13C values (but not δ15N
values) between groups (Levene’s test p = 0.014) is invoked as more evidence for dietary differ-
ence or change [4]. We argue that caution should also be exercised when interpreting the vari-
ance data, particularly given the lack of difference in mean δ13C values. Drawing upon our
earlier discussion of group construction, it is clear that Group 1 and Group 2/3 are not equiva-
lent, with the latter consisting of all burials across the site not assigned to Group 1. Under such
conditions we should expect Group 2/3 to be more variable than Group 1 as an artefact of clas-
sification. That a discrete cluster of burials exhibits less dietary variability than the site as a
whole is unsurprising and currently the only reliable inference we can make is that the Group 1
burials exhibit a subset of the range of values exhibited by the population of the site as a whole. Kinaston et al.’s final statistical analysis of dietary isotopes combined the δ15N and δ13C val-
ues with the 87Sr/86Sr ratios in a principal component analysis. They note: “Two principal components were sufficient to explain 58% and 35% respectively (total 92%)
of the variance in the data. Component one reflected high carbon and nitrogen values while
component two reflected high strontium values with a moderate negative carbon loa-
ding. . PLOS ONE | DOI:10.1371/journal.pone.0135214
October 28, 2015 PLOS ONE | DOI:10.1371/journal.pone.0135214
October 28, 2015 Statistical analysis of dietary stable isotopes .Individuals from Group 1 are clearly separable from those in Group 2/3 (even using
only the second component representing mainly strontium values) and one individual from
Group 2 is at the edge of this cluster” ([4]: 8). 3 / 16 PLOS ONE | DOI:10.1371/journal.pone.0135214
October 28, 2015 The First New Zealanders? Fig 1. Location of the Wairau Bar site and the burial groups. Map and burial location information adapte
from Brooks et al. 2011). doi:10.1371/journal.pone.0135214.g001 Fig 1. Location of the Wairau Bar site and the burial groups. Map and burial location information adapte Fig 1. Location of the Wairau Bar site and the burial groups. Map and burial location information adapted
from Brooks et al. 2011). doi:10.1371/journal.pone.0135214.g001 doi:10.1371/journal.pone.0135214.g001 PLOS ONE | DOI:10.1371/journal.pone.0135214
October 28, 2015 4 / 16 The First New Zealanders? While Group 1 does appear distinct in the PCA plot, a careful examination of the compo-
nents shows a lack of support for distinction of groups based on diet alone. Component 1,
reflecting high carbon and nitrogen (i.e. dietary) values, does not separate Group 1. Instead, the
Group 1 individuals fit well within the range of values present in Group 2/3. Kinaston et al. ([4]: 9) remark that the groups are clearly separable “even using only the second component
representing mainly strontium values”. In fact the second component is probably the sole
driver of separation between the groups, even though it accounts for less variance. The separa-
tion evident in the PCA simply reflects a difference in 87Sr/86Sr values (i.e. a proxy for place of
childhood residence) and does not lend further support to the presence of dietary distinctions
between groups. Results: Alternative Statistical Analysis of Dietary Stable Isotopes We suggest that greater statistical certainty can be achieved by maintaining the independence
of new data sets from existing classifications during initial analysis. Cultural or biological fac-
tors, such as burial location or sex, need not be considered predictors of isotopic variation. Instead, stable isotope data may exhibit independent patterns. Considering this, we begin our
analysis by searching for patterns based solely on the δ15N and δ13C values. We carry out a
cluster analysis, which we then cross-check against cultural and biological variables to deter-
mine the most likely factor(s) behind any patterns. Hierarchical cluster analysis The authors consider two possible explana-
tions: that this trend suggests sexual division of labour with respect to the procurement of par-
ticular food types, consistent with similar practices observed in modern day Pacific Islands
communities, or that food consumption was moderated along sexual lines with males enjoying
greater access to higher prestige foods due to higher social status ([14]: 14). Teouma is an inter-
esting parallel to Wairau Bar since both sites were occupied during an early phase of The most important observation from this cluster analysis is that the culturally defined
groups used by Kinaston et al. [4] are not replicated when only the dietary isotopes are consid-
ered. Rather, all of Group 1 (Burials 1–7) is mixed with burials from Group 2/3 in the second,
large cluster. Performing a t-test revealed no significant differences (δ13C p = 0.31; δ15N
p = 0.10) between Group 1 and Group 2/3 within Cluster Two even without correcting for sex. We consider it important that most (n = 4 of 6) of Cluster One are female (Fig 3). This
group falls outside the local rat and dog ranges reported by Kinaston et al. ([4]: their Fig 4) and
is much closer to marine mammal values, perhaps hinting at an increased marine dietary focus
in this group. A t-test of the burial population with confirmed sex estimates found significant
difference in the mean δ15N value of the sexes (p = 0.02) but not δ13C (p = 0.114). A similar
result is noted by Kinaston et al. [14] for the Teouma site in Vanuatu. At Teouma δ15N and not
δ13C were found to be significantly higher in males. The authors consider two possible explana-
tions: that this trend suggests sexual division of labour with respect to the procurement of par-
ticular food types, consistent with similar practices observed in modern day Pacific Islands
communities, or that food consumption was moderated along sexual lines with males enjoying
greater access to higher prestige foods due to higher social status ([14]: 14). Teouma is an inter-
esting parallel to Wairau Bar since both sites were occupied during an early phase of Fig 3. Scatter plot of the Clusters determined by Ward's Linkage hierarchical clustering. doi:10.1371/journal.pone.0135214.g003
PLOS ONE | DOI:10.1371/journal.pone.0135214
October 28, 2015
6 / 16 Fig 3. Scatter plot of the Clusters determined by Ward's Linkage hierarchical clustering. Fig 3. Hierarchical cluster analysis Using the δ15N and δ13C values we carried out a hierarchical agglomerative cluster analysis
using Ward’s linkage [13]. The resulting dendogram (Fig 2) presents two strong clusters. The Fig 2. Ward's cluster analysis of dietary stable isotope (δ15N and δ13C) values from Wairau Bar with associated age, sex and wealth information. doi:10.1371/journal.pone.0135214.g002
PLOS ONE | DOI:10.1371/journal.pone.0135214
October 28, 2015
5 / Fig 2. Ward's cluster analysis of dietary stable isotope (δ15N and δ13C) values from Wairau Bar with associated age, sex and wealth information. doi:10.1371/journal.pone.0135214.g002 Fig 2. Ward's cluster analysis of dietary stable isotope (δ15N and δ13C) values from Wairau Bar with associated age, sex and wealth information. doi:10 1371/journal pone 0135214 g002 doi:10.1371/journal.pone.0135214.g002 5 / 16 PLOS ONE | DOI:10.1371/journal.pone.0135214
October 28, 2015 The First New Zealanders? first, (Fig 2; Cluster One) is a clear group of six burials with high δ15N and δ13C values. The sec-
ond cluster contains the remaining 21 burials and has two weaker, second-order sub-groups. While we will consider these sub-groups separately in the following section, overlap of the
δ15N and/or δ13C values is present (Fig 3). Interpretation of these sub-groups as distinct is,
therefore, made with caution. ,
The most important observation from this cluster analysis is that the culturally defined
groups used by Kinaston et al. [4] are not replicated when only the dietary isotopes are consid-
ered. Rather, all of Group 1 (Burials 1–7) is mixed with burials from Group 2/3 in the second,
large cluster. Performing a t-test revealed no significant differences (δ13C p = 0.31; δ15N
p = 0.10) between Group 1 and Group 2/3 within Cluster Two even without correcting for sex. We consider it important that most (n = 4 of 6) of Cluster One are female (Fig 3). This
group falls outside the local rat and dog ranges reported by Kinaston et al. ([4]: their Fig 4) and
is much closer to marine mammal values, perhaps hinting at an increased marine dietary focus
in this group. A t-test of the burial population with confirmed sex estimates found significant
difference in the mean δ15N value of the sexes (p = 0.02) but not δ13C (p = 0.114). A similar
result is noted by Kinaston et al. [14] for the Teouma site in Vanuatu. At Teouma δ15N and not
δ13C were found to be significantly higher in males. Hierarchical cluster analysis Scatter plot of the Clusters determined by Ward's Linkage hierarchical clustering. doi:10.1371/journal.pone.0135214.g003 PLOS ONE | DOI:10.1371/journal.pone.0135214
October 28, 2015 PLOS ONE | DOI:10.1371/journal.pone.0135214
October 28, 2015 6 / 16 The First New Zealanders? Fig 4. Human bone collagen δ15N and δ13C values from Wairau Bar coded with the sex of the individual. doi:10 1371/journal pone 0135214 g004 Fig 4. Human bone collagen δ15N and δ13C values from Wairau Bar coded with the sex of the individual. doi:10.1371/journal.pone.0135214.g004 doi:10.1371/journal.pone.0135214.g004 colonisation. The observation of sex-based differences in food consumption due to either divi-
sion of labour or differential status provides interesting insight into the social structure of early
period sites. Despite the statistical support for sex-based differences it is also true that there is a large
degree of overlap between females and males (Fig 4). Just over half of the females fall comfort-
ably within the range occupied by most males. Indeed a t-test confirms there are no significant
differences between males and females in Cluster Two (δ13C p = 0.6; δ15N p = 0.073). This sug-
gests that the Cluster One individuals present a distinct group of mostly female outliers from
the population as a whole. Visual assessment of Figs 4–7 suggests that Cluster One may also be
characterised by older individuals with fewer grave goods. However, statistical analysis revealed
no significance in these patterns. Model selection methods Our analysis (above) of the δ15N and δ13C data suggests that other factors such as sex may
have more explanatory power than the burial groupings employed by Kinaston et al. [4]. In
order to assess competing explanations for the patterns exhibited in the isotope data we car-
ried out a model selection of multivariate regression models using the Akaike Information
Criterion (AIC) and Bayesian Information Criterion (BIC). Beginning with Kinaston Groups,
Age, Sex and Grave Goods (Models A and D) and Duff Groups, Age, Sex and Grave Goods
(Models B and C), a stepwise selection of the best explanatory combination of covariates was
carried out (see [15, 16]). During this process AIC punishes model complexity less than BIC 7 / 16 PLOS ONE | DOI:10.1371/journal.pone.0135214
October 28, 2015 The First New Zealanders? Fig 5. Human bone collagen δ15N and δ13C values from Wairau Bar coded with information about individual age at death. doi:10.1371/journal.pone.0135214.g005 Fig 5. Human bone collagen δ15N and δ13C values from Wairau Bar coded with information about individual age at death. Fig 5. Human bone collagen δ15N and δ13C values from Wairau Bar coded with information about individual age at death. doi:10.1371/journal.pone.0135214.g005 doi:10.1371/journal.pone.0135214.g005 doi:10.1371/journal.pone.0135214.g005 [16] and is therefore less likely to produce an overly simplistic model. Because of this relative
conservatism we report AIC results in this paper, but also include the results of BIC model
selection (S2 Table). The BIC analysis produced the same patterns as the AIC. Burial position
was removed from our analysis because of possible bias in the data caused by taphonomy (see
above). Table 1 shows the results of the model selection using AIC, where the best quality model is
represented by low AIC value and high weight. For δ15N, the best model involves the single
covariate sex (Model A [Kinaston Groups], Model B [Duff Groups], Model C [Groups
absent]). This finding agrees with the significant difference in mean δ15N of males and females
reported above, and the fact that Kinaston et al. [4] found no significant difference between
their groups after controlling for sex. For δ13C, analysis of Models D and E finds that a combi-
nation of grave goods and burial group (either Duff or Kinaston) are the best models. Given
that we found no significant difference in mean δ13C values between the sexes, and Kinaston
et al. PLOS ONE | DOI:10.1371/journal.pone.0135214
October 28, 2015 Model selection methods [4] report no significant difference in mean δ13C values between their groups, this finding
probably reflects difference in the variance data. But, as we argue above, the fact that Kinaston
et al’s Group 2/3 has more variation in δ13C values than Group 1 is likely to be a product of
classification. Consequently we must question the validity of this result. Removing burial
group and testing only the objectively derived variables (Model E), we see that the importance
of grave goods is reduced and variation in δ13C is again best explained by the single covariate
sex. PLOS ONE | DOI:10.1371/journal.pone.0135214
October 28, 2015 8 / 16 The First New Zealanders? Fig 6. Human bone collagen δ15N and δ13C values from Wairau Bar coded with grave goods information. Fig 6. Human bone collagen δ15N and δ13C values from Wairau Bar coded with grave goods information. Fig 6. Human bone collagen δ15N and δ13C values from Wairau Bar coded with grave goods information. doi:10.1371/journal.pone.0135214.g006 doi:10.1371/journal.pone.0135214.g006 PLOS ONE | DOI:10.1371/journal.pone.0135214
October 28, 2015 Strontium Isotopes and Population Mobility Kinaston et al. argue for clear separation of Group 1 from Group 2/3 according to 87Sr/86Sr
ratios. In particular, Kinaston et al. ([4]:7) state that: “. . .given that the strontium results from Group 2/3 are closer to those of the local dog sample
than to Group 1, a reasonable interpretation of this pattern is that the Group 1 individuals
were immigrants to the site while some of the Group 2/3 individuals had resided in or near
the Wairau Bar region during childhood”. While it is true that Group 2/3 have, on average, higher values than Group 1, this interpreta-
tion is not consistent with the authors’ advice that: “variations in human strontium isotope values greater than two standard deviations from the
mean of the local range are then considered to be most likely ‘non-local’ to the site” ([4]: 4). Based on the authors figure (reproduced here in Fig 8a), only four individuals fall within
two standard deviations of the mean strontium values from dog samples, which are taken as
the proxy for a local signature. Therefore, while it is correct to say that some of the Group 2/3
individuals may be local, it would be more accurate according to their standard, to argue that
we can be 95% sure that 20 individuals are non-local. Re-organising the data into rank order is revealing (Fig 8b). The resulting distribution
shows that the 87Sr/86Sr ratios may actually form a far more continuous group than Kinaston PLOS ONE | DOI:10.1371/journal.pone.0135214
October 28, 2015 9 / 16 The First New Zealanders? Fig 7. Human bone collagen δ15N and δ13C values from Wairau Bar coded with burial position information. doi:10 1371/journal pone 0135214 g007 Fig 7. Human bone collagen δ15N and δ13C values from Wairau Bar coded with burial position information. Fig 7. Human bone collagen δ15N and δ13C values from Wairau Bar coded with burial position information. doi:10.1371/journal.pone.0135214.g007 Fig 7. Human bone collagen δ15N and δ13C values from Wairau Bar coded with burial position informatio doi:10.1371/journal.pone.0135214.g007 doi:10.1371/journal.pone.0135214.g007 et al. suggest. Moreover it is evident that some Group 2/3 burials (e.g. burials 14 and 33) have
very low values similar to the allegedly immigrant Group 1 (a fact discussed by the authors). Overall there is not an extreme difference between individuals assigned to Group 1 and mem-
bers of Group 2/3 despite differences in their average values, and there are no clear groups of
outliers. Strontium Isotopes and Population Mobility This contrasts with results from Teouma, another Pacific Island colonisation phase
site where the presence of immigrants has been argued. Fig 8c presents the 87Sr/86Sr results pre-
sented by Bentley et al. [17]. Bentley et al. argue that the four individuals on the right of the
graph are outliers from the main population, a likely indication of them being immigrants to
the site. The stepped appearance of the graph presents a strong contrast to the continuous
slope observed at Wairau Bar. We accept that geological variation coupled with the current
lack of an adequate strontium baseline in New Zealand makes the detection of differences as
distinct as those at Teouma unlikely; nevertheless, the comparison is a cautionary note for the
assignment of Burials 1–7 as a discrete group of immigrants. Response Variable
Model Steps
Covariates
AIC
Weights
δ15N (‰)
Model A
1
Kinaston Group, Age, Sex, Grave Goods
26.35
0.046
2
Kinaston Group, Age, Sex
24.5
0.115
3
Age, Sex
22.6
0.297
4
Sex
21.4
0.541
δ15N (‰)
Model B
1
Duff Group, Age, Sex, Grave Goods
26.49
0.043
2
Duff Group, Age, Sex
24.56
0.113
3
Age, Sex
22.6
0.301
4
Sex
21.4
0.541
Model C
1
Age, Sex, Grave Goods
24.58
0.117
2
Age, Sex
22.6
0.315
3
Sex
21.43
0.566
δ13C (‰)
Model D
1
Kinaston Group, Age, Sex, Grave Goods
27.4
0.092
2
Kinaston Group, Sex, Grave Goods
25.43
0.247
3
Kinaston Group, Grave Goods
23.46
0.661
δ13C (‰)
Model E
1
Duff Group, Age, Sex, Grave Goods
28.2
0.133
2
Duff Group, Sex, Grave Goods
26.2
0.362
3
Duff Group, Grave Goods
25.54
0.504
δ13C (‰)
Model F
1
Age, Sex, Grave Goods
28.63
0.112
2
Age, Sex
26.7
0.293
3
Sex
25.28
0.596
doi:10.1371/journal.pone.0135214.t001
The First New Zealanders? Table 1. Model selection using Akaike Information Criterion (AIC). Response Variable
Model Steps
Covariates
AIC
Weights
δ15N (‰)
Model A
1
Kinaston Group, Age, Sex, Grave Goods
26.35
0.046
2
Kinaston Group, Age, Sex
24.5
0.115
3
Age, Sex
22.6
0.297
4
Sex
21.4
0.541
δ15N (‰)
Model B
1
Duff Group, Age, Sex, Grave Goods
26.49
0.043
2
Duff Group, Age, Sex
24.56
0.113
3
Age, Sex
22.6
0.301
4
Sex
21.4
0.541
Model C
1
Age, Sex, Grave Goods
24.58
0.117
2
Age, Sex
22.6
0.315
3
Sex
21.43
0.566
δ13C (‰)
Model D
1
Kinaston Group, Age, Sex, Grave Goods
27.4
0.092
2
Kinaston Group, Sex, Grave Goods
25.43
0.247
3
Kinaston Group, Grave Goods
23.46
0.661
δ13C (‰)
Model E
1
Duff Group, Age, Sex, Grave Goods
28.2
0.133
2
Duff Group, Sex, Grave Goods
26.2
0.362
3
Duff Group, Grave Goods
25.54
0.504
δ13C (‰)
Model F
1
Age, Sex, Grave Goods
28.63
0.112
2
Age, Sex
26.7
0.293
3
Sex
25.28
0.596
/ Table 1. Model selection using Akaike Information Criterion (AIC). Thus, it offers a unique opportunity to apply biological anthropology methods to the study of
New Zealand’s earliest human population. Until recently the analysis of skeletal remains from
the site has focussed on the physical characteristics, lifestyles and pathologies of individuals [8,
19]. Discussion and Implications As the so-called ‘type site’ of the New Zealand Archaic, Wairau Bar has an important explana-
tory role in New Zealand archaeology. As well as providing evidence for a range of activities
that indicate early economic behaviour and settlement strategy [18], the site provides the larg-
est collection of well-provenanced, early koiwi tangata (human remains) in New Zealand. 10 / 16 PLOS ONE | DOI:10.1371/journal.pone.0135214
October 28, 2015 PLOS ONE | DOI:10.1371/journal.pone.0135214
October 28, 2015 The First New Zealanders? Thus, it offers a unique opportunity to apply biological anthropology methods to the study of
New Zealand’s earliest human population. Until recently the analysis of skeletal remains from
the site has focussed on the physical characteristics, lifestyles and pathologies of individuals [8,
19]. Variation within the cemetery population has also been considered, with sex, relative
wealth and/or status, and temporal change being invoked to account for differences [10, 20,
21]. An early component of these analyses was the distinction drawn between Burial Group 1
and the remaining burials based primarily on burial location within the site and grave goods. Kinaston et al. [4] use this distinction as a starting point for their analysis of stable isotopes,
searching for differences between Group 1 and the rest of the cemetery. Doing so, we argue,
gives too much weight to the spatial location of burials, and less to factors such as the age or
sex of the individuals. As an alternative, we have treated the stable isotope data independently
and have sought to test a broad range of models to come up with the best explanation of
variation. The alternative analysis conducted in this paper suggests two important things about diet:
(1) that sex is the best explanatory variable for dietary variation and (2) that there is little evi-
dence of dietary distinction based on burial location at Wairau Bar. Our analysis showed the
most dietarily-distinct individuals to be those with high δ15N and δ13C values consistent with a
diet containing a higher marine component. These individuals formed a cluster of predomi-
nantly female burials with relatively few grave goods. Table 1. Model selection using Akaike Information Criterion (AIC). Variation within the cemetery population has also been considered, with sex, relative
wealth and/or status, and temporal change being invoked to account for differences [10, 20,
21]. An early component of these analyses was the distinction drawn between Burial Group 1
and the remaining burials based primarily on burial location within the site and grave goods. Kinaston et al. [4] use this distinction as a starting point for their analysis of stable isotopes,
searching for differences between Group 1 and the rest of the cemetery. Doing so, we argue,
gives too much weight to the spatial location of burials, and less to factors such as the age or
sex of the individuals. As an alternative, we have treated the stable isotope data independently
and have sought to test a broad range of models to come up with the best explanation of
variation. The alternative analysis conducted in this paper suggests two important things about diet:
(1) that sex is the best explanatory variable for dietary variation and (2) that there is little evi-
dence of dietary distinction based on burial location at Wairau Bar. Our analysis showed the
most dietarily-distinct individuals to be those with high δ15N and δ13C values consistent with a
diet containing a higher marine component. These individuals formed a cluster of predomi-
nantly female burials with relatively few grave goods. PLOS ONE | DOI:10.1371/journal.pone.0135214
October 28, 2015 11 / 16 The First New Zealanders? Fig 8. Strontium isotope data from Pacific Island samples. A—Reproduction of Kinaston et al. (2013, Fig
5) showing the strontium values of individuals within their Group 1 (black) and Group 2/3 (grey). B. Strontium
values from Wairau Bar individuals organised into rank order, grey bars represent individuals within two
standard deviations of the mean dog value. C—Strontium isotope data from Teouma, Vanuatu (Bentley et al. 2007). Grey bars represent individuals considered to be outliers or immigrants to the site. doi:10.1371/journal.pone.0135214.g008 Fig 8. Strontium isotope data from Pacific Island samples. A—Reproduction of Kinaston et al. (2013, Fig
5) showing the strontium values of individuals within their Group 1 (black) and Group 2/3 (grey). B. Strontium
values from Wairau Bar individuals organised into rank order, grey bars represent individuals within two
standard deviations of the mean dog value. C—Strontium isotope data from Teouma, Vanuatu (Bentley et al. 2007). Grey bars represent individuals considered to be outliers or immigrants to the site. doi:10 1371/journal pone 0135214 g008 doi:10.1371/journal.pone.0135214.g008 PLOS ONE | DOI:10.1371/journal.pone.0135214
October 28, 2015 12 / 16 The First New Zealanders? In the first instance this pattern could be indicative of gendered differences in food procure-
ment activities. Drawing upon a subset of data from the Standard Cross Cultural Sample
(SCCS), Marlowe [22] showed that, within foraging societies, females target reliable foods
while males tend towards energy rich, less reliable food sources. Sexual division of labour dur-
ing food procurement is a consistent pattern throughout Polynesia, although its implementa-
tion varies [23]. In late period Maori society Firth [24] notes gendered subsistence practices
that are generally consistent with Marlowe’s [22] findings: men tended to carry out expeditions
for less reliable food sources, while females targeted more abundant, reliable foods closer to the
domestic base. For example, and in common with most Pacific societies, inshore fishing and
shellfish gathering were preferentially performed by women and accounted for a larger propor-
tion of the diet than the rare, risky and less productive offshore fishing activities of men. Many models of early New Zealand settlement suggest groups actively exploited a wide ter-
ritory via a network of coastal village bases and supplementary restricted function sites [25,
26]. If food procurement amongst early Maori had gender divisions comparable to those of the
later period, it is possible that females and males accumulated different proportions of marine
and terrestrial foods in their diet. In the case of Wairau Bar, the vast estuarine setting would
have provided a range of reliable subsistence resources to exploit. Conversely, hunting activities
may have rapidly depleted local large-bodied game [27] fuelling range expansion, possibly seen
in the establishment of specialist camps within a few days journey of the site [25]. This model
may account for sex-based variation in diet simply due to the proportion of time spent food
gathering in different environmental zones. Although the products of food gathering were
probably pooled, habitual sustenance during food procurement forays in different environ-
ments might in itself be enough to skew isotopic ratios. An alternative hypothesis indicated by Figs 5 and 6, and our AIC analysis of the δ13C data is
that grave goods may have some spatial patterning in the site (see also [11, 20]) and this may
also predict dietary variation. PLOS ONE | DOI:10.1371/journal.pone.0135214
October 28, 2015 Although contentious [28], if we accept that abundance of mor-
tuary offerings represents a proxy of the wealth/status of individuals in life (e.g. [29]), then the
Cluster One dietary outliers appear to have been relatively low wealth or status. Historical evi-
dence attests to the existence of status related dietary divisions in Polynesia [24, 30, 31]. Princi-
pally, oily, fatty meats were considered of higher status [32, 33], although more mundane foods
could gain value through labour-intensive preparation methods [33]. It is possible that meats
like moa, which conform to a generic description of a food with high status in Polynesia, were
less available to individuals such as those in Cluster One due to their social position. However
we were not able to demonstrate this association with robust statistical significance, and prob-
lems with the existing unsystematic spatial categorisation of burials inhibits our ability to inter-
pret the AIC findings. This matter warrants more research. Our reassessment of the strontium isotope data also challenges aspects of the model pro-
posed by Kinaston et al. [4]. While we accept that Burials 1–7 fall in the lower end of the range
of 87Sr/86Sr ratios, we argue that the shape of the data across the site suggests they are part of a
continuum rather than a distinct group (e.g. Fig 8). The range of values expressed in the sample
is very large, and if we strictly follow the guideline that local individuals are those who have
87Sr/86Sr ratios within two standard deviations of the mean value for local dogs then the major-
ity of individuals (20 of 24) interred at Wairau Bar are non-locals. This data, however, is diffi-
cult to interpret without better baseline information on regional variation in biologically
available strontium. The range of geological strontium ratios reported by Kinaston et al. [4]for
the Wairau Valley is so large that it easily encompasses all of the variation in the archaeological
sample. The ratios reported for Pacific Islands [4] are comparatively narrow in range, but
remarkable for being considerably lower than the archaeological samples at Wairau Bar. A
complicating factor is that coastal and island populations have repeatedly been shown to have 13 / 16 PLOS ONE | DOI:10.1371/journal.pone.0135214
October 28, 2015 The First New Zealanders? Acknowledgments We thank Dr Enrico Crema for providing discussions and code that aided the implementation
of model selection methods. Professors Andy Bevan and Stephen Shennan provided valuable
discussions and thoughtful comments on earlier drafts. We thank two anonymous peer review-
ers for their constructive comments. Conclusion We agree with Kinaston et al. that Wairau Bar appears to be a special site in the colonising
phase of New Zealand and that, particularly on the basis of material culture, there are differ-
ences between individuals in the site. However, here we have argued that the stable isotope
data does not support the identification of spatially coherent clusters of individuals with diets
different from the majority of the population at the site. Instead, we find that dietary differ-
ences were most probably influenced by the sex of individuals and that there is no clear isotopic
evidence to support the designation of a group of Wairau Bar individuals as the first immi-
grants. We would argue that these results tell us something potentially more interesting about
the social dimensions of diet and mobility in early New Zealand. At a broad level this study has shown the potential benefits of multi-factorial analyses of sta-
ble isotope data in the Pacific Islands. Traditional single hypothesis-testing approaches often
employ groups that increase the potential of finding positive results, thereby introducing a
degree of circularity and decreasing the likelihood of further tests being carried out. As large-
scale samples like Wairau Bar are relatively rare in Pacific Island archaeology, we advocate a
broad exploration of variables that ensures the maximum amount of information is returned. The development of better strontium baselines would aid in future analysis of mobility, as may
the collection of δ18O data, which, given the variation in precipitation and temperature in New
Zealand is likely to be informative. Author Contributions Conceived and designed the experiments: AB TT. Performed the experiments: AB TT. Ana-
lyzed the data: AB TT. Wrote the paper: AB TT. 87Sr/86Sr ratios approaching that of seawater (0.7092) due to the influence of a marine based
diet, and this can elevate ratios above that of the local geology [17]. In other words, whilst we
agree with Kinaston et al. that it is likely the large variation in 87Sr/86Sr ratios exhibited by the
Wairau Bar sample is indicative of a very mobile population, we believe we do not yet know
enough to make any secure claims about origins. Supporting Information S1 Table. Demographic, burial, wealth and isotopic data from the human remains at
Wairau Bar (adapted from Kinaston et al. 2013: S2 Table). (DOCX) S2 Table. The results of stepwise model selection using both AIC and BIC criterions. (DOCX) References 1. Allen MS, Nagaoka LA (2004) In the footsteps of von Haast. . .the discoveries of something Grand: The
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October 28, 2015 References Oceania 2: 117–135. 31. Firth R (1967) The work of the gods in Tikopia. New York: Humanities Press. 32. Kirch PV,O’Day S (2003) New archaeological insights into food and status: a case study from pre-con-
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Improving partnerships with family members of ICU patients: study protocol for a randomized controlled trial
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Heyland et al. Trials (2018) 19:3
DOI 10.1186/s13063-017-2379-4
STUDY PROTOCOL
Open Access
Improving partnerships with family
members of ICU patients: study protocol
for a randomized controlled trial
Daren K. Heyland1,2,10*, Judy Davidson3, Yoanna Skrobik4, Amanda Roze des Ordons5, Lauren J. Van Scoy6,
Andrew G. Day2, Virginia Vandall-Walker7,8 and Andrea P. Marshall9
Abstract
Background: Over the last decade, health care delivery has shifted to partnering with patients and their families to
improve health and quality of care, and to lower costs. Partnering with family members (FMs) of critically ill patients
who lack capacity is particularly important for improving experiences and outcomes for both patients and FMs.
How best to apply such partnering strategies, however, is yet unknown. The IMPACT trial will evaluate two
interventions that enable partnerships with families of critically ill patients, each in a distinct content area, but
similar in that they empower and support FMs.
Methods: This multi-center, open-label, randomized, phase II clinical trial aims to randomize 150 older, long-stay
ICU patients and their families into one of three groups (50 in each group): (1) The OPTimal nutrition by Informing
and Capacitating FMs of best practices (OPTICs) group, a multi-faceted intervention to engage and empower FMs
to advocate for, and audit, best nutritional practices for their critically ill FMs, (2) A web-based decision-support
intervention called the ICU Workbook (The Canadian Researchers at the End of Life Network (CARENET) ICU
Workbook; https://www.myicuguide.ca/. Accessed 3 Feb 2017.) to support families in shared decision-making
process regarding goals of medical treatments, and (3) Usual care. The main outcomes for this trial include
nutritional adequacy in hospital and hand-grip strength prior to hospital discharge; satisfaction with decisionmaking; decision conflict; and degree of shared decision-making.
Discussion: With the goal of improving the functional recovery of nutritionally high-risk older patients and the
quality of care at the end of life for these patients and their FMs in the ICU, we have proposed two novel family
capacitation strategies. We hope that the nutrition and decision-support interventions implemented and evaluated
in our study will contribute to the evidentiary basis for best family partnered care pathways focused on optimizing
the quality of ICU care for patients with life-threatening illness and their families.
Trial registration: Clinical trials.gov, ID: NCT02920086. Registered on 30 September 2016. Protocol version dated 11
October 2016.
Keywords: Patient and family engagement, Randomized trial, Nutrition, End of life decision-making, Supportive
care, Critical care
* Correspondence: dkh2@queensu.ca
1
Department of Critical Care Medicine, Queen’s University, Kingston, ON,
Canada
2
Clinical Evaluation Research Unit, Kingston General Hospital, Kingston, ON,
Canada
Full list of author information is available at the end of the article
© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Heyland et al. Trials (2018) 19:3
Background
Over the last decade, health care delivery has shifted
to partnering with patients and their families to improve health and quality of care, and to lower costs.
Partnering with family members (FMs, i.e., immediate
family, relatives, friends, and significant others) of
critically ill patients who lack capacity is particularly
important for improving experiences and outcomes
for both patients and FMs [1–3]. Partnering with
FMs decreases patient anxiety, confusion and agitation [4], reduces complications [5], decreases intensive
care unit (ICU) and hospital length of stay [6], and
improves long-term cognitive performance [7].
Overall, partnering with families helps patients feel
more secure and increases patient and family member
satisfaction [8–10]. Independent of its effect on
patient outcomes, partnering with FMs has also been
shown to reduce their anxiety, depression and psychological symptoms [5, 6, 11, 12]. Thus, such strategies that
improve patient and FM outcomes, shorten ICU and hospital length of stay, and have the potential to save billions
of dollars per year in health care costs [11].
How best to apply such partnering strategies, however, is unknown. The IMPACT trial will evaluate
two interventions that enable partnerships with
families of critically ill patients, each in a distinct
content area, but similar in that they empower and
support FMs. The first is a nutritional intervention,
the OPTimal nutrition by Informing and Capacitating family members of best practices (OPTICs), a
multi-faceted intervention to engage and empower
FMs to advocate for, and audit, best nutritional
practices for their critically ill FMs. The second is a
web-based decision-support intervention called the
ICU Workbook [13] to support families in shared
decision-making process regarding goals of medical
treatments.
Herein, we describe the methodological approach to
testing these interventions in the context of a phase II
randomized clinical trial, the IMPACT trial.
Methods
Conceptual frameworks
There are many determinants of the medical care that
patients receive and their subsequent outcomes that include health systems and larger social factors (see Fig. 1).
In an environment where the FM best knows the patient
and the heath care team best knows the patient’s medical
condition, we envision both coming together to make
shared decisions and optimize patient outcomes. However, many FMs do not have the knowledge or confidence to perform this role [14]. Accordingly, we set out
to develop interventions that support families in this key
partnering role.
Page 2 of 11
To inform the development of the interventions, we used
three theoretical frameworks (Lightening our Load [15],
Working to Get Through [16], and Facilitated Sensemaking
[17, 18]) developed by members of our team. These models
recognize the importance of FM involvement and participation in communication, decision-making, and bedside care.
The resultant sense of purpose and control may reduce
post-intensive care syndrome, a series of stress-related
complications experienced by FMs of critically ill patients
(Fig. 2) [19, 20].
The background rationale and prior development work related to these interventions are discussed in Additional file 1.
Overall aims and hypotheses
Our overall aim is to investigate whether, and how, the
tools, knowledge, and skills provided to FMs will increase their satisfaction with care, and their sense of efficacy to act as advocates for best practice. We
hypothesize that the multi-faceted strategies that engage
families in patient care will: (1) increase the patient’s
nutritional intake; (2) optimize physical recovery in older
critically ill patients at high risk of nutritional problems;
(3) reduce FMs’ psychological distress; (4) improve
family satisfaction with decision-making; and (5) reduce
the duration of ICU stay for future decedents. We further hypothesize that the trial will be feasible (as judged
by enrollment rates and compliance with the protocol),
the interventions efficacious, and contamination rates
low (<10% of families in the usual care group will have
been exposed to either or other interventions). The primary and secondary outcomes for each intervention are
different and are explained below.
Study design
This multi-center, open-label, randomized, phase II
clinical trial involves three groups (two active interventions and one usual care, see Fig. 3). We report
the methods of this study according to the Standard
Protocol Items: Recommendations for Interventional
Trials (SPIRIT) Checklist (see Additional file 2). By
comparing the effects of the two treatment groups to
the usual care group, we will understand the overall
treatment effect of each intervention. To control for
the possibility that the extra time and attention
provided to families through the interventions may
alter their perception of care and impact the outcomes of interest [21], we will also compare the treatment effects between two active interventions,
thereby addressing this potential “placebo effect.” The
comparison of the two active interventions groups
will be considered secondary to the comparisons of
each active group to the usual care group. All three
comparisons will be pairwise with no pooling of
groups.
Heyland et al. Trials (2018) 19:3
Page 3 of 11
Fig. 1 Conceptual model of family engagement in the intensive care unit (ICU). Many factors impact medical decisions for a patient.
Patient factors include the patient as a person and their medical condition. Patient as a person represents the person’s prior experiences,
values, preferences and goals. Environmental factors include the health care environment situated within a larger societal context. Family
members may engage in care or decision-making. The family’s role in care gives purpose in crisis and may help family members cope
with the exposure to critical illness. Their direct participation may also improve patient adherence to treatment plan and attainment of
treatment goals. In shared decision-making the family is engaged as a member of the health care team. The family is typically most
familiar with the patient as a person and the patient’s past health status. The clinicians are typically most knowledgeable of the patient’s
critical illness. The decision-support intervention is designed to facilitate communication between the family and clinicians about the
patient as a person and their medical condition. Family engagement in this manner facilitates a shared medical decision that is
consistent with the patient’s values and goals in the context of their illness experience and medical condition, and is congruent with
what the patient would choose if they were competent to make such a decision. Thus, we hypothesize that family engagement can
influence family response to critical illness, and also the treatment plan. Ultimately, both patient and family outcomes are optimized
Setting
Randomization
Eight tertiary ICUs in Canada (n = 4), the United
States (n = 2), and Australia (n = 2) will participate in
this phase II trial to understand important geographical, jurisdictional, or cultural factors that may influence the feasibility and efficacy at this stage of the
program’s development. If the phase II trial is
successful, the findings will be generalizable to a
broader range of ICUs nationally and internationally.
Consent from eligible FMs will be obtained within
72 h following admission to the local ICU after the
RC has explained the study objectives and procedures. Once informed consent is obtained, the RC
will log onto the web-based randomization system.
The randomization system will use a computergenerated randomization schedule allocating patients
1:1:1 by the method of permuted blocks of random
undisclosed size within strata, to either (1) the
OPTICs intervention, (2) the decision-support intervention, or (3) usual care. Randomization will be
stratified by site.
Study population
Given the nature of the study interventions, FMs of
ICU patients who are “nutritionally high-risk” and/or
those at risk of dying in the ICU, or during the subsequent hospitalization will be eligible to participate.
The specific patient and FM eligibility criteria are
presented in Additional file 3. Research coordinators
(RCs) will review census lists and screen hospital
charts daily to identify potentially eligible patients.
Study interventions
Following randomization, the RC will meet the FM,
complete the baseline data collection (see Additional file 4)
and initiate the study procedures (see Fig. 4), as described
below.
Heyland et al. Trials (2018) 19:3
Page 4 of 11
Fig. 2 Post-intensive care syndrome among families of intensive care unit (ICU) survivors. Reprinted with permission from Springer [20]
OPTICs nutrition intervention FMs randomized to
the OPTICs nutrition intervention will meet with a
dietitian early in the patient’s ICU stay (within 72 h
of randomization). The dietitian will collect the patient’s brief nutritional history, verbally communicate
the results of the nutritional risk assessment to the
FM and clinical team, and place copies of the assessment in the hospital record. In addition, they will
educate the FM about nutritional support in the ICU,
focusing on capacitating all FMs to interact with
health care providers (HCPs) and ask them about the
nutrition that their critically ill relative is receiving.
The FM will be provided with a booklet that reiterates the nutritional information and includes materials
to help the family approach and ask questions of
HCPs. The dietitian will assess comprehension using
“talk-back” techniques [22]. In addition to the written
resource, the FM will have ongoing access to videos
that provide similar information as the booklet.
Posters will be placed in the patient’s room with
information about the OPTICs intervention (where
permitted). The dietitian will communicate with the
Family Member of
Critically Ill patients
Family-centered
intervention
Nutrition
Intervention
Randomization
Familycentered
intervention
Decision Support
Intervention
No Family-centered
intervention
Usual Care
Attention
controls for
each other
Improved patient and family-centered outcomes
(Physical recovery, satisfaction with care, etc)
Fig. 3 Study overview
Heyland et al. Trials (2018) 19:3
Page 5 of 11
ICU Admission
Nutrition
Intervention Components
within 72
hours
Family meets with dietician
Nutrition resources provided to family
(posters, handouts)
Nutrition risk communicated to
healthcare team
Decision Support
Intervention Components
Family meets with research assistant to review
MyICUGuide
MyICUGuide handout communicated to
healthcare team
Family meeting with healthcare team
Transfer to Floor
within
week 1
Floor nutrition plan discussed with family
3 day calorie count
MyICUGuide handout communicated to new
healthcare team
Check-in with research assistant
Oral Nutritional Supplementation
48 hr
prior to
discharge
Nutrition discharge plan
Discharge from Hospital
Fig. 4 Timeline of the interventions
FM throughout the ICU stay to answer questions and
identify unmet needs.
At or near the time of the patient’s ICU discharge, the
dietician will provide further education based on the patient’s current nutritional status (i.e., receiving nutritional support, eating by mouth, swallowing difficulties),
information about the nutrition that the patient can expect once on the ward, and will show the FM an educational video specific to nutrition after ICU discharge. As
soon as the patient transitions to oral intake, the
dietitian will introduce the FM to the nutrition diary, an
oral intake audit tool to record the patient’s oral food intake at each meal. The patient will be followed by the
ICU dietitian while in the ICU and by the ward dietitian
once transferred.
Once patients in all groups are taking liquids by
mouth, they will receive two or more Oral Nutritional
Supplements (ONS) per day (approximately 400 kcal/
day) as the existing academic literature suggests a positive impact on patient outcomes [23]. This is considered
to be part of standard of care, but in this nutritional
intervention arm the FM will be encouraged to advocate
for the patient to receive them, coach the patient to take
them as ordered, and monitor oral intake of the supplements using the nutrition diary. A nutrition care plan
for the ward will be developed by the dietitian and communicated to the FM and patient. A 3-day calorie count
will be completed weekly to capture calories and protein
consumed from oral intake, including ONS, while the
patient is on the ward until hospital discharge or for a
maximum of 4 weeks, whichever comes first. Just prior
to hospital discharge, the dietitian will work with the patient and FM to develop a home nutrition plan provided
to them in writing at the time of discharge. All study
materials for the OPTICs intervention can be found on
the Critical Care Nutrition website [24]. All other cointerventions will be permitted; this family engagement
strategy will be provided in addition to usual care.
Decision-support intervention The decision-support
intervention consists of an online resource entitled “ICU
Workbook” [13] that provides information to families on
principles of shared decision-making, experiences that
may be expected during the ICU stay, and advice on
coping with the stress of having a FM in the ICU. This
resource was developed from 19 qualitative interviews of
FMs’ perceived stressors and coping strategies used during the decision-making process, and has been pilot
tested [25]. Integrated into this online resource are several sections and resources listed in Table 1. The RC will
go through the content of the website guide, using talkback techniques to ensure that FMs understand the key
concepts presented on the website [22]. Paper copies of
the electronic content will also be given to FMs and a
link to the website so they can return to review materials
at a later date. After completing the ICU Workbook with
FMs, the RC will verbally communicate the results of
the exercises to the clinical team. The questions and answers will be summarized and copies of the report
placed on the hospital record, and a copy will be given
to the FMs. Next, the RC will work with the health care
team to arrange a family meeting including the attending
physician and bedside nurse to review patient goals of
care within 72 h of completing the ICU Workbook. The
RC will attend this meeting to record its content using
the Observing Patient Involvement (OPTION) tool (described below). The RC will connect with FMs throughout the ICU stay and upon discharge from ICU, to build
Heyland et al. Trials (2018) 19:3
Page 6 of 11
Table 1 Components of the ICU Workbook – the decision-support intervention
Section
Description
1. Orientation and education about the ICU
• Provides a general overview of the intensive care unit (ICU) including key ICU terms, treatments, and
roles of various clinicians who work in the ICU.
• Describes common processes in the ICU, including resuscitation and comfort measures, organ
donation, Power of Attorney, and substitute decision-making (SDM)
• Defines commonly used vocabulary within the ICU.
2. When a loved one is in the ICU
• Provides suggestions for coping strategies for family members of an ICU patient.
• Encourages family member visitation.
• Offers advice about how to ask questions in the ICU.
• Encourages family to seek support or keep a journal during the ICU stay.
3. Looking after yourself
• Reinforces the importance of self-care for families of ICU patients.
• Encourages family members to sleep, eat, and maintain healthy physical activity
• Provides ideas for how to inform and communicate with other family and friends about the
patient’s progress.
4. Making decisions in the ICU
• Describes and encourages shared decision-making.
• Defines the role and responsibilities of family members in SDM.
• Defines the roles of clinicians in decision-making.
• Provides resources available to help with the decision-making process.
5. Help us to get to know you and your
family member
• Family directed questionnaire asking information about the patient’s personal characteristics.
• Assesses family members’ state of mind and emotional status.
• Assesses patient’s clinical status and frailty
6. Informational preferences
• Identifies family member’s desire for information and level of health literacy.
7. Values history tool
• Assesses patients’ values and preferences (as reported by family members).
• Questionnaire helps family members articulate patient’s view of quality of life, value conflicts,
impact of decisions on others, and religious/spiritual/cultural beliefs.
8. Decision preferences
• Elicits family member’s preferences for extent of information sharing and preferred role in
decision-making
• Identifies others who should be involved in decision-making
• Measures residual decisional conflict
and maintain the relationship and answer questions,
and to facilitate communication of the ICU Workbook report to the attending physician and clinical
team on the ward.
Usual care Patients randomized to usual care will not
receive any study interventions. Baseline data collection
and outcome assessment will be performed similarly as
for the intervention groups. Local practices will determine the extent to which families receive support from
allied health care professionals in the management of
nutrition and decision-making for their relative.
Outcomes
The current project is a phase II trial focusing on shortterm efficacy and feasibility outcomes. The future phase
III IMPACT trial will likely have two co-primary outcomes related to the nutritional and decision-support interventions. For the nutritional intervention, the primary
outcome will represent the patient’s physical recovery
long term (i.e., 6-minute walk distance at or before hospital discharge, activities of daily living, and the 36-item
Short Form survey (SF-36) Physical Function at
6 months) as suggested by current experts [26].
Secondary outcomes include adequacy of nutrition in
the ICU, ONS consumption on the wards, time to
discharge alive from hospital (time-to-event analysis
with death as competing risk), ICU and hospital outcomes (mortality and length of stay), 90-day readmission
rates, and the cost-effectiveness of the intervention. We
expect that as a consequence of their involvement and
engagement in patient care, FMs’ psychological wellbeing will be improved as well in this group. For the
decision-support intervention, the primary outcome will
be a measure of family psychological well-being (symptoms of depression and anxiety (Hospital Anxiety and
Depression Scale [27]), and post-traumatic stress disorder (Impact of Events-Revised [28]) 6 months after the
ICU stay). Secondary outcomes will include family satisfaction with decision-making using the Decision-making
component of the Family Satisfaction with ICU Care
(FS-ICU24) subscale and length of ICU stay for decedents (a marker of poor quality end-of-life (EOL) care
from patients’ perspective and that has been responsive
to prior palliative care interventions in the ICU).
For the purpose of this phase II study, we are evaluating more proximal (short-term) outcomes and process
measures. These outcomes will be collected by the RC
and will be assessed 2–3 weeks after ICU-related death
or discharge or prior to hospital discharge, whichever
comes first. Table 2 presents all process measures
(short-term or long-term) planned for the current phase
Compliance with the intervention Met with RC early and went
through the myicuguide website Family meeting to review
intervention output within 72 h of encounter Patient-centered
report from MyICUGuide on chart during ICU and hospital stay
and attending physician aware of contents of this report
Contamination (families exposed to an intervention to which
they were not assigned) Exposed to myicuguide website Exposed
to OPTICs content
Decision-support
intervention
Other
Table of current and projected outcomes for the IMPACT program of research
a
All outcomes listed in this column will be measured in all randomized patients
6MWD, 6-minute walk distance, ICU intensive care unit
Compliance with the intervention Met with dietitian early
in ICU stay and at ICU discharge to review materials
Received a nutrition plan at ICU and ward discharge Used
nutrition audit tool
Nutritional
intervention
Process measures
Table 2 Outcomes considered in the IMPACT protocol program of research
Measure of family member’s long-term
psychological well-being Length of ICU and
hospital stay for decedents
Use of shared-decision making (OPTIONa tool) during
initial family conference Change in decisional conflict at
1 week Family satisfaction with decision-making at ICU
discharge (or 2–3 weeks later for decedents)
Overall family satisfaction with ICU care at ICU discharge
(or 2–3 weeks later for decedents)
Measures of patient’s long-term physical
recovery (e.g., 6 MWD at 3 and 6 months)
Long-term (months following hospital discharge)
outcomes (future phase III)
Nutritional adequacy during ICU stay Consumption of
Oral Nutritional Supplements during hospital stay Intake
on hospital wards (3-day calorie count) Hand-grip
strength at or before hospital discharge
Short-term (hospital) outcomesa (current phase II)
Heyland et al. Trials (2018) 19:3
Page 7 of 11
Heyland et al. Trials (2018) 19:3
II and future III studies. To enable comparisons across
all three groups, all process and outcome measures will
be performed for all patients. With respect to the nutritional intervention, one of the key short-term outcomes
will be nutritional adequacy during ICU stay. We have
previously shown that patients who receive greater
nutritional adequacy support in the ICU have better
long-term outcomes [29]. In the ICU, to assess nutritional adequacy, the total amount of energy or protein
received from either enteral nutrition (EN) or parenteral
nutrition (PN), inclusive of propofol, will be divided by
the amount prescribed in the baseline assessment and
expressed as a percentage for patients in all groups. Unfortunately, no easy method exists to measure nutritional adequacy on the hospital wards where patients are
likely taking nutrition by mouth. We plan to do 3-day
calorie counts weekly for 4 weeks during the hospital
study as well as collect the use of ONS from all study
patients.
For the purposes of this phase II trial, we will assess physical recovery by measuring hand-grip
strength at hospital discharge for patients in all
groups. Impaired muscle strength is an important
outcome because of its association with disability and
functional decline, and reduced health-related quality
of life (QOL) [30]. The OPTICs intervention FMs
who actively engage with nutritional care of their
loved ones may show improved family satisfaction
with care. Additionally, engagement in care is theorized to support FM participation in patient ICU care
[16, 18] and to be protective of the FM’s mental
health by providing a sense of purpose in crisis [17].
We will evaluate the impact of the decision-support
intervention on shared decision-making with FMs using
the Observing Patient Involvement (OPTION) instrument, developed to evaluate communication during
shared decision-making [31]. During the initial family
conference after the decision-support intervention (or
similar time frame for other groups), the RC will identify
key shared decision-making behaviors (such as “did the
clinician present pros and cons of various treatment options?”) and select a response from the 5-point scale ranging from “is not observed” to “observed and executed
to a high standard.” The total summed score ranges
from 0 to 48, with higher scores indicating greater competency in shared decision-making [31].
To assess the impact of the decision-support intervention on FM decision conflict, we will use the 10-item
Decisional Conflict Scale (DCS) [32]. This scale is validated and reliable, and used extensively to evaluate the
effectiveness of shared decision-making interventions
such as patient decisions aids and counseling [33].
Scores range from 0 (no conflict) to 100 (high conflict);
scores greater than 50 are associated with delayed
Page 8 of 11
decisions. This scale has to be linked to a decision made
or an expressed preference. Accordingly, at baseline and
at the end of the first week, we will elicit a preference
for use of life-sustaining treatments in the ICU setting
and administer the DCS to all participating FMs. At the
same two time points, we will ask the FM about their
preference for use of life-sustaining therapies.
We will also use the FS-ICU24 survey to obtain ratings of satisfaction with ICU care from designated FMs
of all patients enrolled in the study. This questionnaire
has been shown to have content and construct validity,
high reliability (correlation coefficient = 0.85), and two
validated subscales (Satisfaction with Overall Care and
Satisfaction with Decision-Making) [34, 35]. We will administer FS-ICU24 to FMs of surviving patients upon
ICU discharge and mail the questionnaire to FMs of decedents 2–3 weeks following death [34].
For this phase II trial, additional outcomes include the
feasibility and fidelity of the implementation and measures of contamination as shown in Table 3. Plans to
manage the data will be consistent with standard operating procedures at the coordinating center.
Sample size
The total sample size for this phase II trial is 150 patients (50 per group). We expect the subsequent phase
III trial will require 20–30 sites with up to 1000 patients
in total. As one of the primary goals of this phase II trial
is to assess the feasibility of implementing the study interventions, we plan to assess feasibility at multiple sites.
With approximately 10–20 patients per site, we expect
to gain enough experience to assess the feasibility of the
study protocol per site.
For each intervention, we have performed a sample size
calculation for one of the key short-term outcomes of each
intervention. From our prior work, we know that the average nutritional adequacy of these patients during their
ICU stay will be 40–50% with a standard deviation of 30%
[36]. We aim to detect a small but clinically meaningful
increase in nutritional adequacy of approximately 20%.
Under these assumptions 50 patients per group will
achieve 92% power at a two-sided alpha = 0.05. For the
decision-support intervention, we will power the trial to
evaluate family satisfaction with decision-making. In our
prior REALISTIC-80 study, the standard deviation of the
FS-ICU24 “Decision-making” domain was 11; we consider
an increase of 5.5 points (a medium effect size) to be
plausible and clinically important. With 50 evaluable
subjects per group, we would achieve 71% power at a twosided alpha = 0.05 to detect a 5.5-point difference between
groups.
Given the other objectives of the study related to feasibility, compliance, and contamination, a sample size of
50 per group will allow us to assess these endpoints with
Heyland et al. Trials (2018) 19:3
Page 9 of 11
Table 3 Additional outcomes for the IMPACT trial
Parameter
Defined as…
Enrollment and consent rate
We will judge the current study protocol feasible if > 75% of eligible families of eligible
patients are approached and > 60% of these consent.
Compliance with the components of the OPTICs
intervention.
The dietitian will keep a log of all FMs with whom the OPTICs intervention materials were
reviewed, the time the intervention was delivered, and whether a nutrition plan was
presented at the end of the ICU stay (survivors only) and ward stay. At baseline, the review of
these materials should occur in > 90% of enrolled FMs and the nutrition plan should be
presented in > 75% of eligible cases for this to be considered feasible.
Compliance with the components of the family
directed decision-support intervention
Review of the website should occur in 100% of the FMs enrolled in this group, and the
family meeting (including the enrolled FM) should occur within 72 h in > 75% of cases for
this intervention to be considered feasible. In addition, the RC will perform a chart review
after ICU death or discharge for all enrolled patients and document evidence that the
components of the decision-support intervention were included in the medical record. We
will consider the protocol successful if > 75% of charts contained such evidence.
Physician Awareness Assessment
One week after enrollment, the RC will administer the Physician’s Awareness Assessment to
the attending physician and/or fellow responsible for the care of the patient during the
period of enrollment to assess the extent to which they were aware of study materials, the
variables captured in the study intervention output (nutritional history, patient pre-morbid
functional state, values, preferences, etc.) and the degree to which this knowledge influenced
their decision-making. If > 75% of them acknowledge exposure to study tools and rate their
impact as substantial, in the respective interventional groups, the intervention will be
considered feasible.
Contamination
We will ask all FMs whether they have had a facilitated review of the myicuguide website
and OPTICs tools. If < 10% of the families of patients in the usual care group acknowledge
that they have seen the study tools and if < 10% of the intervention groups acknowledge
they have been exposed to the other intervention, we will consider this acceptable.
FM family member, RC research coordinator, ICU intensive care unit
reasonable precision, consistent with the sample size of
other phase II studies. For example, with 50 patients per
group there is a 95% chance of estimating a binary
variable (such as loss to follow-up, contamination, compliance, etc.) to within ± 14% and any variable with a
rate < 15% or > 85% (as would be expected for loss to
follow-up, contamination, or compliance) could be estimated to within ± 10% or 19 times out of 20.
Statistical analysis
The feasibility outcomes will be described by group
as rates with 95% confidence intervals. Reasons for
loss-to-follow-up, non-compliance, and contamination
will be tabulated. The distribution of the continuous
outcomes described above will be described by group
and compared among groups using a mixed-effects
model with treatment arm as a fixed effect and site
as a random effect. Due to the limited (six to eight)
number of sites in this phase II trial, we will perform
a sensitivity analysis, treating site as a fixed effect.
For the binary outcomes, we will use the Mantel-Haenszel test stratified by site. At this exploratory
phase II stage we will not formally adjust p values for
multiplicity of tests but will consider the potential
type I and type II errors in our interpretation of results. For key efficacy outcomes with > 5% missing
data, multiple imputation will be used for the primary
analysis supplemented by a complete case sensitivity
analyses. The heterogeneity between sites will be
estimated by the intra-class correlation coefficient for
all outcomes; in case this is needed to inform the design of a subsequent cluster randomized controlled
trial (RCT) [37].
Ethics
We will obtain local ethics approval at each participating
site before commencing. RCs will obtain written informed consent from FMs for their participation in the
trial and proxy consent from the same FM to enable
data collection related to the patient. For surviving patients, as they regain competency, we will consent them
prospectively for their continued involvement in the
study (obtaining hand-grip strength prior to hospital discharge). Given that the interventions are not directed towards the patient (we are only collecting data from the
hospital record), we will not be collecting or reporting
serious adverse events and there will be no Data
Monitoring Committee for this trial. There are no financial or other competing interests for any of the co-authors. Results will be published and posted on the study
team’s internationally renowned websites [38, 39] to aid
in dissemination.
Discussion
With the goal of improving the functional recovery of
nutritionally high-risk older patients and the quality of
care by patients at the end of life and their FMs in the
ICU, we have proposed two novel family capacitation
Heyland et al. Trials (2018) 19:3
strategies. We hope that the nutrition and decisionsupport interventions implemented and evaluated in our
study will contribute to the evidentiary basis for a family
partnered care pathway focused on optimizing the quality of ICU care for patients with life-threatening illness
and their families. We aim to start enrollment in the first
quarter of 2017.
Additional files
Additional file 1: Description of background rationale for the OPTICs
and decision-support intervention. (DOCX 47 kb)
Additional file 2: SPIRIT 2013 Checklist: recommended items to address
in a clinical trial protocol and related documents. (DOCX 74 kb)
Additional file 3: Eligibility criteria. (DOCX 45 kb)
Additional file 4: Baseline data collection. (DOCX 45 kb)
Acknowledgements
Not applicable.
Funding
This project was partially funded from a grant from the Canadian Institutes
of Health Research. The funders played no role in the design, conduct or
analysis of the trial nor has any authority over these activities.
Availability of data and materials
The datasets used and/or analyzed during the current study are available
from the corresponding author on reasonable request.
Authors’ contributions
DKH, JD, YS, ARO, LJV, AD, VVW, and APM contributed substantially to the
study conception and design, drafted the article, or revised it critically for
important intellectual contact. All authors read and approved the final
manuscript.
Ethics approval and consent to participate
This protocol will be reviewed at all participating centers and approved by
local Ethics Boards prior to starting the study at each site. Currently, it has
been approved at all US, Canadian and Australian sites. We will obtain
written informed consent from all participants.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
Author details
1
Department of Critical Care Medicine, Queen’s University, Kingston, ON,
Canada. 2Clinical Evaluation Research Unit, Kingston General Hospital,
Kingston, ON, Canada. 3EBP/Research Nurse Liaison, University of California,
San Diego Health, San Diego, CA, USA. 4Department of Medicine, McGill
University, Montreal, QC, Canada. 5Department of Critical Care Medicine and
Division of Palliative Medicine, University of Calgary, Calgary, AB, Canada.
6
Department of Medicine and Humanities, Division of Pulmonary, Allergy
and Critical Care, Pennsylvania State University, Hershey, PA, USA. 7Faculty of
Health Disciplines, Athabasca University, Athabasca, AB, Canada. 8Faculty of
Nursing, University of Alberta, Edmonton, AB, Canada. 9Menzies Health
Institute Queensland, Griffith University and Gold Coast Health, Southport,
QLD, Australia. 10Kingston General Hospital, Angada 4, Kingston, ON K7L
2 V7, Canada.
Page 10 of 11
Received: 10 March 2017 Accepted: 24 November 2017
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Honor, violencia y poder patriarcal en el proceso mexicano de secularización penal
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HONOR, VIOLENCIA Y PODER PATRIARCAL EN
EL PROCESO MEXICANO DE SECULARIZACIÓN
PENAL
HONOR, VIOLENCE AND PATRIARCHAL POWER IN THE
MEXICAN PROCESS OF CRIMINAL SECULARIZATION
Alejandra Palafox
Universidad de Granada
Recibido: 29/7/2016
Aceptado: 10/09/2016
Para citar este artículo / To cite this article:
Palafox, Alejandra. «Honor, violencia y poder patriarcal en el proceso mexicano de secularización penal». En Nieves Montesinos Sánchez y Beatriz Souto Galván (coords.), Laicidad y creencias. Feminismo/s, 28 (diciembre 2016): 293-313,
DOI: 10.14198/fem.2016.28.12
Para enlazar con este artículo / To link to this article:
http://dx.doi.org/10.14198/fem.2016.28.12
Resumen
A través del análisis de distintos cuerpos jurídicos, manuales, diccionarios, tratados
de derecho y expedientes judiciales, abordamos algunas de las repercusiones que el
proceso de secularización penal, desarrollado en el México decimonónico, tuvo en la
regulación de un conjunto de actos de índole sexual, calificados como delitos de incontinencia, delitos de sensualidad, o delitos contra el orden de las familias, la moral
pública o las buenas costumbres. Evidenciamos al respecto las contradicciones que se
dieron en la paulatina separación de los conceptos de pecado y delito y la supuesta relegación formal del pecado al ámbito de lo interno y lo privado, concluyendo que esta
modernización jurídica conllevó el mantenimiento de un código de valores cristiano
que operó como herramienta de control y sujeción de la población femenina.
Feminismo/s 28, diciembre 2016, pp. 293-313
294
Alejandra Palafox
Palabras clave: Secularización, delitos sexuales, virginidad, honor, siglo XIX.
Abstract
Through the analysis of different juridical bodies, manuals, dictionaries, law treaties
and judicials files, we address some of the effects that the process of criminal secularization in Mexico had in the regulation of set of acts of a sexual nature, classified
as crimes of incontinence, sensuality crimes or crimes against family order, public
morality or decency, during the Nineteenth century. We show about the contradictions that occurred in the gradual separation of the concepts of sin and crime, and the
supposed formal relegation of sin to the internal and private field. We conclude that
this legal modernization entailed the maintenance of a christian values code, which
operated as a tool of control and subjection of the female population.
Keywords: Secularisation, sexual crimes, virginity, honor, XIXth century.
Feminismo/s 28, diciembre 2016, pp. 293-313
1. INTRODUCCIÓN
El concepto de «secularización» está presente en la gran mayoría de la historia
y la historiografía hispanoamericanas y, pese a ello, apenas ha sido cuestionado
y problematizado. Como advierte Joseba Louzao, la polisemia del concepto
de «secularización» y sus variaciones en los diferentes momentos históricos
lo han convertido en una herramienta explicativa o paradigma inoperante
para el estudio de los procesos en perspectiva histórica (335). Al igual que
con otras grandes categorías conceptuales como «racionalismo», «nación» o
«liberalismo», con «secularización» hacemos referencia a una construcción
que, desde finales del siglo XVIII aparece como constitutiva del abstracto y,
también, conflictivo concepto de modernidad estatal y que formó parte del
conjunto ideológico que abogaba por la separación entre el poder real y el
sagrado como requisito irrefutable para la consecución de una nueva organización socio-política.
Hablar de secularización implica hablar necesariamente de un proceso
multidimensional basado en una «recomposición del campo religioso que
caracteriza la modernidad» (Hervieu-Léger 227). Implica aludir a transformaciones, resistencias, cambios, contradicciones, que acompañan la conformación
a lo largo de los siglos XIX y XX de «un sujeto de la modernidad» (Dube
180) o de «una nueva forma de creencia individualizada» en palabras de Elisa
Cárdenas (53), lo que supuso el desarrollo de nuevas formas de apropiación
del espacio y nuevos vínculos sociales1.
En lo que se refiere a nuestro tema de estudio, la secularización penal en
el ámbito concreto de la regulación de los delitos sexuales que tuvo lugar en
México, este proceso, incoado en las postrimerías coloniales, conllevó una
pérdida de peso de las instituciones eclesiásticas en el tratamiento punitivo de
los actos sexuales reprobables así como modificaciones conceptuales ligadas
a la distinción, cada vez más nítida, de las nociones de delito y pecado, junto
1. Por «sujeto de la modernidad», acorde con lo expuesto por Saurabh Dube, entendemos
una categoría conformada por aquellos «actores históricos que han sido participantes
activos en procesos de la modernidad, tanto sujetos a estos procesos como también sujetos
que moldean estos procesos».
Feminismo/s 28, diciembre 2016, pp. 293-313
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Alejandra Palafox
con una relegación formal del pecado al ámbito de lo interno y lo privado.
Ya desde el último tercio del siglo XVIII, se sucedieron diversos conflictos de
competencias dentro de un proceso de paulatina apropiación de la regulación
de las sexualidades prohibidas por parte del poder civil y de un mayor control
secular de las conductas femeninas, lo que culminó con la prohibición en 1746
de que los tribunales eclesiásticos emitiesen sentencia ante casos de delitos
sexuales (Suárez 207-225).
Una vez alcanzada la emancipación política del país en 1821, dentro de
un marco de paulatino desarrollo de un orden jurídico moderno o liberal, en
lo que respecta al ámbito delictivo, este proceso de apropiación civil siguió
desarrollándose a lo largo del siglo XIX mediante el establecimiento del monopolio estatal del derecho, es decir, de la plena identificación entre el derecho
y la ley. Con la expedición del primer código penal mexicano en 1871, el
Estado se convirtió en el máximo organismo responsable de la reglamentación
de los comportamientos y actitudes sexuales de la población en su conjunto.
Durante los primeros cincuenta años de independencia, sin embargo, junto
con las leyes mexicanas y las emanadas anteriormente del poder real o las
cortes españolas, múltiples ordenamientos, incluyendo el derecho canónico,
siguieron definiendo y rigiendo los comportamientos sexuales de la población2.
La fragmentación normativa, junto con la caída en desuso de los severos
castigos contemplados por los códigos coloniales en material sexual, dificultó
el conocimiento de la definición, tratamiento y regulación formal de estos
delitos por parte de los propios juristas. En la práctica, esta situación se tradujo
en un vacío legal y el mantenimiento de una amplia discrecionalidad judicial,
sustentada en el predominio del derecho común y las condiciones específicas
de cada delito a la hora de emitir sentencias frente a la ley escrita y ejercida
a través del recurso continuado a diccionarios y manuales de derecho. Entre
los autores de estas sistematizaciones didácticas, los extranjeros y, en especial,
los españoles, ocuparon un lugar preeminente frente a los juristas mexicanos.
En concreto, a lo largo del siglo XIX, existieron tres obras clave en la sistematización del derecho para el tratamiento de la criminalidad sexual previa a
la codificación: El Febrero Mejicano de Anastasio de la Pascua, el Diccionario
de legislación y jurisprudencia del español Joaquín Escriche y el Novísimo Sala
Mexicano de 1870, a cargo de Manuel Dublán y Luis Méndez.
2. En relación con el derecho canónico, si bien es cierto que este cuerpo normativo perdió
importancia como fuente de derecho positivo con la Reforma juarista, obras doctrinarias
posteriores, como el Novísimo Sala Mexicano, contuvieron referencias que denotan su
vigencia para esos años.
Feminismo/s 28, diciembre 2016, pp. 293-313
Honor, violencia y poder patriarcal en el proceso mexicano de secularización penal 297
En este contexto, los privilegios y la influencia social de la Iglesia fueron
interpretados por la intelectualidad liberal mexicana como lastres del pasado
que la nueva nación independiente debía erradicar. Dentro de una lógica antinómica y evolucionista, los discursos liberales defendieron un concepto de
emancipación y progreso nacional que se oponía, maniqueamente, a la oscuridad clerical y a los vestigios de su poder, aun presentes en las naciones recién
independizadas. No podemos, sin embargo, pensar en estos cambios como
rupturas semánticas unidireccionales e inmediatas, pues como hemos podido
comprobar a través del análisis normativo y de la práctica judicial, los mecanismos reguladores de la sexualidad que se fueron desarrollando en el proceso
de modernización jurídica mexicana mantuvieron un respaldo institucional
a un conjunto de valores originado en la teología moral tomasina y regulado
por un sistema normativo sustentado en una interpretación católica de la vida.
2. DELITO Y PECADO EN LA MODERNIZACIÓN PENAL
Si algo caracterizó al tratamiento institucional de los actos sexuales prohibidos en el orden del Antiguo Régimen fue su conceptualización como pecados
y delitos al mismo tiempo, al no existir una separación del orden divino y
el humano, sino un orden natural fruto de la razón y voluntad divina que,
en términos actuales, estaría formado por una simbiosis entre ambas esferas
(Tomás y Valiente 252). Con la implantación de la secularización penal, tanto
en los discursos recogidos en la práctica judicial como en los nuevos textos
legales, las acciones delictivas, también las de índole sexual, se fueron progresivamente desligando de la noción de pecado que, hasta el momento, las
había acompañado.
El manual de derecho El Febrero Mejicano de Anastasio de la Pascua ofrece
interesantes reflexiones acerca de qué se entendía jurídicamente por delito en la
primera mitad del siglo XIX y su relación con la ley. Un delito era una «transgresión o quebrantamiento de una ley ejercido voluntariamente y a sabiendas,
en daño u ofensa del estado o alguno de sus individuos» (De la Pascua 3-4). El
delito se distinguía de la acción pecaminosa por transgredir la ley civil, a diferencia del pecado, que atentaba contra la moral mediante la infracción de algún
precepto divino o eclesiástico. Los delitos, a su vez, podían ser de naturaleza
pública o privada. Los primeros eran aquellos que, por su gravedad, ofendían
a la sociedad en su conjunto, como un asesinato. Los segundos, sin embargo,
atentaban sólo contra un individuo, sin causar grave daño al conjunto social.
En la práctica judicial, dentro del enfoque casuista, que fijaba la atención en
las condiciones y circunstancias bajo las que se producía el delito más que en
el delito en sí, los abogados defensores aprovecharon el proceso de transición
Feminismo/s 28, diciembre 2016, pp. 293-313
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Alejandra Palafox
secularizadora que atravesaba el derecho, tratando en ocasiones de traducir el
crimen sexual cometido por sus clientes en una falta moral y pecaminosa que
no merecía ser castigada por la justicia civil al pertenecer al fuero interno de la
conciencia. De esta forma, por citar un ejemplo, el estupro o «acto de perder,
como se dice, a una doncella», según el procurador Francisco González, pese a
estar formalmente criminalizado, no era un delito sino «un pecado muy grave
[…] sujeto al fuero sacramental de la penitencia, más no al fuero externo» y,
por tanto, no era digno de recibir un castigo público3. En caso contrario sería
necesario «ensanchar las cárceles», añadía este abogado, dando testimonio de
la frecuencia con la que se cometían este tipo de incontinencias.
Este tipo de argumentos no fueron minoritarios en la práctica judicial
mexicana del siglo XIX, como demuestran, entre otros, el discurso pronunciado por el magistrado Agustín Fernández en la causa incoada en 1864 a raíz
de la demanda por adulterio que Ramón Casares interpuso contra su esposa,
Vicenta Peñafiel, y quien era primo por línea materna del demandante, el pintor
Valentín Coronado. Ambos acusados, tras ser detenidos y puestos a disposición
judicial, reconocieron su delito y trataron de justificar el mismo alegando que
Ramón, el marido ofendido, había maltratado a su mujer durante años para
después abandonarla a su suerte, motivo por el cual ésta había tenido que
refugiarse en los brazos de otro hombre.
Después de varias semanas, en las que las partes implicadas presentaron
diversos documentos y testigos que ratificaban sus respectivas declaraciones,
el demandante decidió presentarse ante el juez para expresar formalmente
que perdonaba a los dos reos, presos en la cárcel Nacional de Belem, desistiéndose así en toda forma de su acusación4. Si bien el adulterio seguía siendo
un delito privado perseguible sólo a instancia de parte, el incesto, además de
ser un delito que ofendía al conjunto de la sociedad, figuraba entre los más
castigados por las leyes y las autoridades judiciales. Pese a ello, el licenciado
Fernández decidió sobreseer la causa, motivando su sentencia con una extensa
e interesante argumentación:
El marido sin género de duda, por su carácter de tal, tiene derecho indisputable
para tener a su lado a su mujer y disfrutar de los goces, conveniencia y ayuda
que proporciona el matrimonio, de los cuales se le privaría con una separación
larga ocasionada por la pena que al fin se le impusiera a su mujer […]. Una
pena innecesaria perjudicaría a la misma sociedad, disolviendo una familia
tal vez para siempre y con riesgo de mayores males, pues una larga separación
3. Archivo General de la Nación de México (AGNM), Tribunal Superior de Justicia del
Distrito Federal (TSJDF), 1838, caja 131, «La causa contra José María Jiménez», f. 46v.
4. A
GNM, TSJDF, 1864, caja 393, s/t, f. 22r.
Feminismo/s 28, diciembre 2016, pp. 293-313
Honor, violencia y poder patriarcal en el proceso mexicano de secularización penal 299
temporal puede resfriar y aun concluir con el cariño y amor conyugal que
ha manifestado Casares por la Peñafiel y tal vez exponerlo a contraer otras
relaciones ilícitas, cuyos males son sin duda de mayor gravedad y perjuicios
para la moral, la sociedad y el interés particular de los mismos cónyuges que
el que resulta de no castigar su delito, el cual por la misma modesta posición
social de los culpables y el marido, no ha producido escándalo que perjudique
la moral pública5.
Este insólito argumento estuvo motivado por la supremacía que el orden
familiar debía ocupar frente a ciertos comportamientos que pese a figurar
legalmente como actos delictivos, entre la opinión de diversos juristas, parecían
considerarse más como faltas pertenecientes al fuero interno. «En ocasiones es
cuerda cosa dejar el castigo de los crímenes a Dios en vez de poner remedio a
mayores males emprendiéndolo el hombre por su cuenta», sostuvo al respecto
el magistrado6. Los acusados fueron así puestos en libertad, apercibidos y bajo
fianza de estar sujetos a derecho hasta la revisión de la sentencia en segunda
instancia. Vicenta, a pesar de los golpes y malos tratamientos que supuestamente había recibido de parte de su marido, y de que hacía ya más de tres años
que no cohabitaba con él, fue «entregada» a éste. La sentencia, pese a tener
un carácter en apariencia extraordinario, ameritó la confirmación de parte del
Tribunal Superior de Justicia del Imperio.
La referencia a Dios en el discurso del juez Fernández, por otro lado, formó
parte de una contradicción que apareció con frecuencia entre los juristas de
la época. A pesar de que ya desde las postrimerías coloniales las instituciones
civiles aumentaron sus espacios de actuación, logrando tras la independencia
política desplazar completamente a las eclesiásticas en la administración de la
justicia criminal, la secularización del derecho en la práctica presentó diferentes ritmos. El aparato simbólico asociado al catolicismo siguió estando muy
presente en los discursos pronunciados en el foro por jueces, fiscales y abogados y tuvo una efectividad incuestionable. Pese a ello, no deja de sorprender
que para la segunda mitad del siglo XIX, uno de los principales magistrados
de la capital mexicana pudiese afirmar, sin ser reconvenido por ello, que en
ciertas circunstancias el sistema de justicia debía ser inoperante frente a actos
criminales, dejando que su resolución dependiera de la voluntad divina, lo que
contradecía abiertamente la separación entre los conceptos de pecado y delito
y dejaba entrever la convivencia entre los juristas de al menos dos tendencias
ideológicas: una orientada a desligar completamente la justicia terrenal de la
celestial y otra interesada en mantener su imbricación.
5. AGNM, TSJDF, 1864, caja 393, s/t, ff. 22v-23r.
6. AGNM, TSJDF, 1864, caja 393, s/t, f. 23r.
Feminismo/s 28, diciembre 2016, pp. 293-313
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Alejandra Palafox
3. HONOR Y CASTIDAD EN LOS DELITOS DE ESTUPRO, FUERZA Y
RAPTO
3.1 Aclaración terminológica
En la práctica judicial decimonónica se entendía por estupro la primera relación sexual que un hombre tenía, mediante el uso de la fuerza o no, con una
mujer virgen (Escriche 642). Para que este acto fuera considerado delito, sin
embargo, debía de haber mediado el empleo de la fuerza física o moral, lo que
aludía a un heterogéneo conjunto de acciones –amenazas, dolo, fraude, seducción o una falsa promesa de matrimonio– mediante los que se suponía que un
hombre podía lograr franquear la honradez de una doncella.
El delito de rapto consistía en el robo que se hacía de una mujer, sacándola
de su casa para llevarla a otro lugar con el fin de casarse o de «corromperla», es
decir, de tener relaciones sexuales con ella (Escriche 1480). Si éste se producía
por la fuerza, era considerado un delito contra la mujer raptada y su familia.
En los casos en los que la mujer consentía por «promesas, halagos o artificios
de su raptor», el delito se consideraba «rapto por seducción» y atentaba sólo
contra los padres, marido o tutor de la mujer seducida.
El término «violación» tuvo distintas acepciones a lo largo del siglo y, hasta
la expedición del código penal de 1871, no fue tipificado en sí como delito. En
la práctica judicial se utilizó tanto como sinónimo de estupro, al referirse a la
acción que atentaba contra la virginidad de una doncella como en su significado
de fuerza carnal, es decir, de «violencia que se hace a una mujer para abusar
de ella contra su voluntad» (Escriche 1538).
La utilización del concepto de mujer honrada como sinónimo de mujer
virgen, casta o sumisa fue común en el tratamiento penal de los delitos sexuales
que se desarrolló en México a lo largo de todo el siglo XIX. Su uso no respondió
sólo a una incorporación semántica, cuasi inconsciente, en la mentalidad colectiva de los juristas, sino que fue parte de los numerosos esfuerzos pedagógicos
que los hombres pertenecientes a la élite intelectual llevaron a cabo para guiar
las actitudes femeninas por el camino de la contención sexual.
Por «honor» en el ámbito jurídico se entendía «la acción o demostración
exterior por la cual se daba a conocer la veneración, respeto o estimación que
alguien tenía por su virtud o mérito» (Escriche 298). Estos méritos y virtudes,
que determinaban la buena reputación de un hombre, dependían en parte del
comportamiento sexual de las mujeres que estuviesen emparentadas con él
(Speckman 1432). Es decir, las mujeres que mantuviesen actitudes de recato,
contención y pudor aseguraban no sólo su propia honra sino el reconocimiento
social de sus familiares varones.
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Honor, violencia y poder patriarcal en el proceso mexicano de secularización penal 301
El origen de esta inequidad en la valoración de los comportamientos sexuales de hombres y mujeres estaba directamente relacionado con un concepto
de honor presente ya en las antiguas sociedades mediterráneas, que fue desarrollado y sistematizado en las principales obras de teología moral cristiana
(Bourdieu 59).
Para el padre de la Iglesia Agustín de Hipona, el honor de una mujer
y de sus familiares varones estaba determinado por su honradez, es decir,
por su conducta sexual. Ésta, sin embargo, no dependía exclusivamente de la
conservación de su himen sino del placer experimentado con la pérdida del
mismo. Como muestra el análisis que el santo presentó en La ciudad de Dios
del mítico suicidio de Lucrecia, tras haber sido violada por Sexto, hijo del rey
Tarquino, la distinción entre cuerpo y alma que caracterizaba a hombres y
mujeres permitía que una mujer que hubiese sido deshonrada lograse reparar
internamente su ofensa mediante la seguridad que le proporcionaba el saber
que no había consentido la violación (De Hipona 100). Si la mujer no había
participado voluntariamente en el acto sexual, no debía de sentirse ofendida en
su persona. La deshonra femenina, por tanto, debía paliarse de manera interna
o ser resarcida junto con el deshonor causado sobre sus familiares varones,
padres y hermanos, quienes eran, ante los ojos de este pensador, los legítimos
encargados de reparar el agravio.
3.2 Virginidad y violencia en la teología cristiana
La teología cristiana, si seguimos lo expuesto por Agustín de Hipona, al venerar
la virginidad como principal cualidad de una mujer, valoró la violencia sexual
en función de la pérdida de esta condición y del agravio que ello suponía contra
sus parientes varones.
El daño corporal, la humillación o el sufrimiento personal que una mujer
podía sentir tras ser víctima de una violación no merecieron la atención del
santo, quien abordó esta temática a través del análisis del episodio mitológico
conocido como «rapto de las Sabinas», según el cual, los primeros fundadores
de la ciudad de Roma, tras haber dejado a sus mujeres en Alba, habrían decidido secuestrar y violar a las mujeres pertenecientes a la tribu de los sabinos
(De Hipona 160).
Este acto violento no fue condenado en sí mismo por el santo, sino en
función del agravio que representaba para los familiares varones de las sabinas.
Como podemos apreciar aquí, ya desde el siglo V, la valorización del rapto
aparecía ligada al concepto de mujer como propiedad del varón encargado de
custodiarla. La acción sancionable que distinguía el rapto de otros males como
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Alejandra Palafox
el estupro, por tanto, residía en el robo que un hombre hacía de una mujer,
sacándola del lugar donde otro varón la tutelaba.
A partir del siglo XIII, la sistematización de la jerarquía dentro de los
actos ilegítimos libidinosos, llevada a cabo por Tomás de Aquino en su Suma
Teológica permeó la regulación civil y eclesiástica de la sexualidad occidental.
Si bien para este padre de la Iglesia la violencia física constituía un agravante
en los casos de rapto o estupro de una virgen o mujer casada, al igual que su
antecesor, no consideró la violación como un crimen de por sí. Los daños que
esos delitos suponían, además, iban dirigidos contra los hombres –maridos,
padres o tutores– que ejercían derechos sobre las mujeres violadas y no sobre
ellas mismas.
Dentro de la escala tomista de los vicios de lujuria, el estupro ocupaba
el penúltimo puesto en orden de gravedad por malicie, antecediendo sólo al
pecado de fornicación. El estupro, denominado también «violación», consistía
para Tomás de Aquino en la «desfloración» ilícita, es decir, fuera de la unión
matrimonial, de una doncella virgen e implicaba un acto de injuria al atentar
directamente contra el padre de la estuprada, quien estaba por ello autorizado
a denunciar judicialmente el agravio (477).
Entre las consecuencias que esta especie de lujuria podía entrañar, según el
santo, se encontraba el riesgo de que la joven estuprada no consiguiera contraer
matrimonio y cayese en la prostitución, actividad que no habría desempeñado
hasta el momento sólo «por temor a perder su integridad virginal» (De Aquino
477). Aunque el estupro no se efectuase a través del uso de la fuerza física,
implicaba siempre un acto de seducción, por lo que Tomás de Aquino defendía
que el responsable fuera condenado a dotar y a casarse con la joven. En caso
de que el padre de ésta rehusara entregársela en matrimonio, el estuprador
debería compensarle mediante un pago económico, además de pagar una dote
de mayor valor a la joven.
El rapto para Tomás de Aquino era una especie de lujuria considerada de
mayor gravedad que el estupro, pues a diferencia de este acto, el rapto siempre
conllevaba el uso de la violencia, ya que se cometía al sacar a una joven de la
casa de su padre o su marido por la fuerza para «corromperla», es decir, para
tener relaciones sexuales con ella (478). El uso de la violencia en este caso
respondía a la vehemencia del deseo, capaz de provocar imprudencias de este
tipo en los varones.
Si la mujer raptada no estaba casada, debía de ser entregada a su padre para
luego, si éste consentía, tomarla en matrimonio. En caso de ya estarlo, debía
de ser devuelta a su esposo, quien tenía derecho sobre ella por el hecho de
estar desposada. «Quien roba algo tiene la obligación de restituirlo» afirmaba
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Honor, violencia y poder patriarcal en el proceso mexicano de secularización penal 303
el santo al respecto (De Aquino 481). La cosificación del cuerpo femenino, en
este punto, era más que evidente: las mujeres no tenían potestad para tomar
decisiones sobre sí mismas, al ser conceptualizadas como bienes sujetos a la
propiedad varonil. El consentimiento de la mujer a la hora de ser sacada de
su casa, por tanto, no intervenía en la definición de este acto pues se interpretaba que el agravio se cometía contra el padre o marido de la raptada y no
ya contra ésta.
3.3 Tipificación delictiva y condenas en la legislación colonial
Las Siete Partidas, conjunto legal más referenciado por los juristas hasta la
expedición del Código Penal, diferenciaron dos tipos criminales referentes a
los delitos que hemos señalado, el «robo» y la «fuerza» pero ambos recibieron un tratamiento conjunto. Pese a no contemplar el concepto de estupro, sí
definían como una «gran maldad» el tener relaciones sexuales con una mujer
virgen, una mujer «de orden», es decir, religiosa, o una mujer viuda, siempre
que ésta viviese de forma honesta, «por halago o por engaño»7. Tener relaciones sexuales con mujeres de las condiciones aludidas conllevaba un atentado
contra la castidad, virtud amada por Dios y que debían amar los hombres, lo
que explicaba su reprobación legal.
Cualquier persona estaba autorizada para acusar a un hombre de haber
cometido este mal ante las autoridades y, en caso de ser probado, las Partidas
contemplaban penas muy distintas que las recogidas por la teología moral,
ordenando la pérdida y confiscación de la mitad de los bienes del hombre
honrado, la pena de azotes y destierro por cinco años del vil y la muerte en la
hoguera del siervo o esclavo8.
En caso de que la mujer con la que se cometiese el acto carnal fuese una
«mujer vil», es decir, no fuese ni virgen, ni viuda honesta, ni religiosa, la acción
en sí, pese a seguir constituyendo un pecado, no conllevaba la imposición de
ningún tipo de pena por parte de la justicia ordinaria. Si el acceso carnal a una
mujer vil se producía a través del uso de la fuerza física, las penas quedaban
sujetas al arbitrio de cada juzgador, quien debía valorar la condición del acusado y de la víctima, así como las circunstancias en las que se produjo el delito9.
En caso de que estos actos se produjesen en mujeres vírgenes, viudas honradas, religiosas o casadas, mediante el uso de la coacción física, con armas o
sin ellas, el responsable debía de ser condenado a muerte, así como a la entrega
7. Partida 7, título 19, ley 1.
8. Partida 7, título 19, ley 2.
9. Partida 7, título 20, ley 3.
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de todos sus bienes a la mujer que hubiese sido «forzada», en caso de que ésta
no quisiera casarse con él.
La fuerza era entendida en la legislación de las Partidas como la ausencia
de consentimiento de la víctima y el uso de la violencia para la realización de
la acción delictiva. Junto con la condición de los implicados, el uso de la fuerza
aparecía como un elemento esencial en la determinación del delito y la condena. Como ha destacado el historiador del derecho José Sánchez-Arcilla, estas
leyes no estaban orientadas a la protección de las mujeres sino a la custodia
de un bien jurídico basado en el honor y la castidad. Las diferentes condenas
contempladas variaban en función de la forma en la que era atacado este bien
mediante «engaño» o mediante «fuerza» (Sánchez-Arcilla 502).
Ya desde el siglo XVIII, los tratadistas de la época, con base en las prácticas
y el derecho consuetudinario, recomendaban en los casos de rapto o estupro
por fuerza conmutar la pena de muerte y confiscación de bienes recogidas en
las Partidas por la de presidio o galeras. En los casos de rapto o estupro por
seducción o engaño, las condenas de confiscación de bienes, azotes y destierro
recogidas en la legislación fueron suplidas, con arreglo al derecho canónico, por
la de contraer matrimonio con la estuprada (siempre que contase con el consentimiento paterno), la dotación y el reconocimiento de la prole si la hubiese.
3.4 La práctica judicial
En relación con el estupro por seducción, a diferencia de lo recogido por la
legislación de Partidas, ya desde los siglos coloniales, las condenas dictadas en
los procesos sobre este tipo de delitos no solían ser corporales y retomaban, de
acuerdo con lo estipulado en el derecho canónico, la pena de dotar o casarse
con la estuprada (De la Pradilla 3).
La dote a la que hacían referencia estas leyes difería de la dote en el sentido
riguroso de la palabra, como advertía Joaquín Escriche, ya que su finalidad no
era garantizar el bienestar de la novia en su futuro matrimonio sino resarcir
el daño causado por el estupro (654). Esta dote no debía restituirse al condenado aun cuando la estuprada no se casase y, además, se contemplaba que
fuese heredada por sus descendientes en caso de que la estuprada falleciera. La
dote simbolizaba, por tanto, una especie de reparación del daño ejercido sobre
la «integridad virginal» de una mujer, conceptualizada por los juristas como
una «prenda de inestimable valor» (Escriche 654) o una «dote preciosísima
de la mujer» (Senén 214), y por tanto, como patrimonio simbólico y material
de la misma.
La cantidad pecuniaria que el estuprador debía pagar, debía establecerse
por las autoridades judiciales «combinando la condición y las facultades del
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Honor, violencia y poder patriarcal en el proceso mexicano de secularización penal 305
estuprador con la calidad de la estuprada y del marido que hubiera podido
tener sin el estupro, de modo que sea suficiente a lo menos para cubrir el daño
que a la estuprada se siguiere»(Escriche 653). Según el jurista español Senén
Vilanova, las mujeres de mayor hermosura, «de dones singulares de naturaleza», o pertenecientes a estratos socioeconómicos más elevados debían recibir
una dote mayor (214).
La conveniencia de aplicar las penas de dotar o casarse con la estuprada,
contempladas por el derecho canónico y extendidas en la práctica judicial
decimonónica, fue cuestionada por varios juristas, como el español José Marcos
Gutiérrez, cuyos argumentos fueron retomados por jueces, fiscales y abogados
para eximir a los acusados de estupro de las penas contempladas.
Según este tratadista, la práctica canónica de obligar al estuprador a contraer matrimonio con su víctima debía de ser abolida ya que castigaba a los
hombres y premiaba a las mujeres que perdieran su virginidad de forma voluntaria como medio para casarse con su supuesto estuprador. De esta manera,
según Gutiérrez, muchas mujeres condescenderían a las pretensiones varoniles
con «lo que más debieran detestar», es decir, perder su honra, con el único fin
de casarse (172). Al mismo tiempo, este jurista denunciaba la frecuencia con la
que muchas mujeres «corrompidas y aun tan abandonadas», habían hecho de sí
mismas «el más infame comercio», engañando o tratando de engañar a muchos
jóvenes honrados fingiendo su virginidad ante los tribunales (Gutiérrez 172).
El jurista Joaquín Escriche, por el contrario, se mostró favorable a que el
estuprado fuera condenado a casarse o a dotar a su víctima, además de reconocer a los hijos habidos del delito. Por otro lado y a diferencia de lo contemplado
por la mayor parte de los tratadistas, Escriche reconoció que el condenado
debía dotar a la mujer también en los casos en los que ésta no fuese virgen en
el momento de la comisión del delito cuando en la opinión común conservase
todavía la reputación de serlo (654).
Esta perspectiva, acorde con la de otros pocos juristas contemporáneos, si
bien seguía relacionando la honestidad femenina con su sexualidad, interpretaba que ésta podía no recaer sólo en la conservación del himen sino también
en la reproducción de comportamientos recatados. Los comentarios al respecto
de Senén Vilanova ofrecen un punto de vista similar, al afirmar que
la condición de ser honesta y recatada es de más mérito que la misma virginidad; pudiendo darse el caso, en prueba de este sentir, que una soltera, que ha
sido desflorada goce las mismas acciones de estupro que aquella que nunca
dejó su virginal entereza (Vilanova 199).
Para los albores de la codificación penal, en atención a los autores del Novísimo
Sala, el estupro, entendido como un delito de sensualidad consistente en el
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Alejandra Palafox
«desfloramiento de una mujer honesta», es decir, virgen, a través de la seducción, no debía castigarse con penas corporales no ya porque no constituyese un
hecho reprobable sino porque no podía ser creíble el testimonio de una mujer
«capaz de vender su pudor, confesar su debilidad y sacarla a la plaza», lo que
la hacía sospechosa de disolución, es decir, de tener una vida y costumbres
relajadas (Dublán y Méndez 149). Por los motivos expuestos, los autores del
Novísimo Sala denunciaron expresamente su desacuerdo con la extendida
práctica judicial de condenar al estuprador, ya fuera a casarse o a dotar a la
ofendida y a reconocer la prole en caso de haberla o a otras penas como el
destierro, según las circunstancias, argumentando en los mismos términos en
los que ya lo había hecho José Marcos Gutiérrez:
nosotros no podemos aplaudir una costumbre que castiga a un cómplice del
mismo delito, que da lugar a que las mujeres hagan su pudor objeto de tráfico,
que hace contraer matrimonios forzosos y desgraciados, y que más de una
vez es el lazo que una mujer astuta y experimentada arma a un joven incauto
(Dublán y Méndez 150).
El delito de fuerza carnal constituía también un delito de estupro cuando se
cometía contra una mujer considerada honrada. Al haberse mitigado el rigor
de las penas recogido en las Partidas, tanto este delito como el rapto solían
condenarse mediante la pena de presidio o galeras, en atención a las circunstancias en las que se cometieran (Escriche 1413).
A diferencia del estupro por seducción o engaño, el delito de fuerza podía
ser perseguido de oficio o a instancia de cualquier persona y no sólo de los
afectados por el mismo. Ninguna ley contemplaba las penas que el delito de
fuerza contra una mujer que no fuera virgen, casada o viuda honrada debía
tener y se dejaba al arbitrio de los jueces la condena a cumplir.
3.5 Demostrar la violación
Para determinar la existencia de un delito de estupro por seducción, es decir,
de acto sexual con una mujer honrada por medio de halagos, falsas promesas
o engaño, los autores de derecho consultados coincidían en la necesidad de
demostrar previamente la honradez de la supuesta víctima. Las sospechas de
conducta desarreglada que podían recaer sobre una mujer eran determinantes para que sus quejas por estupro perdiesen validez ante las autoridades
judiciales.
Entre las pruebas materiales o físicas consideradas por los juristas se
encontraban los «vestigios o señales que deja el estupro en la estuprada, y
que consisten en la desfloración, en las violencias y lesiones sobre los órganos
sexuales u otras partes del cuerpo y en las enfermedades venéreas que a veces
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Honor, violencia y poder patriarcal en el proceso mexicano de secularización penal 307
comunica el delincuente» (Escriche 655). Ante estas posibles marcas el autor
advertía que una desfloración podía ser reciente o antigua y las señales de violencia provenir de otras causas que ninguna relación tuvieran con el estupro.
De la misma manera, los indicios de «mal venéreo» podían ser engañosos. Por
estos motivos, los reconocimientos corporales debían efectuarse sólo cuando
se estimase necesario y por parte de facultativos capacitados.
En relación a los casos de violación o forzamiento, juristas como Anastasio
de la Pascua consideraban que un solo varón no estaba capacitado para agredir
sexualmente a una mujer, supuestamente por contar ésta con más medios para
oponerse a la violencia masculina que el agresor para vencer su resistencia
(251). Esta actitud de desconfianza, muy extendida entre los juristas de la
época, presentaba una continuidad con la actitud que en el siglo V Agustín de
Hipona había manifestado ante el suicidio de Lucrecia, quien se quitó la vida
tras haber sido forzada por Sexto. Al respecto, frente a este mítico acontecimiento, el santo exteriorizó su falta de credibilidad afirmando: «¿Y si (cosa que
ella sólo pudo saber), halagada por su libido, consintió con el mozo que hizo
en ella violenta irrupción, y, castigándolo en sí, fue tanto su dolor que creyó
debía expiarlo con la muerte?» (De Hipona 100).
A pesar de que las leyes castigaban el delito de fuerza sexual ejercido
también sobre mujeres solteras no vírgenes, la extendida reticencia a condenar este tipo de agresiones se tradujo en que, en la práctica judicial, sólo se
persiguieran y sancionaran las cometidas sobre mujeres consideradas honradas
por las autoridades10.
3.6 La codificación penal
Con la entrada en vigor del Código Penal se puso fin a la situación de vacío
jurídico que había acompañado al tratamiento judicial de los delitos de estupro, violación y rapto desde la Independencia. Su inclusión entre los «Delitos
contra el orden de las familias, la moral pública o las buenas costumbres» (CP
1871, lib. III, tít. VI.), evidenció una clara continuidad en la interpretación
de estos actos como atentados contra la virginidad y el honor pero no contra
la integridad de las víctimas. La violencia física ejercida contra éstas siguió
teniendo una consideración supeditada al agravio que estos actos suponían
contra el honor de sus familiares varones. Entre las medidas que confirman lo
afirmado, puede considerarse la indulgencia con la que fueron tratados aquellos
10. Afirmación sustentada en el estudio de 441 expedientes judiciales pertenecientes al
fondo «Tribunal Superior de Justicia del Distrito Federal» del Archivo General de la
Nación de México, relativos a delitos de rapto, estupro o violación entre 1824 y 1880.
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padres que mataran a sus hijas –en caso de que éstas vivieran en su compañía
y estuvieran bajo su potestad– si lo hacían en el momento de hallarlas en acto
carnal o en uno próximo a éste. Al interpretarse que la «corrupción» de una
hija ante los ojos de un padre causaba deshonor, la pena contemplada para
aquel que asesinara en el acto a su descendiente debía ser de cinco años y no
ya de doce, como se recogía para otras variables de homicidio simple11.
El delito de estupro pasó a regularse atendiendo a los cambios en la mentalidad que habían acaecido ya a finales de época colonial. Siguiendo la definición
recogida en esta normativa, según la cual el estupro era «la cópula con una
mujer casta y honesta, empleando la seducción o el engaño para alcanzar
su consentimiento», este delito quedó sancionado con penas que iban desde
ocho años a cinco meses en prisión12. Las penas variaban según la edad de la
víctima, estableciendo ocho años de prisión en el caso de que ésta fuese menor
de diez años. Con la promulgación del Código de Procedimientos Penales, de
15 de diciembre de 1880, quedó establecido que el delito de estupro no podía
perseguirse de oficio. Al igual que lo establecido en el Código Penal para el
delito de rapto, sólo se procedería criminalmente contra los acusados por queja
de la mujer ofendida, de su marido, si ésta fuese casada, o de sus padres, si no
lo fuera, y, a falta de éstos, por queja de sus abuelos, hermanos o tutores13. La
honestidad femenina, como puede advertirse en estos artículos, se estableció
como un concepto directamente relacionado con la sexualidad de las mujeres.
La virginidad, por tanto, siguió siendo un requisito fundamental para poder
realizar una acusación de estupro.
En atención a lo recogido en su exposición de motivos, dirigida al ministro
de Justicia y redactada por Antonio Martínez de Castro, presidente de la comisión encargada de formar el Código, la regulación penal del delito de estupro en
1871 trató de adecuar la misma a lo establecido por las codificaciones europeas
y, en especial, la doctrina de autores y la ejecutoria de tribunales franceses.
Acorde con esta tendencia, el Código eliminó la costumbre canónica de condenar al reo a casarse o a dotar a su víctima, al considerar que una mujer que
contrajera relaciones previas al matrimonio no podía ser una buena esposa ni
una buena madre en el futuro14. Al respecto, Martínez de Castro sostenía que:
un enlace contraído por la fuerza, un matrimonio que ha tenido por origen
la falta de pudor y de recato de una mujer, no puede producir sino desamor
y desprecio en el marido, y la desgracia de ambos cónyuges y de sus hijos;
11. C
P 1871, art. 555.
12. C
P 1871, art. 793.
13. C
PP 1880, art. 814.
14. C
P. 1871, art. 312.
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Honor, violencia y poder patriarcal en el proceso mexicano de secularización penal 309
porque no puede ser casta esposa ni buena madre la que ha sido antes liviana,
como lo tiene acreditado una constante y dolorosa experiencia (40).
Cometía rapto «el que contra la voluntad de una mujer se apodera de ella y se
la lleva por medio de la violencia física o moral, del engaño o de la seducción,
para satisfacer algún deseo torpe o para casarse» (Martínez de Castro 40). La
pena estipulada en el Código al respecto era de entre dos años y nueve meses
y cinco años y tres meses de prisión.
En caso de que el delito se cometiera sin la voluntad de la mujer raptada,
por medio de la violencia o del engaño, o contara con su voluntad pero se
tratara de una víctima menor de dieciséis años, el raptor merecía cuatro años
de prisión y multa de hasta quinientos pesos. El hecho de que la mujer fuese
menor de dieciséis años implicaba el uso de la seducción por parte del raptor
al presumir que, al no haber alcanzado la edad suficiente para tener un juicio
maduro, su consentimiento había sido arrancado a «la timidez y debilidad de
su sexo» era «efecto de ilusiones engañosas», de las que era «fácil rodear la
inexperiencia y credulidad de una joven inexperta y apasionada» (Martínez
de Castro 59).
Frente al delito de violación, entendido como la «cópula con una persona
sin la voluntad de ésta» por medio del uso de la violencia física o moral, sea
cual fuere su sexo, la pena impuesta por el Código osciló entre los diez y los
seis años de prisión y varió en función de la edad de la víctima15. Fungían como
agravantes del delito que el acusado ejerciese autoridad sobre su víctima, fuese
ascendiente, descendiente, padrastro o madrastra del ofendido, así como que el
acto se realizase «contra el orden natural», aumentando la pena en dos años.
En caso de que fuese hermano, la pena se incrementaría en un año y en seis
meses si el reo ejerciera autoridad sobre el ofendido o fuese su tutor, su maestro, o criado asalariado del ofendido, o cometiera la violación abusando de sus
funciones como funcionario público, médico, cirujano, dentista, comadrón, o
ministro de algún culto16. También constituían agravantes del delito, tanto en
los casos de estupro o violación, que resultara alguna enfermedad a la persona
ofendida, lesión o su muerte17.
4. CONSIDERACIONES FINALES
El estudio de la regulación jurídica de la sexualidad ha permitido reflexionar en torno a las continuidades semánticas que los conceptos de «honor» y
15. C
P 1871, art. 795.
16. C
P 1871, arts. 798-802.
17. C
P 1871, art. 802.
Feminismo/s 28, diciembre 2016, pp. 293-313
310
Alejandra Palafox
«virginidad» han arrastrado desde la Antigüedad. Al respecto, hemos considerado pertinente incidir en su valor simbólico y material, dada la centralidad
que ambos elementos ocuparon en los discursos legitimadores de la asimetría de género inherente al proceso mexicano de secularización penal. Hemos
comprobado, además, cómo los fundamentos de las modernas leyes sexuales
mexicanas quedaron, en este sentido, ya establecidos en las doctrinas elaboradas por los principales ideólogos de la teología moral.
Ser considerada ante los ojos de las autoridades judiciales una mujer honrada fue requisito para poder contar con la protección del sistema judicial a
lo largo del siglo XIX. En atención a las leyes y a los autores consultados, el
sistema judicial no contemplaba el sufrimiento de una supuesta víctima de
estupro o violación en la determinación y valoración del delito. El objetivo de
las autoridades judiciales ante estos casos no era otro que el de mantener el
orden social y, para ello, era necesario resarcir el agravio cometido contra el
honor de las mujeres y sus familiares varones, encargados de custodiarlas. La
condena judicial de un estuprador significaba la demostración pública de la
castidad y el buen comportamiento de su víctima, al demostrar que en ningún
momento consintió perder su virginidad.
En términos formales, por tanto, ningún tratadista y ninguna ley hicieron
referencia al padecimiento o las consecuencias psicológicas que, en términos
actuales, una violación podía conllevar para una mujer. Las mujeres víctimas de estupros, raptos o violaciones fueron conceptualizadas por la ley más
como objetos pertenecientes a padres, hermanos o maridos que como sujetos
jurídicos, situación acorde con el eminente carácter masculino del proceso de
individuación que llevaba desarrollándose en México desde las postrimerías
coloniales.
Acorde con la interpretación dada por los padres de la Iglesia, el robo o
la profanación del cuerpo de una mujer virgen siguieron siendo concebidos
como el uso ilícito de algo que le pertenecía a otro. La protección que el sistema judicial brindaba a estas mujeres, por tanto, podía equipararse a la que
las autoridades garantizaban frente a la propiedad privada, paulatinamente
sacralizada en el proceso de modernización estatal impulsado por las élites
liberales. La personalidad jurídica de sus familiares ante la ley, al simbolizar a
los legítimos propietarios del objeto allanado, por tanto, siguió, en términos
formales, contando más que la de ellas mismas.
Al mismo tiempo, la valoración de los daños físicos que este delito podía
causar siguió centrada en la virginidad, siendo ésta el principal objeto jurídico
que las leyes trataban de proteger. La valoración del agravio sufrido por la
mujer se establecía con base en unos parámetros centrados en las consecuencias
Feminismo/s 28, diciembre 2016, pp. 293-313
Honor, violencia y poder patriarcal en el proceso mexicano de secularización penal 311
morales y socioeconómicas que la pérdida de la condición de doncella podía
suponer, ya que se consideraba que al haber tenido «acceso a varón» fuera del
ámbito matrimonial, la víctima se encontraba expuesta al peligro de corromper
sus costumbres y caer en el vicio de la prostitución. De la misma manera, al
haber perdido el bien jurídico de su pureza, el acceso a un buen matrimonio
parecía inalcanzable.
La paulatina sustitución de los teólogos por juristas laicos, primero, y médicos y psiquiatras, desde finales del siglo XIX18, como principales intérpretes y
reguladores de las conductas sociales, no supuso la revocación de los cimientos
sostenedores de la sexualidad prescriptiva. El proceso de secularización que
experimentó la sexualidad como dispositivo discursivo y coercitivo de control
y sujeción, en términos generales, consistió en una re-significación de la teología moral imperante a través de la óptica del moderno derecho natural y de la
medicina científica. Estas continuidades no sólo estuvieron presentes durante
los primeros cincuenta años de independencia del país, periodo en el que la
sexualidad seguía estando regida, formalmente, por la normativa colonial, sino
que lograron permear el proceso de modernización jurídica que culminó con
el Código Penal de 1871. Este proceso no supuso una revocación sino una
apropiación y reconfiguración, en términos liberales, de la doctrina canónica
medieval. Esta evidente continuidad no fue una característica exclusiva del
proceso modernizador mexicano sino que, por el contrario, fue constante en
la formación codificadora de los distintos estados-naciones a uno y otro lado
del Atlántico. A diferencia de otras doctrinas jurídicas medievales, que sufrieron importantes innovaciones como resultado de las modificaciones políticas,
económicas y sociales en el occidente europeo y americano, los fundamentos
canónicos del medioevo respecto a la teoría y a las prácticas sexuales se mantuvieron constantes a lo largo de los siglos posteriores a su sistematización,
permeando los procesos contemporáneos de secularización y modernización
jurídica.
La religión se mantuvo como instrumento de cohesión y control social
al tiempo que se verificó una progresiva separación entre la esfera civil y la
eclesiástica; así como una desacralización del poder político que, en términos
de derecho penal, se tradujo en la separación entre los conceptos de delito y
pecado, y una paulatina sustitución del pecado por el delito en los discursos
procesales. Este cambio fue parte del proceso de adopción de una nueva forma
18. El éxito de la criminología positivista en México, a partir de la publicación en 1880 de
la obra de Cesare Lombroso, estrechó las colaboraciones existentes entre la ciencia del
derecho y la medicina dentro del interés por definir al «delincuente» como sujeto social
desviado (Ver Narváez Hernández).
Feminismo/s 28, diciembre 2016, pp. 293-313
312
Alejandra Palafox
de entender el mundo que no estuvo exento de contradicciones y que implicó
la distinción entre el fuero interno, entendido como la conducta moral, y el
fuero externo, identificado con la conducta pública. El concepto de delito pasó,
así, a constituir un atentado no ya contra un orden legitimado en la figura del
príncipe como representante de Dios en la tierra, sino contra el interés social.
La secularización penal que se experimentó en materia sexual, por tanto,
no fue sinónimo de una «des-cristianización» de la población mexicana sino
que supuso que parte de los mecanismos de regulación de las conductas sexuales de la población capitalina dejaran de pertenecer a la esfera de lo sagrado
y de estar en manos de la Iglesia. Desde las instancias estatales se mantuvo
una evidente continuidad simbólica que operó como instrumento de control
y sujeción de la población en general y de las mayorías subalternas, incluidas
las mujeres, en particular. A pesar de las divergencias frontales que se sucedieron entre la Iglesia y el Estado mexicano en estos años, por tanto, en lo que a
la regulación de la sexualidad femenina se refiere, la reforma liberal operó en
términos de reconfiguración y apropiación más que de rompimiento con los
cimientos católicos.
REFERENCIAS BIBLIOGRÁFICAS
Bourdieu, Pierre. La dominación masculina. Barcelona: Anagrama, 2000.
Cárdenas Ayala, Elisa, «Secularización, laicización: una reflexión pendiente».
Valentina Torres Septién. El impacto de la cultura de lo escrito. México: Consejo
Nacional de Ciencia y Tecnología, 2008.
De Hipona, Agustín. La ciudad de Dios. Madrid: Biblioteca de autores cristianos,
1958.
De la Pascua, Anastasio. Febrero Mejicano, o sea la Librería de Jueces, abogados y
escribanos que, refundida, ordenada bajo nuevo método, adicionada con varios tratados y con el título de Febrero Novísimo dio a luz D. Eugenio de Tapia. Nuevamente
adicionada con otros diversos tratados y las disposiciones del Derecho de Indias y
del Patrio, por el Lic. Anastasio de la Pascua. vol. 7. México: Suprema Corte de
Justicia de la Nación, 2010.
De la Pradilla Barnuevo, Francisco. Suma de todas las leyes penales, canónicas, civiles
y de los Reynos. Sevilla: Luys Estupiñan,1613.
Dube, Saurabh. «Modernidad». Diccionario de Estudios Culturales Latinoamericanos.
Eds. Mónica Szurmuk y Robert Mckee Irwin. México: Instituto Mora, siglo
XXI, 2009, 177-181.
Dublán, Manuel y Luis Méndez. Novísimo Sala mexicano o ilustración al derecho
real de España con las notas del Sr. Lic. D. J. M. de Lacunza. Edición corregida y
considerablemente aumentada con nuevas anotaciones y refundiciones, relativas
a las reformas que ha tenido la legislación de México hasta el año de 1870, por lo
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Honor, violencia y poder patriarcal en el proceso mexicano de secularización penal 313
señores don Manuel Dublán y don Luis Méndez, abogados de los tribunales de la
República. Volumen 2. México: Imprenta del Comercio de N. Chávez, 1870.
Escriche, Joaquín. Diccionario razonado de legislación civil, penal, comercial y
forense, o sea resumen de las leyes, usos, prácticas y costumbres, como asimismo
de las doctrinas de los jurisconsultos, dispuesto por orden alfabético de materias,
con la explicación de los términos del Derecho. Por Don Joaquín Escriche y con
citas del derecho, notas y adiciones por el licenciado Juan Rodríguez de San Miguel.
México: Oficina de Galván, 1837.
Hervieu-Léger, Daniéle. «Secularización y modernidad religiosa». Selecciones de
Teología 103.26 (1987): 217-227.
Louzao Villar, Joseba. «La recomposición religiosa en la Modernidad: un marco
conceptual para comprender el enfrentamiento entre laicidad y confesionalidad
en la España Contemporánea». Hispania Sacra 60 (2008): 331-354.
Gutiérrez, José Marcos. Práctica criminal de España. Publícala el licenciado don José
Marcos Gutiérrez, editor del Febrero Reformado y Anotado, para complemento de
esta obra que carecía de tratado criminal. Madrid: Oficina de don Benito García
y Compañía, 1804.
Martínez de Castro, Antonio. «Exposición de Motivos». Código Penal para el
Distrito Federal y Territorio de la Baja California, sobre delitos del fuero común y
para toda la República Mexicana, sobre delitos contra la Federación. Chihuahua:
Librería de Donato Miramontes, 1883, 7-70.
Narváez Hernández, José Ramón. «Bajo el signo de Caín. El ser atávico y la criminología positivista en México». Anuario Mexicano de Historia del Derecho
17 (2005): 303-322.
Sánchez-Arcilla Bernal, José. «Violación y estupro. Un ensayo para la Historia de
los «tipos del derecho penal». Anuario Mexicano de Historia del Derecho 22
(2010): 485-562.
Speckman Guerra, Elisa. «Los jueces, el honor y la muerte. Un análisis de la justicia (ciudad de México. 1871-1931)». Historia Mexicana 4 (2006): 1411-1466.
Suárez Escobar, Marcela. Sexualidad y norma sobre lo prohibido. La Ciudad de México
y las postrimerías del Virreinato. Tesis de Doctorado en Historia. México:
UNAM, 1994.
Tomás y Valiente, Francisco. «El Derecho Penal como instrumento de gobierno».
Estudis: Revista de Historia Moderna 22 (1996): 249-262.
Vilanova y Mañes, Senén. Materia criminal forense, o tratado universal teórico y
práctico de los delitos y delincuentes en género y especie... Madrid: Imprenta de
don Tomás Albán, 1807,
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Review of: "Mathematics Is Physical"
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Qeios, CC-BY 4.0 · Review, June 24, 2023 Potential competing interests: No potential competing interests to declare. This is a subject in metaphysics and philosophy. I am not an expert but I find this is interesting. The author presented an
interesting claim that "math is physical", which sort of is not standard. Usually, we think math and physics are relatively
independent, one dealing with logic, and one dealing with nature. Physics shall be mathematical so that a physical theory
can be tested by experiment and make prediction. While math does not have to be physical since it only uses abstract
symbols and rules on them. So I suggest the author first to define what it means by "physics", and what it means by "physical". This two terms are not
defined on the first hand. Is there a difference between "physics" and "nature"? Can we say "math is natural?" For a basic setting, it is beneficial to think of the meaning of the number "infinity". Is it physical? Or is it a 'real' number? Does “physical” mean “finite”? This is like the question thought of by Pythagoras that whether irrational numbers, like
square root of 2, are real numbers. Infinity is the central issue in Godel, Turing, Cantor. So it will be good to discuss it to
set the stage. About infinity, in physics a brunch of study is phase transition which relies on the notion of infinity. So, is the theory of
phase transition a valid physical theory? It will be good if the author can study this example to further unveil his concept. About infinity, in physics a brunch of study is phase transition which relies on the notion of infinity. So, is the theory of
phase transition a valid physical theory? It will be good if the author can study this example to further unveil his concept. It is also necessary to briefly discuss the relationship between math and computation theory. Let me ask: is math
computational? is computation physical? Shall all numbers be computable? We know that, according to Turing, not all
numbers are computable, but all numbers are mathematical. This seems imply that, computation, as a process with a
certain amount of spacetime cost, must be physical, but math does not. However, if the author does not agree with this, it
is then necessary to explain more on this. I can not see the relation between Gödel and Landauer. Did Landauer mentioned Gödel before in his paper? Review of: "Mathematics Is Physical" Dongsheng Wang Potential competing interests: No potential competing interests to declare. Qeios ID: WG9RDB · https://doi.org/10.32388/WG9RDB Potential competing interests: No potential competing interests to declare. The author stated “Mathematics is intrinsically connected to physics in two fundamental ways. First, it is performed by
physical entities such as human mathematicians or machines, both of which have finite resources.” Sure, but I believe
Plato would not agree with this logic. Platonists believe there are Ideas, and math is a study of Ideas, but physics is not. Poisson brackets in Eq 5 can be explained in detail. For Eq 6, there is also a global constraint which is the normalization
as we know. For Eq 11, cf classical vs quantum, there is also a no-hiding theorem, Phys. Rev. Lett. 98, 080502, 2007, stating that
quantum information cannot exist in the form of correlations. This might be relevant here. Qeios ID: WG9RDB · https://doi.org/10.32388/WG9RDB 1/2 Qeios, CC-BY 4.0 · Review, June 24, 2023 Using the Hamilton form, or Geometric quantum mechanics, people has considered it for quantum computing. Say, in a
paper https://arxiv.org/abs/1606.04998. I do not have suggestion on the technical parts. My suggestions are mainly on the conceptual aspects of this thoughtful
article. Hope my suggestion and confusion can help to improve the manuscript. I do not have suggestion on the technical parts. My suggestions are mainly on the conceptual aspects of this thoughtful
article. Hope my suggestion and confusion can help to improve the manuscript. Qeios ID: WG9RDB · https://doi.org/10.32388/WG9RDB 2/2
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METODOLOGI PENULISAN SYEKH ‘ABD AL-HAY AL-LAKNAWI DALAM KITAB ZAFAR AL-AMANI
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Taqaddumi: Journal of Quran and Hadith Studies
Vol. 1, No. 2
DOI Prefix 10.12928 Taqaddumi: Journal of Quran and Hadith Studies
Vol. 1, No. 2
DOI Prefix 10.12928 Keyword: al-Laknawi, figure, ‘ulum al-hadith, works, hadith Keyword: al-Laknawi, figure, ‘ulum al-hadith, works, hadith METODOLOGI PENULISAN SYEKH ‘ABD AL-HAY AL-LAKNAWI
DALAM KITAB ZAFAR AL-AMANI Mohd. Khafidz Soroni
Kolej Universiti Islam Antarabangsa Selangor, Malaysia
mohdkhafidz@kuis.edu.my Kata kunci: al-Laknawi, tokoh, ‘ulum al-hadith, karya, hadis Taqaddumi: Journal of Quran and Hadith Studies
Vol. 1, No. 2
DOI Prefix 10.12928 Abstract Throughout the history of the tradition of Islamic studies, there have been various works of ‘ulum al-
hadith that have been produced by hadith scholars. These works differ in their writing methodology
from one work to another. To understand the differences, a study of each work and the background
of the author is very necessary to be conducted to reveal and explain it. Thus, this study aims to
introduce one of the figures in the discipline of ‘ulum al-hadith named Sheikh ‘Abd al-Hay al-
Laknawi (d. 1304H) with a focus on one of his monumental works, namely Zafar al-Amani fi Sharh
Mukhtasar al-Jurjani. This qualitative study uses content analysis methods to identify and
comprehensively describe the methodology of al-Laknawi's writing in the work. From the results of
the study it can be concluded that the methodology of writing al-Laknawi is seen as quite systematic
and loaded with scientific discussions on issues that are quite complex in the science of hadith such
as the issue of hadith hasan, the problem of doing good deeds with weak hadith, the issue of hadith
mursal, issues munkar hadith and so on, along with his interpretation of the various views of the
scholars presented. According to the research, this book of Zafar al-Amani is quite worthy to be
placed in line with the great works of the discipline of ‘ulum al-hadith which should be studied and
used as a reference by scholars and students. Abstrak Di sepanjang sejarah tradisi pengajian Islam, terdapat berbagai karya ‘ulum al-hadith yang telah
dihasilkan oleh ulama hadis. Karya-karya ini berbeda metodologi penulisannya antara satu karya
dengan satu karya yang lain. Kajian terhadap setiap karya serta latar belakang pengarangnya sangat
perlu dilakukan dengan tujuan untuk memperkenalkan salah seorang tokoh dalam disiplin ‘ulum al-
hadith tersebut bernama Syekh ‘Abd al-Hay al-Laknawi (w. 1304H) dengan tumpuan terhadap salah
satu karya monumentalnya, yaitu Zafar al-Amani fi Sharh Mukhtasar al-Jurjani. Kajian berbentuk
kualitatif ini menggunakan metode analisis isi kandungan sehingga dapat menguraikan secara
komprehensif metodologi penulisan al-Laknawi di dalam karya tersebut. Hasil kajian menyimpulkan
bahwa metodologi penulisan al-Laknawi ini cukup sistematis dan sarat dengan perbincangan ilmiah
mengenai masalah-masalah yang kompleks dalam ilmu hadis seperti isu hadis hasan, masalah
beramal dengan hadis daif, isu hadis mursal, isu hadis munkar dan sebagainya, berserta pentarjihan
beliau terhadap berbagai pandangan ulama yang disajikan. Kitab Zafar al-Amani ini menurut
pengkaji cukup layak untuk diletakkan sebaris dengan karya-karya agung disiplin ‘ulum al-hadith
lainnya yang wajar dipelajari dan dijadikan rujukan oleh para ilmuwan dan para penuntut ilmu. 1 MOHD. KHAFIDZ SORONI Taqaddumi: Journal of Quran and Hadith Studies
Vol. 1, No. 2
DOI Prefix 10.12928 Taqaddumi: Journal of Quran and Hadith Studies
Vol. 1, No. 2
DOI Prefix 10.12928 Taqaddumi: Journal of Quran and Hadith Studies
Vol. 1, No. 2
DOI Prefix 10.12928 Taqaddumi: Journal of Quran and Hadith Studies
Vol. 1, No. 2
DOI Prefix 10.12928 Pendahuluan Keperluan dan kepentingan ‘ulum al-hadith dalam tradisi pengajian Islam telah menarik
ramai ulama hadis mengarang karya khusus mengenainya. Terdapat pelbagai karya ‘ulum al-
hadith yang dikarang oleh ulama hadis di sepanjang zaman, sama ada berbentuk karya asli, syarah,
adaptasi atau gubahan nazam. Dalam wacana kesarjanaan, karya-karya ini perlu dibedakan
metodologi penulisannya agar dapat difahami secara lebih mapan, diklasifikasikan mengikut
peringkat dan dimanfaatkan kandungannya sesuai mengikut tahap pembelajaran. Kajian ini
bertujuan untuk memperkenalkan salah seorang tokoh ulama hadis dalam abad ke 14 Hijrah
bernama Syekh ‘Abd al-Hay bin ‘Abd al-Halim al-Laknawi serta sumbangannya dalam bidang
‘ulum al-hadith dengan tumpuan terhadap salah satu karya monumentalnya, iaitu Zafar al-Amani
fi Sharh Mukhtasar al-Jurjani. Setakat ini menurut hemat pengkaji, belum ditemukan penelitian
yang telah dilakukan terhadap kitab ini sebelumnya. Justru dalam kajian ini, pengkaji akan
mengupas khusus mengenai metodologi penulisan yang diguna pakai oleh beliau dalam
menyajikan materi perbahasan kitabnya itu. Bagaimanapun, perbincangan mengenainya secara
umum akan dibagikan kepada dua bagian iaitu; pertama, mengenai biografi Syekh ‘Abd al-Hay
al-Laknawi dan kedua, mengenai metodologi penulisan karya beliau yang menjadi fokus utama
kajian. Kajian berbentuk kualitatif ini menggunakan metode kajian kepustakaan bagi menelusuri
riwayat hidup dan sumbangan ilmiah al-Laknawi dalam dunia pengajian Islam, serta metode
analisis isi kandungan bagi mengenal pasti dan menghuraikan secara komprehensif metodologi
penulisan al-Laknawi di dalam kitabnya Zafar al-Amani. Biografi Syekh ‘Abd Al-Hay Al-Laknawi Nama penuh beliau ialah ‘Abd al-Hay bin ‘Abd al-Halim bin Amin Allah bin Akbar al-
Ansari al-Sihalawi al-Laknawi al-Hanafi. Nama beliau juga adakala ditulis dengan tambahan nama
'Muhammad' di awalnya sepertimana berikut; Muhammad ‘Abd al-Hay bin Muhammad ‘Abd al-
Halim bin Muhammad Amin Allah bin Muhammad Akbar al-Ansari al-Laknawi. Gelaran ‘al-
Ansari’ adalah nisbah kepada seorang sahabat Nabi SAW bernama Abu Ayyub al-Ansari RA
kerana nasab beliau sampai kepadanya. Nasab ini dijaga turun temurun oleh kaum keluarga beliau 1 al-Laknawi, al-Raf‘ wa al-Takmil fi al-Jarh wa al-Ta‘dil (Halab: Maktab al-Matbu‘at al-Islamiyyah, 1987), hlm
19; Waliy al Din al Nadwi al Imam ‘Abd al Hayy al Laknawi (Dimasyq: Dar al Qalam 1995) hlm 57 60 1 al-Laknawi, al-Raf‘ wa al-Takmil fi al-Jarh wa al-Ta‘dil (Halab: Maktab al-Matbu‘at al-Islamiyyah, 1987), hlm
19; Waliy al-Din al-Nadwi, al-Imam ‘Abd al-Hayy al-Laknawi (Dimasyq: Dar al-Qalam, 1995), hlm 57-60.
2 al-Laknawi, ibid. hlm 19. 19; Waliy al-Din al-Nadwi, al-Imam ‘Abd al-Hayy al-Laknawi (Dimasyq: Dar al-Qalam, 1995),
2 al-Laknawi, ibid. hlm 19. 4 al-Laknawi, op.cit. hlm 20-21. p
5 Waliy al-Din, op.cit. hlm 68-70. 3 Waliy al-Din, op.cit. hlm 68. 1 al-Laknawi, al-Raf‘ wa al-Takmil fi al-Jarh wa al-Ta‘dil (Halab: Maktab al-Matbu‘at al-Isla y
6 al-Laknawi, op.cit. hlm 21. MOHD. KHAFIDZ SORONI
3
1 al-Laknawi, al-Raf‘ wa al-Takmil fi al-Jarh wa al-Ta‘dil (Halab: Maktab al-Matbu‘at al-Islamiyyah, 1987), hlm
19; Waliy al-Din al-Nadwi, al-Imam ‘Abd al-Hayy al-Laknawi (Dimasyq: Dar al-Qalam, 1995), hlm 57-60.
2 al-Laknawi, ibid. hlm 19.
3 Waliy al-Din, op.cit. hlm 68.
4 al-Laknawi, op.cit. hlm 20-21.
5 Waliy al-Din, op.cit. hlm 68-70.
6 al-Laknawi, op.cit. hlm 21. MOHD. KHAFIDZ SORONI MOHD. KHAFIDZ SORONI 2 Taqaddumi: Journal of Quran and Hadith Studies
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DOI Prefix 10.12928
yang merupakan zuriat keturunan sahabat besar tersebut.1 Nama panggilan (kunyah) beliau pula
ialah Abu al-Hasanat yang diberikan kepada beliau oleh bapanya sendiri setelah beliau mencapai
usia baligh.2 Taqaddumi: Journal of Quran and Hadith Studies
Vol. 1, No. 2
DOI Prefix 10.12928 yang merupakan zuriat keturunan sahabat besar tersebut.1 Nama panggilan (kunyah) beliau pula
ialah Abu al-Hasanat yang diberikan kepada beliau oleh bapanya sendiri setelah beliau mencapai
usia baligh.2 yang merupakan zuriat keturunan sahabat besar tersebut.1 Nama panggilan (kunyah) beliau pula
ialah Abu al-Hasanat yang diberikan kepada beliau oleh bapanya sendiri setelah beliau mencapai
usia baligh.2 Beliau dilahirkan pada hari Selasa, 26 Zulkaedah tahun 1264H di sebuah tempat bernama
Banda. Bapa beliau merupakan seorang alim dan semasa al-Laknawi lahir, bapanya bertugas
sebagai seorang guru di Madrasah al-Nawwab Dhi al-Faqqar al-Dawlah di Banda, Uttar Pradesh,
India.3 al-Laknawi mula menghafal al-Quran ketika berusia 5 tahun. Gurunya pada masa ini
bernama Hafiz Qasim ‘Ali al-Laknawi. Kemudian beliau mengikut bapanya berpindah ke Jounpur
untuk mengajar di Madrasah al-Haj Imam Bakhsy al-Marhum. Di sana, al-Laknawi menyambung
hafalan al-Quran dengan seorang guru bernama Hafiz Ibrahim. Selain beliau juga berguru secara
tetap dengan bapanya sendiri. Beliau secara semula jadi dianugerahkan oleh Allah memiliki daya
ingatan yang kuat. Justru, ketika berusia 10 tahun, beliau telah berjaya menghafal keseluruhan al-
Quran. Malah, beliau turut menjadi imam bagi solat tarawih menurut adat kebiasaan masyarakat
pada masa itu.4 Sejak kecil, bapanya amat mengambil berat terhadap pendidikannya. Beliau banyak
membimbing serta mengajarnya berbagai ilmu alat dan asas. Kemudian al-Laknawi mula
mempelajari pelbagai bidang ilmu, seperti saraf, nahu, ma‘ani, bayan, mantik, hikmah, tibb, fiqh,
usul al-fiqh, ilmu kalam, hadis, tafsir dan sebagainya hingga dapat menyelesaikan kesemuanya
dalam usia 17 tahun. Guru dan pembimbing utama di sepanjang pengajian beliau ini adalah
bapanya sendiri, Syekh ‘Abd al-Halim.5 Selepas kewafatan bapanya, beliau mempelajari ilmu riyadiyyah daripada datuk
saudaranya, Mawlana Ni‘mat Allah ibn Nur Allah al-Laknawi dan ilmu hisab daripada murid
bapanya, al-Mulwi Khadim Husayn al-Muzaffar puri al-‘Azim abadi.6 3 Taqaddumi: Journal of Quran and Hadith Studies
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DOI Prefix 10.12928 Beliau turut berpeluang dapat pergi menunaikan haji sebanyak dua kali. Haji kali pertama
bersama bapanya pada tahun 1279H ketika beliau berusia 15 tahun. Haji kali kedua pula pada
tahun 1292H ketika beliau berusia 28 tahun. 7 Waliy al-Din, op.cit. hlm 74-76. 10 Ibid. hlm 94-111. y
8 Ibid. hlm 76-77. 9 Ibid. hlm 79-80. MOHD. KHAFIDZ SORONI 8 Ibid. hlm 76-77. MOHD. KHAFIDZ SORONI Beliau turut mengambil manfaat ilmu daripada para
alim ulama yang berada di Makkah dan Madinah pada masa itu.7 Setelah tamat pengajian, beliau telah menumpukan masanya untuk mengajar, mendidik,
menelaah dan menulis di tempat asalnya, Lucknow. Demikian adalah rutin kehidupan beliau
sehinggalah ke akhir hayatnya. Beliau pernah ditawarkan jawatan sebagai pengarah Mahkamah
Tinggi Hyderabad untuk menggantikan tempat bapanya. Namun, beliau telah menolak tawaran
tersebut. Beliau sudah berpuas hati dengan elaun sebanyak 250 Rupee sebulan serta dapat memberi
tumpuan terhadap kerja-kerja mengajar, menulis dan berkhidmat kepada masyarakat.8 Dalam kesibukan menyumbang jasa bakti terhadap masyarakat dan umat, beliau mula jatuh
sakit pada pertengahan tahun 1303H dan kerap jatuh pengsan. Penyakit beliau berterusan
sehinggalah akhirnya beliau meninggal dunia pada 30 Rabiulawwal tahun 1304H disebabkan
serangan jantung. Beliau telah meninggal dunia dalam usia yang agak muda, iaitu kira-kira 39
tahun. Menurut al-Bandawi dalam Kanz al-Barakat, seramai kira-kira 20,000 orang telah datang
untuk menyembahyangi jenazahnya.9 Sebagaimana yang telah disinggung sebelum ini, jumlah guru-guru al-Laknawi agak
sedikit dan tidak begitu ramai. Secara ringkas, guru-guru utama beliau ialah:10 1. Bapanya sendiri, al-‘Allamah ‘Abd al-Halim bin Amin Allah al-Laknawi. 2. al-Mufti Syekh Ni‘mat Allah ibn Nur Allah al-Laknawi. 3. al-Mulwi Khadim Husayn al-Muzaffar puri al-‘Azim abadi. Ketiga-tiga mereka merupakan guru utama yang mengajar ilmu-ilmu naqli dan aqli kepada
beliau, terutama sekali bapanya, al-‘Allamah ‘Abd al-Halim. Justru, al-Laknawi begitu
menyanjung dan memandang tinggi kedudukan bapanya di mana beliau menulis: “Bapaku,
Mawlana Muhammad ‘Abd al-Halim ialah pengarang karya-karya yang masyhur dan empunya
limpahan ilmu yang banyak, yang berasa bangga dengan kewujudannya para cendekiawan India,
Arab dan juga Ajam, yang bersandar dan merujuk kepadanya para pemuka alam, yang mengatasi 4 Taqaddumi: Journal of Quran and Hadith Studies
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DOI Prefix 10.12928 Taqaddumi: Journal of Quran and Hadith Studies
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DOI Prefix 10.12928 para rakan dan para pendahulunya dari segi pengajaran dan penulisan yang baik, yang bijaksana
lagi mendahului ahli zamannya dan orang sebelumnya dari segi penerimaan hasil karyanya, yang
wafat pada tahun 1285H”. Malah, beliau telah mengabadikan biografi bapanya di dalam sebuah
kitab berjudul: Hasrah al-‘Alam bi Wafah Marji‘ al-‘Alam, yang bermaksud “Kerugian dunia
dengan kewafatan pakar rujuk dunia”.11 Ketokohan bapanya dapat dilihat pada karya-karya yang dihasilkan olehnya. 11 al-Laknawi, op.cit. hlm 19-20. yy
Dar Ibn Hazm, 1999), hlm 1005. , p
12 al-Hasani, ‘Abd al-Hayy al-Nadwi, Nuzhah al-Khawatir wa Bahjah al-Masami‘ wa al-Nawazir, Jilid 7 (Beirut:
Dar Ibn Hazm, 1999), hlm 1005. )
13 Waliy al-Din, op.cit. hlm 120-145. MOHD. KHAFIDZ SORONI Dar Ibn Hazm, 1999), hlm 1005.
13
li
l
i
hl
120 145 al Laknawi, op.cit. hlm 19 20.
12 al-Hasani, ‘Abd al-Hayy al-Nadwi, Nuzhah al-Khawatir wa Bahjah al-Masami‘ wa al-Nawazir, Jilid 7 (Beirut:
Dar Ibn Hazm, 1999), hlm 1005.
13 Waliy al-Din, op.cit. hlm 120-145. 14 al-Laknawi, op.cit. hlm 22-27; Waliy al-Din, ibid. hlm 165-298; Salah Muhammad Abu al-Haj, al-Manhaj al-
Fiqhi li al-Imam al-Laknawi (Amman: Dar al-Nafa’is, 2002), hlm 93-133. 16. Dan ramai lagi. al-Laknawi merupakan seorang ulama yang prolifik dan telah menghasilkan banyak
karangan yang bermutu dan bernilai. Ini kerana sejak remaja, beliau sudah gemar menulis dan
mengarang kitab. Beliau telah menyusun banyak karya dalam pelbagai bidang ilmu seperti akidah,
hadis, usul al-fiqh, fiqh, fara’id, fatwa, sejarah, sirah, biografi, mantik, nahu, saraf dan lain-lain. Kebanyakan karangannya di dalam bahasa Arab. Waliy al-Din al-Nadwi (1995) menyatakan
bahawa karya beliau berjumlah sebanyak 120 buah karya. Namun, Salah Muhammad Abu al-Haj
(2002) telah menambah sejumlah karya lain, termasuk yang belum disempurnakan, menjadikan
kesemuanya berjumlah sebanyak 129 buah karya. Antara karya-karya beliau yang pengkaji telah bagikan menurut bidang-bidang ilmu adalah
seperti berikut:14 MOHD. KHAFIDZ SORONI ‘Abd al-Baqi bin ‘Ali Muhammad al-Ansari. 12. ‘Abd al-Halim bin Isma‘il al-Madrasi. 13. ‘Abd al-Halim bin Tafaddal Husayn al-‘Abbasi. 14. ‘Abd al-Hamid bin ‘Abd al-Karim al-Farahi. 15. ‘Abd al-‘Aziz bin ‘Abd al-Rahim al-Ansari. 16. Dan ramai lagi. MOHD. KHAFIDZ SORONI Antaranya
ialah al-Tahqiqat al-Mardiyyah li Hall Hashiyah al-Sayyid al-Zahid ‘ala al-Risalah al-Qutbiyyah,
al-Qawl al-Aslam li Hall Sharh al-Sulam li Mulla Hasan, Hall al-Ma‘aqid fi Sharh al-‘Aqa’id li
al-Jalal al-Dawwani, Mu‘in al-Gha’isin fi Radd al-Mughalitin, al-Idahat li Mabhath al-
Mukhtalitat, Kashif al-Zulmah fi Bayan Aqsam al-Hikmah, Nazm al-Durar fi Silk Shaqq al-Qamar,
Nur al-Iman fi Athar Habib al-Rahman, Qamar al-Aqmar Hashiyah Nur al-Anwar, Ta‘liqat ‘ala
al-Hidayah (al-Si‘ayah li ‘Atshan al-Hidayah) dan sebagainya.12 Ketokohan bapanya dapat dilihat pada karya-karya yang dihasilkan olehnya. Antaranya
ialah al-Tahqiqat al-Mardiyyah li Hall Hashiyah al-Sayyid al-Zahid ‘ala al-Risalah al-Qutbiyyah,
al-Qawl al-Aslam li Hall Sharh al-Sulam li Mulla Hasan, Hall al-Ma‘aqid fi Sharh al-‘Aqa’id li
al-Jalal al-Dawwani, Mu‘in al-Gha’isin fi Radd al-Mughalitin, al-Idahat li Mabhath al-
Mukhtalitat, Kashif al-Zulmah fi Bayan Aqsam al-Hikmah, Nazm al-Durar fi Silk Shaqq al-Qamar,
Nur al-Iman fi Athar Habib al-Rahman, Qamar al-Aqmar Hashiyah Nur al-Anwar, Ta‘liqat ‘ala
al-Hidayah (al-Si‘ayah li ‘Atshan al-Hidayah) dan sebagainya.12 Manakala guru-guru al-Laknawi yang pernah memberikan ijazah sanad kepada beliau
ialah: Manakala guru-guru al-Laknawi yang pernah memberikan ijazah sanad kepada beliau
ialah: 4. al-‘Allamah al-Sayyid Ahmad ibn Zayni Dahlan al-Makki, mufti ulama Syafii dan tenaga
pengajar di Masjid al-Haram, Makkah. 5. Syekh ‘Ali bin Yusuf al-Hariri al-Madani, Syaykh al-Dala’il. 6. al-Muhaddith Syekh ‘Abd al-Ghani ibn Abi Sa‘id al-Dihlawi. 7. Syekh Muhammad ibn ‘Abd Allah ibn Humayd al-Hanbali al-‘Amiri al-Najdi, mufti ulama
Hanbali di Makkah. 8. Syekh Muhammad ibn Muhammad al-Gharb al-Syafi‘i. 8. Syekh Muhammad ibn Muhammad al-Gharb al-Syafi‘i. al-Laknawi telah melahirkan ramai anak murid yang turut menuruti jejak langkahnya. Antara murid-murid beliau ialah;13 1. Idris bin ‘Abd al-‘Aliy al-Hanafi. 2. Ifham Allah bin In‘am Allah al-Ansari al-Laknawi. 3. Anwar Allah bin Syuja‘ al-Din al-‘Umari. 4. al-Sayyid Amin bin Taha bin Zayn al-Hasani. 5 Taqaddumi: Journal of Quran and Hadith Studies
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DOI Prefix 10.12928
5. Badi‘ al-Zaman bin Masih al-Zaman al-Laknawi. 6. Hafiz Allah bin Din ‘Ali al-Bandawi. 7. Syir ‘Ali bin Rahm ‘Ali al-Husayni. 8. Zuhur Islam bin Hasan ‘Ali al-Kazimi. 9. Zahir Ahsan bin Subhan ‘Ali al-Nimwi. 10. ‘Abd al-Bari bin Talattuf Husayn al-Bakri. 11. ‘Abd al-Baqi bin ‘Ali Muhammad al-Ansari. 12. ‘Abd al-Halim bin Isma‘il al-Madrasi. 13. ‘Abd al-Halim bin Tafaddal Husayn al-‘Abbasi. 14. ‘Abd al-Hamid bin ‘Abd al-Karim al-Farahi. 15. ‘Abd al-‘Aziz bin ‘Abd al-Rahim al-Ansari. 16 Dan ramai lagi 8. Zuhur Islam bin Hasan ‘Ali al-Kazimi. 9. Zahir Ahsan bin Subhan ‘Ali al-Nimwi. 10. ‘Abd al-Bari bin Talattuf Husayn al-Bakri. 11. MOHD. KHAFIDZ SORONI Ilmu saraf: 1. Imtihan al-Talabah fi al-Siyagh al-Mushkilah, yang merupakan kitab pertama susunan
beliau. 1. Imtihan al-Talabah fi al-Siyagh al-Mushkilah, yang merupakan kitab pertama susunan
beliau. 2. al-Tibyan fi Sharh al-Mizan. 2. al-Tibyan fi Sharh al-Mizan. Ilmu akidah: 3. Hashiyah ‘ala Hawashi al-Khayali ‘ala Sharh al-‘Aqa’id al-Nasafiyyah. Ilmu hadis: 7 Taqaddumi: Journal of Quran and Hadith Studies
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4. Hashiyah ‘ala Sharh al-Mawaqif.
5. Hashiyah ‘ala Sharh ‘Aqa’id al-Nasafi.
Ilmu mantik:
6. ‘Alam al-Huda.
7. Hall al-Mughlaq fi Bahth al-Majhul al-Mutlaq.
8. al-Kalam al-Matin fi Tahrir al-Barahin.
Ilmu sejarah atau tarikh:
9. Hasrah al-‘Alam bi Wafah Marji‘ al-‘Alam
10. al-Fawa’id al-Bahiyyah fi Tarajim al-Hanafiyyah
11. Khayr al-‘Amal bi Dhikr Tarajim ‘Ulama’ Farangi Mahal
12. al-Nasib al-Awfar fi Tarajim ‘Ulama’ al-Mi’ah al-Thalithah ‘Ashar.
13. Inba’ al-Khillan bi Anba’ ‘Ulama’ Hindustan
14. Ibraz al-Ghayy al-Dafi‘ fi Shifa’ al-‘Iyy
15. Tarb al-Amathil bi Tarajim al-Afadil
16. Farhah al-Mudarrisin bi Asma’ al-Mu’allafat wa al-Mu’allifin
Ilmu fiqh:
17. Iqamah al-Hujjah ‘ala Ann al-Ikthar min al-Ta‘abbud Lays bi Bid‘ah,
18. Sibahah al-Fikr fi al-Jahr bi al-Dhikr,
19. Tuhfah al-Akhyar bi Ihya’ Sunnah Sayyid al-Abrar,
20. Nukhbah al-Anzar ‘ala Tuhfah al-Akhyar (yang kesemua kitab ini ditahkik oleh Syekh
‘Abd al-Fattah Abu Ghuddah)
21. Majmu‘ah al-Fatawa
22. Naf‘ al-Mufti wa al-Sa’il bi Jam‘ Mutafarriqat al-Masa’il
23. al-Si‘ayah fi Kashf ma fi Sharh al-Wiqayah
24. al-Nafi‘ al-Kabir li Man Yutali‘ al-Jami‘ al-Saghir
25. Akam al-Nafa’is fi Ada’ al-Adhkar bi Lisan Faris
26. al-Kalam al-Mubram fi Naqd al-Qawl al-Muhaqqaq al-Muhkam
27 al-Fulk al-Mashhun fi ma Yata‘allaq bi Intifa‘ al-Murtahin bi al-Ruhun 4. Hashiyah ‘ala Sharh al-Mawaqif. 2. al-Tibyan fi Sharh al-Mizan.
Ilmu akidah: 3. Hashiyah ‘ala Hawashi al-Khayali ‘ala Sharh al-‘Aqa’id al-Nasafiyyah. MOHD. KHAFIDZ SORONI
6
14 al-Laknawi, op.cit. hlm 22-27; Waliy al-Din, ibid. hlm 165-298; Salah Muhammad Abu al-Haj, al-Manhaj al-
Fiqhi li al-Imam al-Laknawi (Amman: Dar al-Nafa’is, 2002), hlm 93-133. 6 Taqaddumi: Journal of Quran and Hadith Studies
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DOI Prefix 10.12928
4. Hashiyah ‘ala Sharh al-Mawaqif. 5. Hashiyah ‘ala Sharh ‘Aqa’id al-Nasafi. Ilmu mantik:
6. ‘Alam al-Huda. 7. Hall al-Mughlaq fi Bahth al-Majhul al-Mutlaq. 8. al-Kalam al-Matin fi Tahrir al-Barahin. Ilmu sejarah atau tarikh:
9. Hasrah al-‘Alam bi Wafah Marji‘ al-‘Alam
10. al-Fawa’id al-Bahiyyah fi Tarajim al-Hanafiyyah
11. Khayr al-‘Amal bi Dhikr Tarajim ‘Ulama’ Farangi Mahal
12. al-Nasib al-Awfar fi Tarajim ‘Ulama’ al-Mi’ah al-Thalithah ‘Ashar. 13. Inba’ al-Khillan bi Anba’ ‘Ulama’ Hindustan
14. Ibraz al-Ghayy al-Dafi‘ fi Shifa’ al-‘Iyy
15. Tarb al-Amathil bi Tarajim al-Afadil
16. Farhah al-Mudarrisin bi Asma’ al-Mu’allafat wa al-Mu’allifin
Ilmu fiqh:
17. Iqamah al-Hujjah ‘ala Ann al-Ikthar min al-Ta‘abbud Lays bi Bid‘ah,
18. Sibahah al-Fikr fi al-Jahr bi al-Dhikr,
19. Tuhfah al-Akhyar bi Ihya’ Sunnah Sayyid al-Abrar,
20. Nukhbah al-Anzar ‘ala Tuhfah al-Akhyar (yang kesemua kitab ini ditahkik oleh Syekh
‘Abd al-Fattah Abu Ghuddah)
21. Majmu‘ah al-Fatawa
22. Naf‘ al-Mufti wa al-Sa’il bi Jam‘ Mutafarriqat al-Masa’il
23. al-Si‘ayah fi Kashf ma fi Sharh al-Wiqayah
24. al-Nafi‘ al-Kabir li Man Yutali‘ al-Jami‘ al-Saghir
25. Akam al-Nafa’is fi Ada’ al-Adhkar bi Lisan Faris
26. al-Kalam al-Mubram fi Naqd al-Qawl al-Muhaqqaq al-Muhkam
27. al-Fulk al-Mashhun fi ma Yata‘allaq bi Intifa‘ al-Murtahin bi al-Ruhun
28. Hadiyah al-Abrar fi Subhah al-Adhkar. Ilmu hadis: y
16 al-Laknawi, op.cit. hlm 28. MOHD. KHAFIDZ SORONI Taqaddumi: Journal of Quran and Hadith Studies
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DOI Prefix 10.12928 29. al-Raf‘ wa al-Takmil fi al-Jarh wa al-Ta‘dil, 30. al-Ajwibah al-Fadilah li al-As’ilah al-‘Asyarah al-Kamilah,
31. Zafar al-Amani bi Sharh Mukhtasar al-Sayyid al-Syarif al-Jurjani (yang ketiga-tiga kitab
ini ditahkik oleh Syekh ‘Abd al-Fattah Abu Ghuddah)
32. al-Athar al-Marfu‘ah fi al-Akhbar al-Mawdu‘ah
33. al-Ta‘liq al-Mumajjad ‘ala Muwatta’ al-Imam Muhammad. Ilmu akhlak: 30. al Ajwibah al Fadilah li al As ilah al Asyarah al Kamilah,
31. Zafar al-Amani bi Sharh Mukhtasar al-Sayyid al-Syarif al-Jurjani (yang ketiga-tiga kitab
ini ditahkik oleh Syekh ‘Abd al-Fattah Abu Ghuddah)
32. al-Athar al-Marfu‘ah fi al-Akhbar al-Mawdu‘ah
33. al-Ta‘liq al-Mumajjad ‘ala Muwatta’ al-Imam Muhammad. Ilmu akhlak: 33. al-Ta‘liq al-Mumajjad ‘ala Muwatta’ al-Imam Muhammad 33. al-Ta‘liq al-Mumajjad ‘ala Muwatta’ al-Imam Muhammad. Ilmu akhlak: 15 Waliy al-Din, ibid. hlm 165-298. MOHD. KHAFIDZ SORONI Ilmu akhlak: 34. Zajr al-Shubban wa al-Shibah ‘an Irtikab al-Ghibah
35. ‘Umdah al-Nasa’ih bi Tark al-Qaba’ih 34. Zajr al-Shubban wa al-Shibah ‘an Irtikab al-Ghibah 35. ‘Umdah al-Nasa’ih bi Tark al-Qaba’ih Waliy al-Din al-Nadwi (1995) telah menghuraikan secara terperinci setiap satu karya
beliau ini di dalam bukunya yang khusus mengenai biografi Imam al-Laknawi.15 al-Laknawi jelas berpegang dengan mazhab Hanafi sebagaimana yang tertera pada gelaran
‘al-Hanafi’ yang sering beliau letakkan pada namanya. Malah, karya-karya fiqhnya menunjukkan
kepada mazhab pegangannya ini tanpa ragu-ragu lagi. Namun, beliau bukanlah seorang yang
sangat taksub kepada mazhabnya kerana beliau berpegang dengan prinsip mengutamakan dalil
yang kuat di dalam setiap pandangannya. Beliau pernah menyatakan: ومن مِنَحِ ه تعالى أنه رزقني التوجه إلى فن الحديث وفقهه، وال أعتمد على مسألة ما لم يوجد لها أصل من آية أو حديث. وما كان
خالف الحديث الصحيح الصريح أتركه، وأظن المجتهد فيه معذوراً، بل مأجوراً، ولكني لستُ ممن يشوش العوام الذين هم
كاألنعام، بل أتكلم بالناس على قدر عقولهم. ومن مِنَحِ ه تعالى أنه رزقني التوجه إلى فن الحديث وفقهه، وال أعتمد على مسألة ما لم يوجد لها أصل من آية أو حديث. وما كان
خالف الحديث الصحيح الصريح أتركه، وأظن المجتهد فيه معذوراً، بل مأجوراً، ولكني لستُ ممن يشوش العوام الذين هم
كاألنعام، بل أتكلم بالناس على قدر عقولهم. Terjemahan: “Dan antara kurniaan Allah Taala kepadaku ialah Dia memberikan aku
kecenderungan terhadap ilmu hadis dan fiqh hadis, dan aku tidak akan berpegang pada sesuatu
masalah selagi mana tidak ditemui baginya dalil asal daripada ayat al-Quran mahu pun hadis. Dan
apa yang bercanggah dengan hadis yang sahih lagi sarih, aku akan tinggalkannya, dan aku
menganggap mujtahid yang mengeluarkan hukum tersebut adalah ma‘dhur (dimaafkan), bahkan
diberikan pahala. Tetapi, aku bukanlah dari kalangan orang yang gemar mengganggu orang awam,
yang mana mereka itu adalah seperti haiwan, bahkan aku berbicara dengan orang ramai menurut
kadar akal fikiran mereka”.16 8 Taqaddumi: Journal of Quran and Hadith Studies
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DOI Prefix 10.12928 Meskipun al-Laknawi seorang yang sangat alim dalam ilmu hadis dan fiqh serta mampu
berijtihad mengeluarkan hukum sendiri, walaupun ijtihadnya kadangkala bercanggah dengan
mazhab Hanafi, namun beliau sama sekali tidak mengganggu dan mengelirukan pegangan yang
sudah sedia ada di kalangan masyarakatnya pada masa itu. 17 al-Hasani, op.cit. Jilid 8, hlm 1268. p
18 Waliy al-Din, op.cit. hlm 84. MOHD. KHAFIDZ SORONI Ilmu akhlak: Ia merupakan suatu tindakan yang betul
dan langkah yang tepat diambil oleh beliau dalam menjaga kepentingan awam lebih daripada
kepentingan peribadi. Di samping beliau sedar bahawa pandangan yang mereka pegang juga masih
di dalam ruang lingkup ijtihad para ulama. Ramai ulama yang memuji dan menyanjung sahsiah dan ketokohan al-Laknawi. Antaranya
adalah daripada tokoh-tokoh berikut: i- al-‘Allamah al-Sayyid ‘Abd al-Hayy al-Hasani al-Nadwi (w. 1341H) di dalam kitabnya Nuzhah
al-Khawatir wa Bahjah al-Masami‘ wa al-Nawazir, berkata: “Seorang yang bijak lagi cerdik,
tajam ingatan, menjaga kehormatan, bersifat lemah lembut, seorang khatib yang fasih, yang melaut
dalam pelbagai ilmu ma‘qul dan manqul, mengetahui perkara-perkara syarak yang halus dan sulit,
menguasai pelbagai bidang ilmu, berhati-hati dalam menukilkan hukum hakam dan memperhalusi
masalah-masalah, menjadi tokoh tunggal di India dengan ilmu fatwa, hingga para kafilah berjalan
dengan menyebarkan namanya, sehingga para ulama setiap iklim turut menunding jari pada
kehebatannya. Beliau mempunyai kekuatan lengkap, kemampuan sempurna, kelebihan penuh
serta cakupan menyeluruh di dalam bab usul dan furuk. Juga mempunyai seni kemahiran dalam
penyampaian ilmu yang baik, yang tidak mampu ditandingi oleh orang lain... Walhasil, beliau
merupakan antara keajaiban zaman dan juga antara keindahan benua India. Ucapan pujian
terhadapnya merupakan satu kata sepakat dan pengiktirafan terhadap wibawanya bukanlah lagi
suatu perselisihan.”17 ii- al-‘Allamah al-Sayyid Siddiq Hasan Khan al-Qinnawji (w. 1307H) yang meskipun berbeza
pandangan dengan al-Laknawi dalam beberapa isu, namun ketika beliau mendengar berita
kewafatannya, beliau telah mengalirkan air mata, mengucapkan doa dan memohon rahmat
kepadanya serta berkata: “Pada hari ini telah terbenamnya matahari ilmu”.18 iii- al-‘Allamah Syekh Ibrahim bin ‘Uthman al-Samannudi al-Misri (w. selepas 1326H) dalam
kitabnya Sa‘adah al-Darayn kata beliau: “Tokoh sezaman kami dan salah seorang golongan mulia 9 Taqaddumi: Journal of Quran and Hadith Studies
Vol. 1, No. 2
DOI Prefix 10.12928 Taqaddumi: Journal of Quran and Hadith Studies
Vol. 1, No. 2
DOI Prefix 10.12928 zaman kami di India, mawlana, ‘allamah zaman ini, matahari ahli ‘irfan, yang soleh kamil dan
cendekiawan mulia, pengarang banyak karya-karya bermanfaat dan kitab-kitab susunan yang
berfaedah, yang wafat pada tahun 1304 H رحمه هللا تعالى, sedang beliau adalah ‘allamah dunia”.19
iv- al-‘Allamah al-Sayyid ‘Abd al-Hayy bin ‘Abd al-Kabir al-Kattani (w. Ilmu akhlak: 1382H) dalam Fihris al-
Faharis kata beliau: “Penutup ulama India, yang paling banyak menyusun karya antara kalangan
mereka, serta yang paling sempurna kupasan, telaah, seimbang dan sederhananya di antara
mereka… Beliau adalah pemilik hemah yang tidak mengenal rasa jemu, serta empunya
penumpuan jitu dalam mencatat, mengumpul dan menelaah yang tidak disentuh rasa penat lelah,
di samping sifat kebijaksanaan dan kesejahteraan akal fikirannya”.20 Terdapat banyak lagi pujian dan sanjungan para ulama dan ahli ilmu terhadap beliau yang
membuktikan ketokohan dan kedudukan tinggi beliau di sisi mereka. f
23 Seperti cetakan Dar al-Hadith, Kaherah pada tahun 1405H yang ditahkik oleh ‘Abd al-Ghaffar Sulayman al-
Bandari dengan judul: al-Dibaj al-Mudhahhab. 8.
l-Laknawi, Zafar al-Amani fi Mukhtasar al-Jurjani (Dubai: Dar al-Qalam, 1995), hlm 17-18. , p
20 al-Kattani, ‘Abd al-Hayy bin ‘Abd al-Kabir, Fihris al-Faharis, Jilid 2 (Beirut: Dar al-Gharb al-Islami, 1982),
hlm 728.
21 l L k
i Z f
l A
i fi M kht
l J
j
i (D b i D
l Q l
1995) hl
17 18 19 al-Laknawi, op.cit. hlm 39. eperti cetakan Dar al-Rasyid, Riyadh pada tahun 1407H yang ditahkik oleh ‘Ali Zuwayn dengan judu
al-Hadith. p
l-Kattani, ‘Abd al-Hayy bin ‘Abd al-Kabir, Fihris al-Faharis, Jilid 2 (Beirut: Dar al-Gharb al-Isla
8. MOHD. KHAFIDZ SORONI
10
19 al-Laknawi, op.cit. hlm 39.
20 al-Kattani, ‘Abd al-Hayy bin ‘Abd al-Kabir, Fihris al-Faharis, Jilid 2 (Beirut: Dar al-Gharb al-Islami, 1982),
hlm 728.
21 al-Laknawi, Zafar al-Amani fi Mukhtasar al-Jurjani (Dubai: Dar al-Qalam, 1995), hlm 17-18.
22 Seperti cetakan Dar al-Rasyid, Riyadh pada tahun 1407H yang ditahkik oleh ‘Ali Zuwayn dengan judul: Risalah
fi Usul al-Hadith.
23 Seperti cetakan Dar al-Hadith, Kaherah pada tahun 1405H yang ditahkik oleh ‘Abd al-Ghaffar Sulayman al-
Bandari dengan judul: al-Dibaj al-Mudhahhab. MOHD. KHAFIDZ SORONI
24 al-Laknawi, op.cit. hlm 5-6. Metodologi Penulisan Kitab Zafar al-Amani Sebagaimana yang jelas dalam mukadimah kitab, al-Laknawi memberikan judul kitabnya
ini: Zafar al-Amani fi Mukhtasar al-Jurjani. Namun di tempat lain, beliau memberikannya judul:
Zafar al-Amani bi Sharh al-Mukhtasar al-Mansub ila al-Jurjani dengan tambahan kalimah ‘al-
Mansub ila’ (yang disandarkan kepada). Namun pentahkik kitab, Syekh ‘Abd al-Fattah Abu
Ghuddah telah mencetaknya dengan judul: Zafar al-Amani bi Sharh Mukhtasar al-Sayyid al-Sharif
al-Jurjani tanpa kalimah ‘al-Mansub ila’ kerana beliau berpendapat ianya lebih tepat kerana kitab
Mukhtasar sememangnya karya al-Jurjani.21 Kitab al-Laknawi ini merupakan syarah terhadap Mukhtasar, sebuah kitab ‘ulum al-hadith
karya al-‘Allamah al-Sayyid al-Syarif ‘Ali bin Muhammad bin ‘Ali al-Husayni al-Jurjani al-Hanafi
(w. 816H). Judul kitab al-Jurjani ini berbeza antara satu versi dengan satu versi yang lain. Antara
judulnya; al-Mukhtasar fi ‘Ilm Mustalah al-Hadith atau Mukhtasar fi ‘Ulum al-Hadith atau
Risalah fi Usul al-Hadith22 atau juga disebut dengan judul al-Dibaj al-Mudhahhab.23 Oleh kerana
kemasyhurannya, kitab al-Jurjani ini juga mempunyai banyak versi cetakan di pelbagai negara. 10 MOHD. KHAFIDZ SORONI Taqaddumi: Journal of Quran and Hadith Studies
Vol. 1, No. 2
DOI Prefix 10.12928 Taqaddumi: Journal of Quran and Hadith Studies
Vol. 1, No. 2
DOI Prefix 10.12928 Secara ringkas, kitab al-Jurjani sebenarnya merupakan ringkasan daripada dua buah karya Secara ringkas, kitab al-Jurjani sebenarnya merupakan ringkasan daripada dua buah karya
susunan Imam al-Tibi (w. 743H), iaitu kitab al-Khulasah fi Usul al-Hadith dan mukadimah kitab
al-Kashif ‘an Haqa’iq al-Sunan. Imam al-Tibi pula meringkaskannya daripada kitab al-Taqrib
oleh Imam al-Nawawi (w. 676H) dan kitab al-Manhal al-Rawi fi Mukhtasar ‘Ulum al-Hadith al-
Nabawi oleh Imam Badr al-Din ibn Jama‘ah (w. 733H), yang mana kedua-duanya merupakan
ringkasan daripada kitab al-Muqaddimah oleh Imam Ibn al-Salah (w. 643H).24 Gambar 1: Susur galur asal bagi kitab Zafar al-Amani Gambar 1: Susur galur asal bagi kitab Zafar al-Amani 11 Taqaddumi: Journal of Quran and Hadith Studies
Vol. 1, No. 2
DOI Prefix 10.12928 Taqaddumi: Journal of Quran and Hadith Studies
Vol. 1, No. 2
DOI Prefix 10.12928 Kitab Mukhtasar al-Jurjani menurut al-Laknawi merupakan sebuah kitab usul al-hadith Kitab Mukhtasar al-Jurjani menurut al-Laknawi merupakan sebuah kitab usul al-hadith
ringkas yang terbaik sehinggakan ia menjadi masyhur dan tersebar dengan luas. Beliau melihat di
zamannya, orang turut memberi tumpuan dalam mempelajari kitab ini dan juga mengajarkannya. Namun begitu, beliau melihat tidak ada satu kitab syarah pun yang dapat menerangkan dan
menghuraikan dengan lebih lanjut ibarat-ibarat kitab ini, sama ada ibaratnya yang jelas mahu pun
ibaratnya yang samar. 25 al-Laknawi op.cit. hlm 28-29. p
26 Ibid. hlm 39. MOHD. KHAFIDZ SORONI p
26 Ibid. hlm 39. 27 Ibid. hlm 326. Metodologi Penulisan Kitab Zafar al-Amani Lantas, beliau pun mengambil inisiatif dan tanggungjawab untuk memenuhi
keperluan tersebut.25 Selain disebabkan kitab Mukhtasar al-Jurjani yang disusun dalam bentuk yang amat
ringkas, pengkaji berpendapat ada faktor dorongan yang lain bagi al-Laknawi menyusun
syarahnya. Iaitu kitab Mukhtasar al-Jurjani ini tidak disusun untuk menonjolkan mazhab Hanafi,
sedangkan pengarangnya bermazhab Hanafi dan kebanyakan orang yang mempelajarinya dan juga
mengajarkannya di benua India pada waktu itu adalah bermazhab Hanafi. Malah dalam sesetengah
tempat, al-Jurjani seperti hanya mengikut dan menyokong mazhab Syafii seperti membagikan
khabar hanya kepada khabar mutawatir dan khabar ahad sahaja, tanpa mengeluarkan khabar
masyhur secara berasingan daripada khabar ahad,26 menyatakan bahawa khabar mawquf bukanlah
hujah menurut pandangan yang paling sahih27 dan meletakkan hadis mursal dalam kategori hadis
daif tanpa sebarang perincian dan huraian lanjut tentang kehujahannya.28 Bahkan, terdapat juga
sedikit kesalahan di dalamnya, seperti memasukkan khabar mawquf dan maqtu‘ ke dalam kategori
hadis daif, sedangkan yang menentukan status kedua-duanya ialah sanad. Maka secara zahir, jika
sahih sanadnya, maka sahihlah khabar mawquf dan maqtu‘ tersebut, dan jika daif sanadnya, maka
daiflah khabar mawquf dan maqtu‘ tersebut.29 Justru dengan kedudukan kitab seperti ini, al-Laknawi menyedari bahawa dengan
mensyarahkannya akan membantu para pelajar mahu pun pengajar lebih memahami dan
mendalami kandungan kitab Mukhtasar al-Jurjani yang ringkas itu. Di samping lebih meraikan
usul mazhab Hanafi berkaitan hadis di dalamnya. Justru pengkaji berpendapat, kemungkinan di
atas faktor-faktor ini semua yang mendorong al-Laknawi bertekad untuk mensyarahkannya. 28 Ibid. hlm 343. 29 Ibid. hlm 325. 12 Taqaddumi: Journal of Quran and Hadith Studies
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DOI Prefix 10.12928 Taqaddumi: Journal of Quran and Hadith Studies
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DOI Prefix 10.12928 Taqaddumi: Journal of Quran and Hadith Studies
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DOI Prefix 10.12928 al-Laknawi mula menyusun syarahnya pada tahun 1285H ketika beliau menetap di
Hyderabad. Beliau telah menyiapkan separuh daripadanya, iaitu sehingga perbahasan hadis al-
musalsal. Kemudian penyusunannya terhenti lama kerana berbagai kesibukan dan faktor lain. Beliau tidak menyudahkannya sehinggalah semakin banyak dorongan untuk beliau
menyiapkannya yang datang daripada kalangan para pelajar dan para ilmuwan. 30 Ibid. hlm 524. MOHD. KHAFIDZ SORONI MOHD. KHAFIDZ SORONI
30 Ibid. hlm 524. Metodologi Penulisan Kitab Zafar al-Amani Oleh kerana
desakan yang berterusan, beliau akhirnya menyambut saranan mereka hinggalah beliau berjaya
menyiapkan syarah tersebut pada hari Selasa 12 Safar tahun 1304H, iaitu kira-kira sebulan lebih
sebelum kewafatannya.30 ni telah dicetak beberapa kali dan butir-butir keterangannya sepertimana berikut: Kitab ini telah dicetak beberapa kali dan butir-butir keterangannya sepertim • Ia pertama kali dicetak selepas kewafatan pengarangnya di al-Matba‘ Cisymah Fayd di
Lucknow, India pada tahun 1304H. Penerbitannya telah diusahakan oleh al-Mulwi Khadim
Husayn al-‘Azim abadi, Nadir Husayn dan Syekh Muhammad ‘Abd al-Hamid bin
Muhammad ‘Abd al-Halim bin Muhammad ‘Abd al-Hakim al-Laknawi. • Kemudian ia dicetak oleh Dar al-Qalam, Dubai, Emiriah Arab Bersatu pada tahun 1994
(1414H) dengan ditahkik oleh Dr. Taqiy al-Din al-Nadwi. • Kemudian ia dicetak oleh Dar al-Qalam, Dubai, Emiriah Arab Bersatu pada tahun 1994
(1414H) dengan ditahkik oleh Dr. Taqiy al-Din al-Nadwi. • Setelah itu, muncul pula cetakan Maktab al-Matbu‘at al-Islamiyyah, Halab, Syria yang
ditahkik oleh Syekh ‘Abd al-Fattah Abu Ghuddah pada tahun 1995 (1416H). Bersama
cetakan ini disertakan sebuah lampiran berjudul: Akhta’ al-Duktur Taqiy al-Din al-Nadwi
fi Tahqiq Kitab Zafar al-Amani li al-Laknawi yang mengkritik cetakan yang ditahkik oleh
Dr. Taqiy al-Din al-Nadwi di atas. • Kitab ini juga telah dicetak oleh Dar al-Kutub al-‘Ilmiyyah, Beirut dengan ditahkik oleh
Khalil al-Mansur pada tahun 1998. • Kitab ini juga telah dicetak oleh Dar al-Kutub al-‘Ilmiyyah, Beirut dengan ditahkik oleh
Khalil al-Mansur pada tahun 1998. Kitab syarah al-Laknawi dianggap antara kitab terpenting dalam ilmu mustalah al-hadith
yang dihasilkan oleh ulama mutaakhirin. al-Laknawi mempersembahkan isi perbahasan kitab ini
secara lengkap dan detail. Beliau turut menghuraikan di dalamnya permasalahan-permasalahan
ilmu hadis yang rumit dan menjadi perselisihan di kalangan para ulama. Sebagaimana yang telah diterangkan, kitab Zafar al-Amani ini merupakan syarah kepada
kitab Mukhtasar al-Jurjani, maka keseluruhan susunan kitab syarah ini sudah pastinya adalah 13 Taqaddumi: Journal of Quran and Hadith Studies
Vol. 1, No. 2
DOI Prefix 10.12928
sama dengan susunan kitab asal berkenaan. Susunannya secara umum, dibagikan kepada dua
bagian, iaitu bagian al-Muqaddimah dan bagian al-Maqasid. sama dengan susunan kitab asal berkenaan. Susunannya secara umum, dibagikan kepada dua
bagian, iaitu bagian al-Muqaddimah dan bagian al-Maqasid. sama dengan susunan kitab asal berkenaan. Susunannya secara umum, dibagikan kepada dua
bagian, iaitu bagian al-Muqaddimah dan bagian al-Maqasid. Bagian al-Muqaddimah adalah berkaitan penjelasan mengenai usul dan beberapa istilah
hadis, iaitu al-matn, al-hadith, al-sanad, al-isnad, al-mutawatir dan al-ahad. Manakala bagian al-
Maqasid pula terdiri daripada empat buah bab, iaitu: 1. MOHD. KHAFIDZ SORONI
14 Metodologi Penulisan Kitab Zafar al-Amani Bab pertama, mengenai bagian-bagian hadis dan jenis-jenisnya, yang merupakan bab yang
paling panjang dalam kitab ini. Ia mengandungi tiga fasal, iaitu fasal pertama mengenai
hadis sahih, fasal kedua mengenai hadis hasan dan fasal ketiga mengenai hadis daif serta
pembagiannya secara terperinci. 2. Bab kedua, mengenai al-jarh wa al-ta‘dil. Ia mengandungi dua fasal, iaitu fasal pertama
mengenai al-‘adalah wa al-dabt dan fasal kedua mengenai al-jarh. 3. Bab ketiga, mengenai proses periwayatan hadis (tahammul al-hadith). Ia membincangkan
tentang tujuh kaedah dalam proses periwayatan hadis. 4. Dan bab keempat, mengenai nama-nama para perawi hadis (asma’ al-rijal). Ia didahului
dengan perbincangan mengenai takrif al-sahabi dan al-tabi‘i. 4. Dan bab keempat, mengenai nama-nama para perawi hadis (asma’ al-rijal). Ia didahului
dengan perbincangan mengenai takrif al-sahabi dan al-tabi‘i. Di dalam setiap bagian dan bab, al-Laknawi telah menambah banyak perbahasan tambahan
lain dan contoh-contoh penjelasan bagi melengkapkan kitab syarahannya. Jadual di bawah berikut
dapat menunjukkan susunan dan perbandingan kandungan antara kedua-dua buah kitab, antara
kitab yang mensyarah, iaitu Zafar al-Amani dan kitab yang disyarahkan, iaitu Mukhtasar al-
Jurjani. Susunan dan Perbandingan Kandungan Antara Kitab Zafar al-Amani dan Kitab
Mukhtasar al-Jurjani
Bil. Zafar al-Amani
Mukhtasar al-Jurjani
1. المقدمة في بيان أصول الحديث ومصطلحاته
المقدمة في بيان أصول الحديث
ومصطلحاته
2. - تعريف المتن
- تعريف المتن
3. -
تعريف الحديث
-
تعريف الحديث
4. السنة واألثر والخبر
-
5. -
السند
- السند Mukhtasar al-Jurjani 14 MOHD. KHAFIDZ SORONI MOHD. KHAFIDZ SORONI MOHD. KHAFIDZ SORONI
15 , N
DOI Prefix 10.12928
6. -
اإلسناد
-
اإلسناد
7. - الخبر المتواتر
- الخبر المتواتر
8. أبحاث متعلقة
بالخبر
:المتواتر
1
-
في تقسيم الكالم إلى الخبر واإلنشاء
-
9. 2
-
في
معنى الصدق والكذب
-
10. 3
-
في تقسيم الخبر إلى الصادق والكاذب واحتماله
الصدق والكذب
-
11. 4
- في ذكر المتواتر والمشهور واآلحاد
-
12. 5
- في عدد رواة التواتر
-
13. 6
- في شروط التواتر
-
14. 7
-
كون العلم الحاصل بالمتواتر ضروريا أو نظريا
-
15. 8
-
في عالمة تواتر الخبر
-
16. 9
-
في الفرق بين المتواتر والمشهور
-
17. 10
-
في إفادة المشهور وخبر اآلحاد العلم
-
18. بحث وجود مثال المتواتر وعدمه وجود مثال المتواتر وعدمه
19. -
خبر اآلحاد
-
خبر اآلحاد
20. إيجاب خبر اآلحاد العمل دون العلم
-
21. تنبيهات متعلقة بخبر اآلحاد :
1
-
في معنى قولهم: خبر الواحد موجب للعمل
-
22. 2
-
في تقسيمه إلى المقبول وغيره
-
23. 3
- فيما يشترط لقبوله وما ال يشترط
-
24. بحث المستفيض والمشهور والعزيز والغريب
-
25. إبطال أن كون الحديث عزيزاً شرط البخاري
-
26. الرد على من قال: إن العزيز ال يوجد
-
27. بحث كثرة األحاديث والطرق
-
كثرة األحاديث والطرق
28. )المقاصد: (مرتبة على أربعة أبواب
)المقاصد: (مرتبة على أربعة أبواب
29. ذكر ألفاظ التعديل ومراتبه
-
30. ذكر ألفاظ الجرح ومراتبه
-
31. إطالق المنكر ومنكر الحديث
- 15 Taqaddumi: Journal of Quran and Hadith Studies DOI Prefix 10.12928
32. أبحاث أسماء الرواة وأنسابهم وما يتعلق بها :
- بحث المهمل
-
33. - بحث المؤتلف والمختلف
-
34. - بحث المتشابه
-
35. -
معرفة طبقات الرواة
-
36. - معرفة المواليد والوفيات
-
37. - معرفة البالد واألوطان
-
38. -
معرفة أسماء المكنيين
-
39. - معرفة كنى المسمين
-
40. - معرفة من اسمه كنيته
-
41. -
معرفة من اختلف في اسمه أو
كنيته
-
42. -
معرفة من كثرت كناه أو نعوته
-
43. -
معرفة من وافقت كنيته كنية زوجته أو وافق اسمه
كنية أبيه ونحو ذلك
-
44. - معرفة من وافق اسم أبيه واسم شيخه
-
45. -
معرفة من نسب إلى غير أبيه
-
46. - معرفة من نسب إلى أمه
-
47. -
معرفة من نسب إلى جده
-
48. - معرفة من نسب إلى جدته
-
49. MOHD. KHAFIDZ SORONI MOHD. KHAFIDZ SORONI
16 - معرفة من نسب إلى غير ما يسبق إلى الفهم
-
50. -
معرفة من اتفق اسمه مع اسم أبيه وجده
-
51. - معرفة من اتفق اسمه مع اسم شيخه وشيخ شيخه
-
52. -
معرفة من وافق اسم شيخه مع اسم تلميذه
-
53. -
معرفة أحوال األسماء المجردة
-
54. -
معرفة األسماء المفردة
-
55. -
معرفة األلقاب
-
56. -
معرفة األنساب
-
57. ( أ ) الباب األول: في أقسام الحديث ( أ ) الباب األول: في أقسام الحديث
58. 1
-
الفصل األول: في الصحيح
1
-
الفصل األول: في الصحيح 16 MOHD. KHAFIDZ SORONI
17 DOI Prefix 10.12928
MOHD. KHAFIDZ SORONI
17
59. -
أقسام الصحيح
-
أقسام الصحيح
60. بحث أصح األسانيد
-
61. ذكر المتفق عليه
-
62. - شرط الشيخين
- شرط الشيخين
63. أخذ الصحيح من غير الصحيحين
-
64. -
بحث التعليق
-
التعليق
65. 2
-
:الفصل الثاني في الحسن
2
-
:الفصل الثاني في الحسن
66. -
أقسام الحسن
-
أقسام الحسن
67. -
الفرق بين الصحيح والحسن
-
الفرق بين الصحيح والحسن
68. تعاريف الحسن مع مالها وما عليها
-
69. ذكر شروط األئمة الستة وغيرهم
-
70. -
بحث قول الترمذي: حسن صحيح ونحوه
- قول الترمذي: حسن صحيح ونحوه
71. - الصحيح لغيره
- الصحيح لغيره
72. - بحث الضعيف الذي ال يحتج به والذي يحتج به
- الضعيف الذي ال يحتج به والذي يحتج به
73. ذكر بعض األمثلة
-
74. 3
- الفصل الثالث: في الضعيف
3
- الفصل الثالث: في الضعيف
75. - التساهل في رواية الضعيف
- التساهل في رواية الضعيف
76. بحث نفيس في قبول الضعيف في فضائل األعمال
-
77. معنى عدم قبول خبر اآلحاد في العقائد
-
78. :عدة عبارات من أقسام الحديث
(
1
) الضرب األول: ما يشترك فيه الصحيح والحسن
:والضعيف
1
-
المسند
:عدة عبارات من أقسام الحديث
(
1
) الضرب األول: ما يشترك فيه الصحيح
:والحسن والضعيف
1
- المسند
79. 2
-
المتصل
2
-
المتصل
80. 3
- المرفوع
3
- المرفوع
81. بحث قول
الصحابي: أمرنا بكذا
-
82. قول الصحابي: من السنة كذا
-
83. قول الصحابي: كنا نفعل كذا
-
84. 4
-
المعنعن
4
-
المعنعن 17 Taqaddumi: Journal of Quran and Hadith Studies
Vol. 1, No. 2
DOI Prefix 10.12928 MOHD. KHAFIDZ SORONI
18 MOHD. KHAFIDZ SORONI
19 Vol. 1, No. 2
DOI Prefix 10.12928
MOHD KHAFIDZ SORONI
18
85. 5
-
المعلق
5
-
المعلق
86. 6
-
األفراد
6
-
األفراد
87. 7
- المدرج
7
- المدرج
88. ذكر بعض األمثلة
-
89. 8
-
)المشهور (اللغوي
8
-
)المشهور (اللغوي
90. ذكر بعض
األمثلة
-
91. 9
- الغريب
9
- الغريب
92. 10
-
العزيز
10
-
العزيز
93. 11
-
المصحف
11
-
المصحف
94. 12
-
المسلسل
12
-
المسلسل
95. ذكر بعض المسلسالت من مرويات المؤلف
-
96. -
ذكر االعتبار
-
ذكر االعتبار
97. (
2
:) الضرب الثاني: ما يختص بالضعيف
1
- الموقوف
(
2) الضرب الثاني:
:ما يختص بالضعيف
1
- الموقوف
98. بحث في حجية قول الصحابي وغيره
-
99. بحث في تفسير الصحابة
-
100. 2
-
المقطوع
2
-
المقطوع
101. 3
-
المرسل
3
-
المرسل
102. بحث قبول المرسل
-
103. 4
-
المنقطع
4
-
المنقطع
104. 5
-
المعضل
5
-
المعضل
105. 6
-
الشاذ
6
-
الشاذ
106. 7
- المنكر
7
- المنكر
107. إطالق المنكر
-
108. 8
-
المعلل
8
-
المعلل
109. ذكر بعض األمثلة
-
110. 9
-
المدلس
9
-
المدلس
111. -
ذكر أقسام التدليس
-
ذكر أقسام التدليس
112. أسامي المدلسين
- 18 Manakala metode syarahan atau huraian yang digunakan oleh al-Laknawi di dalam
syarahnya dapat dilihat sebagaimana keterangan berikut: Manakala metode syarahan atau huraian yang digunakan oleh al-Laknawi di dalam
syarahnya dapat dilihat sebagaimana keterangan berikut: Manakala metode syarahan atau huraian yang digunakan oleh al-Laknawi di dalam
syarahnya dapat dilihat sebagaimana keterangan berikut:
i. Bagi menghuraikan kitab Mukhtasar al-Jurjani, al-Laknawi akan menuliskan terlebih dahulu
sebagian frasanya, kemudian baru diikuti dengan syarahan daripada beliau, sebagaimana metode
yang popular dipilih oleh kebanyakan ulama dalam menyusun sesebuah kitab syarah. Ini kerana
metode sebegini memudahkan pembaca untuk memahami teks yang disyarahkan satu persatu. Secara umumnya, kedudukan kitab Mukhtasar al-Jurjani adalah sebagai matan, manakala
kedudukan kitab Zafar al-Amani pula adalah sebagai syarahnya. ii. Kadar syarah yang disampaikan oleh al-Laknawi tidak sama dari segi ukuran panjang
pendeknya. Adakalanya beliau mensyarahkan sesuatu frasa dengan kadar yang sederhana dan
adakalanya pula beliau mensyarahkannya dengan syarahan yang agak panjang lebar. Ini berlaku
berdasarkan keperluan perbahasan bagi sesuatu topik yang dibincangkan. Al-Laknawi turut
menambah perbahasan-perbahasan tambahan lain bagi melengkapkan syarahnya. Dalam jadual di
atas, menunjukkan secara jelas perbahasan-perbahasan tambahan yang dibuat oleh al-Laknawi ke
atas kitab Mukhtasar al-Jurjani. Secara kasar, mungkin dapat dianggarkan kitab syarah al-
Laknawi ini adalah lebih besar sebanyak tujuh atau lapan kali ganda daripada kitab asal yang
disyarah. i. Bagi menghuraikan kitab Mukhtasar al-Jurjani, al-Laknawi akan menuliskan terlebih dahulu
sebagian frasanya, kemudian baru diikuti dengan syarahan daripada beliau, sebagaimana metode
yang popular dipilih oleh kebanyakan ulama dalam menyusun sesebuah kitab syarah. Ini kerana
metode sebegini memudahkan pembaca untuk memahami teks yang disyarahkan satu persatu. Secara umumnya, kedudukan kitab Mukhtasar al-Jurjani adalah sebagai matan, manakala
kedudukan kitab Zafar al-Amani pula adalah sebagai syarahnya. ii. Kadar syarah yang disampaikan oleh al-Laknawi tidak sama dari segi ukuran panjang
pendeknya. Adakalanya beliau mensyarahkan sesuatu frasa dengan kadar yang sederhana dan
adakalanya pula beliau mensyarahkannya dengan syarahan yang agak panjang lebar. Ini berlaku
berdasarkan keperluan perbahasan bagi sesuatu topik yang dibincangkan. Al-Laknawi turut
menambah perbahasan-perbahasan tambahan lain bagi melengkapkan syarahnya. Dalam jadual di
atas, menunjukkan secara jelas perbahasan-perbahasan tambahan yang dibuat oleh al-Laknawi ke
atas kitab Mukhtasar al-Jurjani. Secara kasar, mungkin dapat dianggarkan kitab syarah al-
Laknawi ini adalah lebih besar sebanyak tujuh atau lapan kali ganda daripada kitab asal yang
disyarah. 20 DOI Prefix 10.12928
MOHD. KHAFIDZ SORONI
19
113. 10
-
المضطرب
10
-
المضطرب
114. ذكر األحاديث المضطربة
-
115. 11
- المقلوب
11
- المقلوب
116. ذكر بعض المقلوبات
-
117. 12
- الموضوع
12
- الموضوع
118. بحث نفيس في حكم ما اختلف الحفاظ في تصحيحه
وتحسينه وضعفه ووضعه
-
119. ذكر بعض األمثلة في اختالفهم
-
120. بحث ما يعرف به الوضع
-
ما يعرف به الوضع
121. ذكر بعض األخبار الموضوعات
-
122. ذكر أقسام الوضاعين
-
ذكر أقسام الوضاعين
123. ذكر بعض الموضوعات
-
ذكر بعض الموضوعات
124. ذكر كتاب الموضوعات البن الجوزي
- ذكر كتاب الموضوعات البن الجوزي
125. ذكر من صنف في الموضوعات
-
126. (ب) الباب الثاني: في الجرح والتعديل
(ب) الباب الثاني: في الجرح والتعديل
127. ذكر المتعنتين في الجرح
-
128. 1
- الفصل األول: في العدالة والضبط
1
-
الفصل
األول: في العدالة والضبط
129. بحث البدعات والفسق
-
130. بحث الرواية بالمعنى
-
131. وجوه الفرق بين الرواية والشهادة
-
132. 2
-
الفصل الثاني: في الجرح
2
-
الفصل الثاني: في الجرح
133. (جـ) الباب الثالث: في تحمل الحديث (جـ) الباب الثالث: في تحمل الحديث
134. -
بحث رواية
الصبيان وغيرهم
-
رواية الصبيان وغيرهم
135. -
:طرق تحمل الحديث
1
- السماع
-
:طرق تحمل الحديث
1
- السماع
136. 2
-
القراءة على الشيخ
2
-
القراءة على الشيخ
137. "الفرق بين "حدثنا" و"أخبرنا
-
138. ذكر أرفع أقسام الرواية
-
139. 3
- اإلجازة
3
- اإلجازة 19 Taqaddumi: Journal of Quran and Hadith Studies
Vol. 1, No. 2
DOI Prefix 10.12928
140. 4
- المناولة
4
- المناولة
141. 5
- المكاتبة
5
- المكاتبة
142. طريقة كتاب المكاتيب
-
143. 6
-
اإلعالم
6
-
اإلعالم
144. 7
-
الوجادة
7
-
الوجادة
145. (د) الباب الرابع: في أسماء الرجال
(د) الباب الرابع: في أسماء الرجال
146. 1
- تعريف الصحابي
1
- تعريف الصحابي
147. بحث نفيس متعلق بالصحابة ، فيه عشرون .فائدة
-
148. 2
-
تعريف التابعي
2
-
تعريف التابعي
149. .ًكون اإلمام أبي حنيفة تابعيا
-
150. 3
- مواليد ووفيات بعض األئمة
3
- مواليد ووفيات بعض األئمة Taqaddumi: Journal of Quran and Hadith Studies
Vol. 1, No. 2
DOI Prefix 10.12928 31 al-Laknawi, ibid. hlm 269. MOHD. KHAFIDZ SORONI 32 Ibid. hlm 450. bid. hlm 450.
l-Laknawi, Zafar al-Amani, ditahkik oleh Dr. Taqiy al-Din al-Nadwi (Dubai: Dar al-Qalam, 1995). MOHD. KHAFIDZ SORONI Taqaddumi: Journal of Quran and Hadith Studies
Vol. 1, No. 2
DOI Prefix 10.12928 Taqaddumi: Journal of Quran and Hadith Studies
Vol. 1, No. 2
DOI Prefix 10.12928 iii. Selain menjelaskan makna, al-Laknawi juga membawakan banyak contoh penjelasan dalam
topik-topik yang dibincangkan. Ini kerana kandungan kitab Mukhtasar al-Jurjani yang asal terlalu
ringkas hingga contoh yang diberi dalam sesuatu topik adakalanya hanya dalam bentuk makna
hadis sahaja dan adakalanya tidak ada contoh sama sekali. Maka, al-Laknawi telah
menyempurnakan dan menambah contoh-contoh penjelasan mengenainya di dalam syarahan
beliau. iv. Dalam memetik hadis, al-Laknawi secara konsisten akan menyatakan sumber asal hadis, jika
ianya mempunyai sumber asal serta menyebutkan statusnya sama ada sahih, hasan, daif dan
sebagainya. Misalnya dalam membawakan contoh hadis-hadis yang masyhur di kalangan
masyarakat, al-Laknawi menyebutkan:31 منها حديث: "الوضوء على الوضوء نور على نور"، اشتهر عند ال فقهاء وذكروه في كتبهم، وال اعتبار له عند المحدثين، فقد قال
الحافظ العراقي في تخريج أحاديث اإلحياء: لم أجد له أصالً، انتهى. وكذا قال العالمة محمد طاهر الفتني في آخر كتابه مجمع
البحار: إنه لم يوجد، ونقل الحافظ السخاوي في المقاصد الحسنة عن شيخه ابن حجر أنه.حديث ضعيف، رواه رزين في مسنده Terjemahan: “Antaranya hadis: “Wuduk di atas wuduk merupakan cahaya di atas cahaya”. Ia
cukup masyhur di sisi para fuqaha’ dan disebut dalam kitab-kitab mereka. Sedangkan ianya tidak
boleh diambil iktibar menurut para muhaddithin. Kata al-Hafiz al-‘Iraqi dalam Takhrij Ahadith al-
Ihya’: “Aku tidak menemukan asal baginya”. Demikian jua kata al-‘Allamah Muhammad Tahir
al-Futtani di akhir kitabnya Majma‘ al-Bihar; bahawa ianya tidak wujud. al-Hafiz al-Sakhawi
menukilkan dalam al-Maqasid al-Hasanah daripada gurunya, Ibn Hajar bahawa ianya hadis daif,
diriwayatkan oleh Razin dalam Musnadnya”. v. Antara kebiasaan al-Laknawi sebagaimana dalam kebanyakan karyanya yang lain ialah
membuat catatan nota kaki di bagian bawah kitab. Begitu juga dengan karyanya ini, al-Laknawi
turut melengkapi syarahannya dengan catatan nota kaki jika perlu bagi menjelaskan sesuatu
pernyataan, masalah atau isu yang perlu kepada penjelasan lebih lanjut. Namun, kebanyakannya
adalah berbentuk kritikan terhadap tulisan Syekh Siddiq Hasan Khan al-Qinnawji (w. 1307H)
yang disamarkan namanya oleh al-Laknawi sebagai (غير ملتزم ال
صحة من أفاضل عصرنا) yang
bermaksud “orang yang tidak melazimi perkara sahih dari kalangan tokoh zaman kita”. Catatan
nota kaki tersebut akan diakhiri dengan lafaz (منه) yang bermaksud: “Daripadanya”, iaitu daripada 21 Taqaddumi: Journal of Quran and Hadith Studies
Vol. 1, No. 2
DOI Prefix 10.12928 Taqaddumi: Journal of Quran and Hadith Studies pensyarah kitab. MOHD. KHAFIDZ SORONI MOHD. KHAFIDZ SORONI Misalnya, dalam mengulas tentang nama al-Khattabi sebagai Hamd, bukannya
Ahmad, al-Laknawi berkata:32
ال أحمد، كما وقع في "إتحاف النبالء" لغير ملتزم الصحة
من أفاضل عصرنا عند ذكر "إصالح غلط المحدثين" للخطابي، فإنه
خطأ فاحش )... (منه pensyarah kitab. Misalnya, dalam mengulas tentang nama al-Khattabi sebagai Hamd, bukannya
Ahmad, al-Laknawi berkata:32 pensyarah kitab. Misalnya, dalam mengulas tentang nama al-Khattabi sebagai Hamd, bukannya pensyarah kitab. Misalnya, dalam mengulas tentang nama al-Khattabi sebagai Hamd, bukannya
Ahmad, al-Laknawi berkata:32
ال أحمد، كما وقع في "إتحاف النبالء" لغير ملتزم الصحة
من أفاضل عصرنا عند ذكر "إصالح غلط المحدثين" للخطابي، فإنه ال أحمد، كما وقع في "إتحاف النبالء" لغير ملتزم الصحة
من أفاضل عصرنا عند ذكر "إصالح غلط المحدثين" للخطابي، فإنه
خطأ فاحش )... (منه Terjemahan: “Bukan Ahmad, sebagaimana yang terdapat dalam Ithaf al-Nubala’ oleh orang yang
tidak melazimi perkara sahih dari kalangan tokoh zaman kita ketika beliau menyebut kitab Islah
Ghalat al-Muhaddithin oleh al-Khattabi. Sesungguhnya ia merupakan satu kesilapan yang teruk... (Daripadanya).” vi. Di dalam huraiannya, al-Laknawi lebih banyak memberi tumpuan kepada perbincangan isi
kandungan kitab Mukhtasar al-Jurjani secara ilmiah dan kurang menyentuh tentang aspek
linguistiknya kecuali jika ia perlu sahaja. Misalnya, agar pembaca dapat membaca dengan betul
lafaz al-mudraj (المدرج), al-Laknawi menerangkan kedudukannya (al-Laknawi 1995: 248):
.بصيغة اسم المفعول من اإلفعال Terjemahan: “Dengan sighah ism maf‘ul daripada kata al-if‘al”. 1. al-Adhkar oleh al-Nawawi (hlm. 214, 322) 32 Ibid. hlm 450.
33 al-Laknawi, Zafar al-Amani, ditahkik oleh Dr. Taqiy al-Din al-Nadwi (Dubai: Dar al-Qalam, 1995). Terjemahan: “Dengan sighah ism maf‘ul daripada kata al-if‘al”. Secara amnya, al-Laknawi tidak memberatkan pembaca dengan perbincangan struktur
bahasa atau sintaksis kalimahnya kerana mungkin mahu mengekalkan fokus utama kitab ini yang
khusus mengenai disiplin ‘ulum al-hadith. Pengkaji mendapati al-Laknawi tidak menyenaraikan secara jelas sumber-sumber rujukan
yang digunakan oleh beliau. Namun, berdasarkan kaedah petikan teks yang dibuat oleh beliau, kita
dapat menduga sumber-sumber rujukan yang diguna pakai. Ini kerana dalam memetik teks
daripada sumber rujukan, al-Laknawi secara konsisten akan menyebut nama pengarang berserta
judul kitab atau salah satu daripadanya, diikuti oleh petikan teks berkenaan yang diakhiri dengan
perkataan ‘انتهى’ (tamat). Berdasarkan penelitian pengkaji, didapati sumber-sumber rujukan yang digunakan oleh al-
Laknawi dapat disenaraikan sepertimana berikut (senarai susunan disusun menurut urutan huruf
abjad berserta nombor halaman di dalam kitab Zafar al-Amani cetakan Dar al-Qalam yang ditahkik
oleh Dr. Taqiy al-Din al-Nadwi):33 1. al-Adhkar oleh al-Nawawi (hlm. 214, 322) 22 MOHD. KHAFIDZ SORONI Taqaddumi: Journal of Quran and Hadith Studies
Vol. 1, No. 2
DOI Prefix 10.12928 2. al-Ansab oleh Abu Sa‘d al-Sam‘ani (hlm. 518) 3. al-Arba‘in oleh Imam Fakhr al-Din al-Razi (hlm. 46) 4. al-Ashbah wa al-Naza’ir oleh Ibn Nujaym (hlm. 268) 5. Bulugh al-Maram min Adillah al-Ahkam oleh al-Hafiz Ibn Hajar (hlm. 394, 40 6. al-Burhan fi ‘Ulum al-Qur’an oleh Badr al-Din al-Zarkasyi (hlm. 341) 7. al-Bustan oleh al-Faqih Abu al-Layth Nasr al-Samarqandi (hlm. 479) 8. al-Daw’ al-Lami‘ fi A‘yan al-Qarn al-Tasi‘ oleh al-Sakhawi (hlm. 523) 9. Dirasat al-Labib fi al-Uswah al-Hasanah bi al-Habib oleh Mu‘in al-Sindi al-Hindi (hlm. 506) 10. al-Durar al-Kaminah fi A‘yan al-Mi’ah al-Thaminah oleh al-Hafiz Ibn Hajar (hlm. 521) 11. al-Durar al-Saniyyah fi ma ‘Ala min al-Asanid al-Shanawaniyyah oleh Syekh Muhammad
bin ‘Ali al-Syanawani (hlm. 301) 12. al-Durr al-Manthur oleh al-Suyuti (hlm. 440, 441) 13. Fatawa Qasim bin Qutlubugha (hlm. 333) 13. Fatawa Qasim bin Qutlubugha (hlm. 333) 14. Fath al-Baqi Sharh Alfiyyah al-‘Iraqi oleh Zakariyya al-Ansari (hlm. 328) 15. Fath al-Bari Sharh Sahih al-Bukhari oleh al-Hafiz Ibn Hajar (hlm. 58, 60, 89, 106, 135,
152, 163, 265, 362, 412, 459, 474, 477, 479) 16. Fath al-Ghaffar Sharh al-Manar oleh Ibn Nujaym al-Misri (hlm. 342) 17. Fath al-Mannan fi Ta’yid Madhhab al-Nu‘man oleh Syekh ‘Abd al-Haq al-Dihlawi (hlm. 335) 18. al-Fath al-Mubin Sharh al-Arba‘in oleh Ibn Hajar al-Makki (hlm. 215) 19. Fath al-Qadir Hashiyah al-Hidayah oleh Ibn al-Humam (hlm. 200, 214, 254, 281, 333
509) 20. al-Fawa’id al-Majmu‘ah fi al-Ahadith al-Mawdu‘ah oleh al-Syawkani (hlm. 457) 21. Terjemahan: “Dengan sighah ism maf‘ul daripada kata al-if‘al”. al-Fayd al-Tari Sharh Sahih al-Bukhari oleh Ja‘far bin Muhammad Maqsud ‘Alam al-
Ridawi (hlm. 499) 22. al-Futuhat al-Makkiyyah oleh Syekh Ibn ‘Arabi (hlm. 506) 22. al-Futuhat al-Makkiyyah oleh Syekh Ibn ‘Arabi (hlm. 506) 23. Hashiyah al-Mishkah oleh al-Syarif al-Jurjani (hlm. 439) . Hashiyah al-Mishkah oleh al-Syarif al-Jurjani (hlm. 439) 24. Hasr al-Sharid oleh Mawlana ‘Abid al-Sindi (hlm. 291, 293, 295) 25. Hawashi Sharh al-‘Aqa’id al-Nasafiyyah oleh al-Maula al-Khayali (hlm. 41) MOHD. KHAFIDZ SORONI
23 23 51. Lata’if al-Ma‘arif oleh Ibn Rajab (hlm. 417) MOHD. KHAFIDZ SORONI MOHD. KHAFIDZ SORONI 26. Hayah al-Hayawan oleh al-Kamal al-Damiri (hlm. 436) 26. Hayah al-Hayawan oleh al-Kamal al-Damiri (hlm. 436) 27. al-Hidayah oleh al-Mirghinani (hlm. 252, 254) 27. al-Hidayah oleh al-Mirghinani (hlm. 252, 254) 28. al-Idah wa al-Bayan li ma Ja’a fi Laylah al-Nasf min Sha‘ban oleh Ibn Hajar al-Makki
(hlm. 416) 29. Ihya’ ‘Ulum al-Din oleh Imam al-Ghazali (hlm. 34) 30. al-‘Ilal oleh al-Tirmidhi (hlm. 174, 194) 30. al-‘Ilal oleh al-Tirmidhi (hlm. 174, 194) 31. al-Ilmam bi Ahadith al-Ahkam oleh Taqiy al-Din Ibn Daqiq al-‘Id (hlm. 395) 32. al-Imam fi Ma‘rifah Ahadith al-Ahkam oleh Taqiy al-Din Ibn Daqiq al-‘Id (hlm. 395) 33. al-Iqtirah oleh Taqiy al-Din Ibn Daqiq al-‘Id (hlm. 121, 194, 250, 423) 34. Irshad al-Sari Sharh Sahih al-Bukhari oleh al-Qastallani (hlm. 56, 268) 35. Al-Isabah fi Ahwal al-Sahabah oleh al-Hafiz Ibn Hajar (hlm. 65) 36. al-Isha‘ah fi Ahwal al-Sa‘ah oleh Siddiq Hasan Khan (hlm. 285) 37. al-Istidhkar Sharh al-Muwatta’ oleh Ibn ‘Abd al-Barr (hlm. 365) 38. Itmam al-Dirayah li Qurra’ al-Niqayah oleh al-Suyuti (hlm. 332) 39. al-Itqan fi ‘Ulum al-Qur’an oleh al-Suyuti (hlm. 339, 341) 39. al-Itqan fi ‘Ulum al-Qur’an oleh al-Suyuti (hlm. 339, 341) 40. Jam‘ al-Jawami‘ oleh al-Suyuti (hlm. 394) = lihat: al-Jami‘ al-Kabir 41. al-Jami‘ li Akhlaq al-Rawi wa Adab al-Sami‘ oleh al-Khatib al-Baghdadi (hlm. 338) 42. al-Jami‘ al-Saghir fi Hadith al-Bashir al-Nadhir oleh al-Suyuti (hlm. 394) 43. Jami‘ al-Usul oleh Ibn al-Athir (hlm. 40, 46) 44. al-Kaf al-Shaf fi Takhrij Ahadith al-Kashshaf oleh al-Hafiz Ibn Hajar (hlm. 431, 445) 45. Kashf al-Asrar Sharh Usul al-Bazdawi oleh ‘Abd al-‘Aziz bin Ahmad al-Bukhari (hlm. 334) 334) 46. Al-Kashf al-Hathith ‘am man Rumiya bi Wad‘ al-Hadith oleh Burhan al-Din al-Halabi
(hlm. 59, 423, 432) 47. al-Kashif ‘an Haqa’iq al-Sunan oleh al-Tibi (hlm. 437, 438) = Hashiyah al-Mishkah 48. al-Kawakib al-Darari Sharh Sahih al-Bukhari oleh al-Karmani (hlm. 265) 49. al-Khulasah fi Usul al-Hadith oleh Imam al-Tibi (hlm. 178, 183, 238, 324, 332, 360, 374,
439, 494) 50. al-La’ali’ al-Masnu‘ah fi al-Ahadith al-Mawdu‘ah oleh al-Suyuti (hlm. 422, 427, 452) 50. al-La’ali’ al-Masnu‘ah fi al-Ahadith al-Mawdu‘ah oleh al-Suyuti (hlm. 422, 427, 452)
51. Lata’if al-Ma‘arif oleh Ibn Rajab (hlm. 417) 50. al-La ali al-Masnu ah fi al-Ahadith al-Mawdu ah oleh al-Suyuti (hlm. 422, 427, 452)
51. Lata’if al-Ma‘arif oleh Ibn Rajab (hlm. 417) 51. Lata’if al-Ma‘arif oleh Ibn Rajab (hlm. 417) 24 MOHD. KHAFIDZ SORONI Taqaddumi: Journal of Quran and Hadith Studies
Vol. 1, No. 2
DOI Prefix 10.12928 Taqaddumi: Journal of Quran and Hadith Studies
Vol. 1, No. 2
DOI Prefix 10.12928 52. Ma‘alim al-Sunan oleh al-Khattabi (hlm. 120) 53. Madarij al-Isnad oleh al-Qadi ‘Ali Khan al-Kupamawi (hlm. 37) 54. Ma’khadh al-‘Ilm oleh Ibn Faris al-Lughawi (hlm. 486) 55. Majma‘ al-Bihar oleh Muhammad Tahir al-Futtani (hlm. 269, 270) 56. al-Maqasid al-Hasanah oleh al-Sakhawi (hlm. 201, 202, 205, 269, 272, 415) 57. al-Mawdu‘at oleh Ibn al-Jawzi (hlm. 414, 426, 451) 57. al-Mawdu‘at oleh Ibn al-Jawzi (hlm. 414, 426, 451) 58. al-Mawdu‘at oleh ‘Ali al-Qari (hlm. 416) 58. al-Mawdu‘at oleh ‘Ali al-Qari (hlm. 416) 59. al-Minah al-Makkiyyah Sharh al-Qasidah al-Hamziyyah oleh Ibn Hajar al-Makki al-
Haytami (hlm. 272, 447) 59. al-Minah al-Makkiyyah Sharh al-Qasidah al-Hamziyyah oleh Ibn Hajar al-Makki al-
Haytami (hlm. 272, 447) 60. Mir’ah al-Jinan oleh al-Yafi‘i (hlm. 49) 61. Mir’ah al-Usul Sharh Mirqah al-Wusul oleh Mulla Khusru (hlm. 334) 62. al-Mirqah Hashiyah al-Mishkah oleh ‘Ali al-Qari (hlm. 522) 63. Mirqah al-Su‘ud Sharh Sunan Abi Dawud oleh al-Suyuti (hlm. 274) 64. Misbah al-Zujajah ‘ala Sunan Ibn Majah oleh al-Suyuti (hlm. 431) 65. Mizan al-I‘tidal oleh al-Dhahabi (hlm. 503) 65. Mizan al-I‘tidal oleh al-Dhahabi (hlm. 503) 66. al-Mizan al-Kubra oleh Syekh ‘Abd al-Wahhab al-Sya‘rani (hlm. 225) 67. al-Muhalla oleh Ibn Hazm (hlm. 160) 67. al-Muhalla oleh Ibn Hazm (hlm. 160) 68. Mukhtasar ‘Ulum al-Hadith oleh al-Qadi Badr al-Din Ibn Jama‘ah (hlm. 127, 178, 237,
281, 486) = lihat: al-Manhal al-Rawi 69. al-Muntakhab al-Hussami oleh Hussam al-Din al-Akhsikati (hlm. 459) 70. Mushtabih al-Nisbah oleh ‘Abd al-Ghani bin Sa‘id al-Azdi (hlm. 100) 71. Muqaddimah fi Usul al-Tafsir oleh Ibn Taymiyyah (hlm. 334) 342) 71. Muqaddimah fi Usul al-Tafsir oleh Ibn Taymiyyah (hlm. 342) 72. al-Muqizah oleh al-Dhahabi (hlm. 175) 73. Nasb al-Rayah Takhrij Ahadith al-Hidayah oleh al-Zayla‘i (hlm. 366) 74. Nasim al-Riyad Sharh Shifa’ ‘Ayyad oleh al-Syihab al-Khafaji (hlm. 217) 75. Nazm al-Durar fi Silk Shaqq al-Qamar oleh ‘Abd al-Halim bin Amin Allah al-Laknawi
(hlm. 504) 76. al-Nukat ‘ala Muqaddimah Ibn al-Salah oleh al-Hafiz Ibn Hajar (hlm. 124, 37 76. al-Nukat ‘ala Muqaddimah Ibn al-Salah oleh al-Hafiz Ibn Hajar (hlm. 124, 379, 421)
77. al-Nukat ‘ala Muqaddimah Ibn al-Salah oleh al-Zarkasyi (hlm. 175) 77. al-Nukat ‘ala Muqaddimah Ibn al-Salah oleh al-Zarkasyi (hlm. 175) 25 511) = lihat: Fath al-Mughith 88. Sharh Alfiyyah al-‘Iraqi oleh Zayn al-Din al-‘Iraqi (hlm. 59, 121, 122, 124, 131, 152, 157,
162, 177, 191, 195, 207, 226, 239, 246, 250, 262, 319, 326, 374, 377, 390, 481, 486, 510) 89. Sharh al-Hidayah oleh Badr al-Din al-‘Ayni (hlm. 199, 229, 254, 271, 509) = lihat: al-
Binayah 90. Sharh Jam‘ al-Jawami‘ oleh al-Mahalli (hlm. 44) 91. Sharh Jami‘ al-Tirmidhi oleh Abu al-Fath Muhammad bin Muhammad bin Sayyid al-Nas
(hlm. 191, 195) 92. Sharh Kanz al-Daqa’iq oleh al-‘Ayni (hlm. 229) 93. Sharh al-Manar oleh Ibn Malak (hlm. 32, 41, 44, 66, 334) = lihat: Sharh Mana 93. Sharh al-Manar oleh Ibn Malak (hlm. 32, 41, 44, 66, 334) = lihat: Sharh Manar al-Usul
94. Sharh al-Maqasid oleh al-Taftazani (hlm. 223, 508) 94. Sharh al-Maqasid oleh al-Taftazani (hlm. 223, 508) 94. Sharh al-Maqasid oleh al-Taftazani (hlm. 223, 508) 95. Sharh al-Muhadhdhab oleh al-Nawawi (hlm. 233, 352) 96. Sharh Mukhtasar Ibn al-Hajib oleh al-Qadi ‘Adud al-Din al-Syafi‘i (hlm. 66) 97. Sharh Mukhtasar al-Manar oleh Qasim bin Qutlubugha (hlm. 333) 98. Sharh Mukhtasar al-Muzani oleh al-Dawudi (hlm. 233) 99. Sharh al-Muwatta’ oleh al-Qadi Ibn al-‘Arabi (hlm. 70) 26 MOHD. KHAFIDZ SORONI MOHD. KHAFIDZ SORONI 78. al-Nukat al-Wafiyyah Sharh al-Alfiyyah oleh Burhan al-Din Ibrahim bin ‘Umar al-Biqa‘i
(hlm. 190) 79. Nukhbah al-Fikar oleh al-Hafiz Ibn Hajar (hlm. 41, 49, 52, 413, 498) 80. Qamar al-Aqmar li Nur al-Anwar oleh ‘Abd al-Halim bin Amin Allah al-Laknawi (hlm. 509) 509) 81. al-Qawl al-Badi‘ fi al-Salah ‘ala al-Habib al-Shafi‘ oleh al-Sakhawi (hlm. 210, 217) 82. al-Qawl al-Musaddad fi al-Dhabb ‘an Musnad Ahmad oleh al-Hafiz Ibn Hajar (hlm. 459) 83. Rasa’il al-Qari oleh ‘Ali al-Qari al-Makki (hlm. 270, 271) 84. Sanad al-Anam Sharh Musnad al-Imam oleh ‘Ali al-Qari al-Makki (hlm. 206) 85. al-Sayf al-Maslul ‘ala Man Sabb al-Rasul oleh al-Subki (hlm. 223) 86. Shann al-Gharah ‘ala Man Azhara Ma‘arrah oleh Ibn Hajar al-Makki (hlm. 332) 87. Sharh Alfiyyah al-‘Iraqi oleh al-Sakhawi (hlm. 43, 81, 85, 88, 121, 264, 328, 343, 362,
374, 378, 380, 389, 391, 392, 401, 424, 455, 457, 460, 462, 469, 481, 486, 488, 492, 507, 87. Sharh Alfiyyah al-‘Iraqi oleh al-Sakhawi (hlm. 43, 81, 85, 88, 121, 264, 328, 343, 362,
374, 378, 380, 389, 391, 392, 401, 424, 455, 457, 460, 462, 469, 481, 486, 488, 492, 507,
511) = lihat: Fath al-Mughith MOHD. KHAFIDZ SORONI Taqaddumi: Journal of Quran and Hadith Studies
Vol. 1, No. 2
DOI Prefix 10.12928 100. Sharh Nukhbah al-Fikar oleh al-Hafiz Ibn Hajar (hlm. 47, 49, 71, 82, 96, 196, 242, 262,
277, 331, 359, 362, 375, 423) = lihat: Nuzhah al-Nazar 101. Sharh Sahih Muslim oleh Imam al-Nawawi (hlm. 59, 106, 236, 238, 343, 408, 461) 102. Sharh Sharh Nukhbah al-Fikar oleh al-Fadil Akram bin ‘Abd al-Rahman al- 102. Sharh Sharh Nukhbah al-Fikar oleh al-Fadil Akram bin ‘Abd al-Rahman al-Sindi (hl
42, 43, 64, 71, 86, 176, 249, 263, 266) = lihat: Im‘an al-Nazar 102. Sharh Sharh Nukhbah al-Fikar oleh al-Fadil Akram bin ‘Abd al-Rahman al-Sindi (hlm. 42 43 64 71 86 176 249 263 266)
lih t I ‘
l N 42, 43, 64, 71, 86, 176, 249, 263, 266) = lihat: Im‘an al-Nazar 103. Sharh Sharh Nukhbah al-Fikar oleh ‘Ali al-Qari (hlm. 36, 41, 71, 266, 278) 104. Sharh Tahrir al-Usul oleh Bahr al-‘Ulum al-Laknawi (hlm. 333) 105. Shifa’ al-Saqqam fi Ziyarah Khayr al-Anam oleh Taqiy al-Din al-Subki (hlm. 416)
106. al-Sihah oleh al-Jawhari (hlm. 510) 107. al-Siqayah li ‘Atshan al-Hidayah oleh ‘Abd al-Halim al-Laknawi (hlm. 321, 372) 108. Sirah al-Amin al-Ma’mun oleh Nur al-Din al-Halabi al-Syafi‘i (hlm. 209) 109. Tabaqat al-Shafi‘iyyah oleh Taj al-Din al-Subki (hlm. 468) 110. Tabaqat al-Shafi‘iyyah oleh Taqiy al-Din Ibn Syahbah al-Dimasyqi (hlm. 49, 520) 111. Tabsir al-Muntabih bi Tahrir al-Mushtabih oleh al-Hafiz Ibn Hajar (hlm. 10 112. Tabyid al-Sahifah fi Manaqib al-Imam Abi Hanifah oleh al-Suyuti (hlm. 515) 113. Tabyin al-‘Ajab fi ma Warada fi Fadl Rajab oleh al-Hafiz Ibn Hajar (hlm. 417) 114. al-Tabyin li Asma’ al-Mudallisin oleh Burhan al-Din Ibrahim al-Halabi (hlm. 373, 377,
378, 380) 115. al-Tabyin Sharh al-Muntakhab al-Hussami oleh Amir Katib bin Amir ‘Umar al-Farabi
al-Itqani (hlm. 334) 116. Tadrib al-Rawi Sharh Taqrib al-Nawawi oleh al-Suyuti (hlm. 210, 266, 324, 332, 357,
358, 363, 389, 425, 453, 466, 490) 117. Tahdhib al-Tahdhib oleh al-Hafiz Ibn Hajar (hlm. 65, 172) 118. al-Tahqiq Sharh al-Muntakhab al-Hussami oleh ‘Abd al-‘Aziz bin Ahmad al-Bukhari
(hlm. 334) 119. Tahrir al-Usul oleh Ibn al-Humam (hlm. 333, 509) 119. Tahrir al-Usul oleh Ibn al-Humam (hlm. 333, 509) 120. Tajrid al-Sahabah oleh al-Dhahabi (hlm. 499) 121. Takhrij Ahadith al-Ihya’ oleh al-‘Iraqi (hlm. 269, 363, 417) 122. Tanqih al-Usul oleh Sadr al-Syari‘ah (hlm. 459) 122. Tanqih al-Usul oleh Sadr al-Syari‘ah (hlm. 459) 123. Taqrib al-Tahdhib oleh al-Hafiz Ibn Hajar (hlm. 108, 504) 27 MOHD. KHAFIDZ SORONI MOHD. KHAFIDZ SORONI MOHD. KHAFIDZ SORONI
27 Taqaddumi: Journal of Quran and Hadith Studies
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124. al-Taqrib wa al-Taysir oleh Muhyi al-Din al-Nawawi (hlm. 146, 355, 357, 467, 492) =
lihat: Mukhtasar ‘Ulum al-Hadith Taqaddumi: Journal of Quran and Hadith Studies
Vol. 1, No. 2
DOI Prefix 10.12928 124. al-Taqrib wa al-Taysir oleh Muhyi al-Din al-Nawawi (hlm. 146, 355, 357, 467, 492) =
lihat: Mukhtasar ‘Ulum al-Hadith 124. al-Taqrib wa al-Taysir oleh Muhyi al-Din al-Nawawi (hlm. 146, 355, 357, 467, 492) =
lihat: Mukhtasar ‘Ulum al-Hadith 125. al-Targhib wa al-Tarhib oleh al-Mundhiri (hlm. 206) 126. al-Tawshih Hashiyah Sahih al-Bukhari oleh al-Suyuti (hlm. 282) 127. al-Taysir fi Sharh al-Jami‘ al-Saghir oleh ‘Abd al-Ra’uf al-Munawi (hlm. 48 128. Tazyin al-Ara’ik bi Irsal al-Nabi SAW ila al-Mala’ik oleh al-Suyuti (hlm. 499) 129. al-Ta‘zim wa al-Minnah fi Anna Abaway Rasul Allah SAW fi al-Jannah oleh al-Suyuti
(hlm. 417) 130. al-Tuhfah al-Ithna ‘Ashariyyah oleh ‘Abd al-‘Aziz al-Dihlawi (hlm. 507) 131. Tulu‘ al-Thurayya bi Izhar ma Kana Khafiyya oleh al-Suyuti (hlm. 331) 132. ‘Ulum al-Hadith oleh al-Hakim (hlm. 69, 132, 318, 338) = lihat: Ma‘rifah ‘Ulum al-
Hadith 133. ‘Ulum al-Hadith oleh Ibn al-Salah (hlm. 142, 150, 160, 174, 182, 187, 190, 226, 228,
360, 452, 483, 484, 496) = lihat: Muqaddimah Ibn al-Salah 134. ‘Umdah al-Qari Sharh Sahih al-Bukhari oleh al-Qadi Badr al-Din al-‘Ayni (hlm. 145,
504) 135. Unmudhaj al-‘Ulum oleh Jalal al-Din al-Dawwani al-Syafi‘i (hlm. 215) 136. Al-Uns al-Jalil fi Tarikh al-Quds wa al-Khalil oleh Mujir al-Din al-Hanbali (hlm. 49)
137. al-Usul oleh al-Bazdawi (hlm. 459) 138. Wafayat al-A‘yan oleh Ibn Khallikan (hlm. 49) 138. Wafayat al-A‘yan oleh Ibn Khallikan (hlm. 49) 139. al-Wishah wa Tathqif al-Rimah fi Radd Tawhim al-Majd fi al-Sihah oleh ‘Abd al-
Rahman bin ‘Abd al-‘Aziz al-Maghribi (hlm. 511) Rahman bin ‘Abd al-‘Aziz al-Maghribi (hlm. 511) d al-Ma‘ad oleh Ibn al-Qayyim (hlm. 400, 406) 140. Zad al-Ma‘ad oleh Ibn al-Qayyim (hlm. 400, 406) Senarai rujukan yang banyak ini jelas membuktikan bahawa sumber yang digunakan oleh
al-Laknawi di dalam syarahnya ini cukup pelbagai, daripada ‘ulum al-hadith, fiqh, usul al-fiqh,
sirah, akidah, tafsir, ‘ulum al-Qur’an, tasawuf, sejarah dan sebagainya. Ia juga membuktikan
bahawa al-Laknawi merupakan seorang ilmuwan yang mempunyai medan pembacaan dan
penelaahan yang cukup luas. Justru, tidak hairanlah jika sumber rujukan yang digunakan oleh
beliau di dalam syarahnya ini cukup banyak bilangannya serta pelbagai. 28 a. Beberapa kesilapan teknikal Menurut Syekh ‘Abd al-Fattah Abu Ghuddah, terdapat beberapa kesalahan teknikal yang
dilakukan oleh al-Laknawi semasa menyusun kitab ini. Ia berlaku disebabkan beberapa faktor,
antaranya yang utama ialah beliau menyusun kitab ini dalam keadaan kesihatannya terganggu dan
sangat kurang memuaskan. Ini sebagaimana yang telah dinyatakan bahawa beliau hanya dapat
menyiapkan kitab ini kira-kira sebulan lebih sahaja sebelum tarikh kewafatannya. Sudah tentu jika
ada sebarang kekurangan dan kesilapan padanya selepas itu, tidak akan dapat dibaiki oleh beliau. Antara kesalahan teknikal tersebut ialah seperti beliau menisbahkan kata-kata kritikan yang
sepatutnya daripada Ibn Rusyayd (w. 721H) ditukar menjadi kata-kata Ibn Hibban (w. 354H) di
awal kitab Sahihnya.34 Beliau juga menisbahkan kata-kata yang sepatutnya daripada al-Hafiz al-
Zanjani (w. 471H) ditukar menjadi kata-kata al-Hakim (w. 405H) dan al-Khatib al-Baghdadi (w. 463H).35 MOHD. KHAFIDZ SORONI MOHD. KHAFIDZ SORONI Taqaddumi: Journal of Quran and Hadith Studies
Vol. 1, No. 2
DOI Prefix 10.12928 Taqaddumi: Journal of Quran and Hadith Studies
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DOI Prefix 10.12928 Taqaddumi: Journal of Quran and Hadith Studies
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DOI Prefix 10.12928 Kitab Zafar al-Amani secara umumnya merupakan sebuah kitab syarah ‘ulum al-hadith
yang amat baik, namun tidak dinafikan bahawa masih terdapat beberapa kelemahan dan
kekurangan padanya. Antaranya berdasarkan penelitian pengkaji ialah: 34 al-Laknawi, Zafar al-Amani bi Sharh Mukhtasar al-Sayyid al-Sharif al-Jurjani (Halab: Maktab al-Matbu‘at al- 36 Ibid. hlm 22. 34 al-Laknawi, Zafar al-Amani bi Sharh Mukhtasar al-Sayyid al-Sharif al-Jurjani (Halab: Ma a
a
aw ,
afa al
mani bi Sha h
ukhtasa al Sayyid al Sha if al Ju jani ( a ab:
a
Islamiyyah, 1996), hlm 70.
35 MOHD. KHAFIDZ SORONI 34 al-Laknawi, Zafar al-Amani bi Sharh Mukhtasar al-Sayyid al-Sharif al-Jurjani (Halab: Maktab al-Matbu‘at al-
Islamiyyah 1996) hlm 70 yy
,
),
35 Ibid. hlm 205. 38 Ibid. hlm 269-318. c. Sorotan perbahasan yang terlalu panjang al-Laknawi juga adakalanya membuat sorotan yang terlalu panjang mengenai sesuatu topik
yang sebenarnya boleh diminimakan. Misalnya, dalam menyorot perbahasan tentang hadis al-
musalsal, al-Laknawi menghimpunkan begitu banyak riwayat hadis al-musalsal dan tidak sekadar
hanya memberi sebagian contoh-contohnya sahaja.38 Seolah-olah beliau ingin memaparkan
kesemua sanad hadis al-musalsal yang diperolehi daripada guru-gurunya. Sedangkan fokus utama
kitab ini ialah bagi membincangkan ilmu mustalah al-hadith. al-Laknawi telah menggunakan halaman kitab yang banyak sekitar 49 muka untuk
memuatkan hadis-hadis al-musalsal berkenaan. Sedangkan menurut hemat pengkaji, sanad-sanad
hadis al-musalsal ini sebaiknya diasingkan ke dalam sebuah kitab khusus mengenai sanad-sanad
keilmuan beliau yang biasanya disebut sebagai kitab thabat. b. Sebagian penjelasan yang kurang mantap Dalam mensyarahkan kitab Mukhtasar al-Jurjani, al-Laknawi hampir menghuraikan setiap
inci kandungan kitab tersebut dengan terperinci sekali tanpa meninggalkan ruang-ruang yang
boleh menunjukkan kelompongan. Bagaimanapun, ada terdapat sedikit huraian beliau yang
mungkin dilihat kurang mantap. Misalnya, beliau mendefinisikan ilmu hadith atau ilmu usul al-
hadith hanya terbatas kepada sanad sahaja, tidak termasuk matan sekali. Katanya:
ال االنقطاع اإلر ال
اف إ ناده ن االت
حة الض ف، أ
ل هللاﷺ ن حيث ال
ر
ه عل يُ رف به أح ال ال ند إل هو علم يُعرف به أحوال السند إلى رسول هللا ﷺ من حيث الصحة والضعف، وأوصاف إسناده من االتصال واالنقطاع واإلرسال
والرفع والوقف وغير .ذلك، وأحوال رجاله من الجرح والتعديل Terjemahan: “Iaitu suatu ilmu yang dapat diketahui melaluinya hal ehwal sanad sampai kepada
Rasulullah SAW dari segi sahih dan daif, serta ciri-ciri sanadnya sama ada bersambung, terputus,
irsal, raf‘, waqf dan sebagainya, serta hal ehwal rijalnya dari segi al-jarh wa al-ta‘dil”.36 34 al-Laknawi, Zafar al-Amani bi Sharh Mukhtasar al-Sayyid al-Sharif al-Jurjani (Halab: Maktab al-Matbu‘at al-
Islamiyyah, 1996), hlm 70. 29 MOHD. KHAFIDZ SORONI Taqaddumi: Journal of Quran and Hadith Studies
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DOI Prefix 10.12928 Taqaddumi: Journal of Quran and Hadith Studies
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DOI Prefix 10.12928 Definisi ini jelas hanya berkaitan dengan sanad hadis sahaja, tidak termasuk matan hadis. Sedangkan definisi yang lengkap mencakupi sanad dan matan sekaligus. Buktinya yang jelas,
terdapat bagian-bagian ilmu hadis yang turut menyentuh perbincangan tentang matan, iaitu dalam
membincangkan hadis al-mawdu‘, al-shadh, al-mudraj, al-maqlub, ziyadah al-thiqah, al-
mudtarib, al-musahhaf, al-muharraf dan al-mu‘al (al-mu‘allal). Definisi ini jelas hanya berkaitan dengan sanad hadis sahaja, tidak termasuk matan hadis. al-Laknawi juga meletakkan penjelasan tentang al-asma’ wa al-kuna secara panjang lebar
di bagian pendahuluan al-Maqasid sebelum masuk bab pertama.37 Sedangkan, sebaik-baiknya
penjelasan tentang al-asma’ wa al-kuna ini diletakkan di dalam bab empat yang memang khusus
mengenai nama-nama para perawi hadis (asma’ al-rijal). al-Laknawi juga meletakkan penjelasan tentang al-asma’ wa al-kuna secara panjang lebar
di bagian pendahuluan al-Maqasid sebelum masuk bab pertama.37 Sedangkan, sebaik-baiknya
penjelasan tentang al-asma’ wa al-kuna ini diletakkan di dalam bab empat yang memang khusus
mengenai nama-nama para perawi hadis (asma’ al-rijal). 37 Ibid. hlm 85-105. MOHD. KHAFIDZ SORONI 39 Perbandingan dibuat secara khusus dengan kitab Manhaj Dhawi al-Nazar oleh Syekh al-Tarmasi (w. 1338H).
Ini kerana beliau hidup sezaman dengan Syekh al-Laknawi serta kitabnya dianggap antara kitab ‘ulum al-hadith
mutakhir yang terlengkap. Lihat: al-Tarmasi, Muhammad Mahfuz, Manhaj Dhawi al-Nazar fi Sharh Manzumah ‘Ilm
al-Athar (Surabaya: al-Haramayn, t.t.). d. Terdapat sub-sub topik yang ditinggalkan Antara kekurangan kitab Zafar al-Amani juga ialah tidak membincangkan di dalamnya
sejumlah bagian ilmu hadis seperti al-mazid fi muttasil al-asanid, kitabah al-hadith wa dabtuhu,
sifah riwayah al-hadith wa adabuha, adab al-muhaddith, adab talib al-hadith, al-‘ali wa al-nazil,
gharib alfaz al-hadith, al-muharraf, al-nasikh wa al-masukh, mukhtalif al-hadith, asbab wurud al-
hadith, tawarikh al-mutun, riwayah al-akabir ‘an al-asaghir, riwayah al-sahabah ‘an al-tabi‘in
‘an al-sahabah, riwayah al-aqran, riwayah al-ikhwah wa al-akhawat, riwayah al-aba’ ‘an al-
abna’, riwayah al-abna’ ‘an al-aba’, al-sabiq wa al-lahiq, man rawa ‘an syaykh thumma rawa 30 Taqaddumi: Journal of Quran and Hadith Studies
Vol. 1, No. 2
DOI Prefix 10.12928
‘anhu bi wasitah, al-wuhdan, man lam yarwi illa hadithan wahidan, man lam yarwi illa ‘an wahid,
man asnada ‘anhu SAW hadithan min al-sahabah alladhina matu fi hayatihi, al-mushtabih al-
maqlub, al-mubhamat, ma‘rifah man khalata min al-thiqat (al-mukhtalitun) dan al-mawali. ‘anhu bi wasitah, al-wuhdan, man lam yarwi illa hadithan wahidan, man lam yarwi illa ‘an wahid,
man asnada ‘anhu SAW hadithan min al-sahabah alladhina matu fi hayatihi, al-mushtabih al-
maqlub, al-mubhamat, ma‘rifah man khalata min al-thiqat (al-mukhtalitun) dan al-mawali. ‘anhu bi wasitah, al-wuhdan, man lam yarwi illa hadithan wahidan, man lam yarwi illa ‘an wahid,
man asnada ‘anhu SAW hadithan min al-sahabah alladhina matu fi hayatihi, al-mushtabih al-
maqlub, al-mubhamat, ma‘rifah man khalata min al-thiqat (al-mukhtalitun) dan al-mawali. Ini merupakan bagian-bagian ilmu hadis yang dikesan tidak disentuh oleh al-Laknawi di
dalam syarahnya berdasarkan perbandingan dengan kitab-kitab mustalah al-hadith yang lain.39
Secara jelas, ia berpunca daripada keterikatan beliau dengan kitab susunan al-Jurjani yang
sememangnya tidak menyentuh bagian-bagian tersebut. al-Laknawi sebenarnya menyedari hal ini
kerana al-Jurjani telah meringkaskan kitabnya daripada karya al-Tibi yang turut menyentuh
sejumlah daripada bagian-bagian tersebut dalam karyanya. Lantas beliau memberi komentar: “Ada
pun pengarang (al-Jurjani), beliau telah meringkaskannya dengan ringkasan yang kurang
sempurna dan meninggalkan banyak perkara yang sewajarnya disebutkan...”.40 y
y
40 al-Laknawi, op.cit. hlm 528. (
y
y ,
40 al-Laknawi, op.cit. hlm 528. DAFTAR PUSTAKA al-Hasani, ‘Abd al-Hayy al-Nadwi. Nuzhah al-Khawatir wa Bahjah al-Masami‘ wa al-
Nawazir. Beirut: Dar Ibn Hazm, 1999. Ibn Jama‘ah, Badr al-Din. al-Manhal al-Rawi fi Mukhtasar ‘Ulum al-Hadith al-Nabawi,
cet. 2. Dimasyq: Dar al-Fikr, 1986. Ibn al-Salah, ‘Uthman bin ‘Abd al-Rahman. ‘Ulum al-Hadith. Dimasyq: Dar al-Fikr, 1986. al-Jurjani, ‘Ali bin Muhammad bin ‘Ali al-Husayni. al-Dibaj al-Mudhahhab. ditahkik oleh
‘Abd al-Ghaffar Sulayman al-Bandari. Kaherah: Dar al-Hadith, 1405 H. al-Jurjani, ‘Ali bin Muhammad bin ‘Ali al-Husayni. Risalah fi Usul al-Hadith. ditahkik
oleh ‘Ali Zuwayn. Riyadh: Dar al-Rasyid, 1407 H. al-Kattani, ‘Abd al-Hayy bin ‘Abd al-Kabir. Fihris al-Faharis. cet. 2. Beirut: Dar al-Gharb
al-Islami, 1982. al-Laknawi, Muhammad ‘Abd al-Hayy. Zafar al-Amani bi Sharh Mukhtasar al-Sayyid al-
Sharif al-Jurjani. Halab: Maktab al-Matbu‘at al-Islamiyyah, 1996. al-Laknawi, Muhammad ‘Abd al-Hayy. Zafar al-Amani fi Mukhtasar al-Jurjani. Dubai:
Dar al-Qalam, 1995. al-Laknawi, Muhammad ‘Abd al-Hayy. al-Raf‘ wa al-Takmil fi al-Jarh wa al-Ta‘dil. Halab: Maktab al-Matbu‘at al-Islamiyyah, 1987. al-Nawawi, Yahya bin Syaraf. al-Taqrib wa al-Taysir. Beirut: Dar al-Kitab al-‘Arabi,
1985. Salah Muhammad Abu al-Haj. al-Manhaj al-Fiqhi li al-Imam al-Laknawi. Amman: Dar
al-Nafa’is, 2002. al-Tarmasi, Muhammad Mahfuz. Manhaj Dhawi al-Nazar fi Sharh Manzumah ‘Ilm al-
Athar. Surabaya: al-Haramayn, t.t. al-Tibi, al-Husayn bin ‘Abd Allah. al-Khulasah fi Usul al-Hadith. Beirut: ‘Alam al-Kutub,
1985. Waliy al-Din al-Nadwi. al-Imam ‘Abd al-Hayy al-Laknawi. Dimasyq: Dar al-Qalam, 1995. al-Zirikli, Khayr al-Din. 2002. al-A‘lam. Beirut: Dar al-‘Ilm li al-Malayin. 32 MOHD. KHAFIDZ SORONI Kesimpulan al-Laknawi merupakan tokoh ulama benua India yang cukup terkenal di persada
antarabangsa dalam awal abad ke 14 Hijrah. Dalam sejarah penulisan ‘ulum al-hadith beliau telah
membuka satu lembaran baru dalam disiplin ilmu hadis di kalangan ulama mazhab Hanafi secara
khusus melalui kitabnya Zafar al-Amani. Secara umum, metodologi penulisan al-Laknawi dilihat
cukup sistematis dan sarat dengan perbincangan ilmiah mengenai masalah-masalah yang agak
kompleks dalam ilmu hadis seperti isu hadis hasan, masalah beramal dengan hadis daif, isu hadis
mursal, isu hadis munkar dan sebagainya, berserta pentarjihan beliau terhadap berbagai pandangan
ulama yang disajikan dan diperbincangkan. Kupasannya dalam sesuatu masalah cukup mendetail
dan melacak hingga ke akar umbinya melalui pelbagai rujukan yang cukup bervariasi. Justru, tidak
hairan apabila karyanya ini telah mendapat tumpuan yang besar daripada kalangan ulama mazhab
Hanafi secara khusus, di samping para ilmuwan mazhab yang lain. Secara ringkas, meski
kekurangan yang ada, kitab Zafar al-Amani ini menurut pengkaji cukup layak untuk diletakkan
sebaris dengan karya-karya agung dalam disiplin ‘ulum al-hadith yang wajar dipelajari dan
dijadikan rujukan penting dalam tradisi pengajian Islam. (
y
y ,
40 al-Laknawi, op.cit. hlm 528. 31 MOHD. KHAFIDZ SORONI Taqaddumi: Journal of Quran and Hadith Studies
Vol. 1, No. 2
DOI Prefix 10.12928
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Triterpenes from Hoya paziae Kloppenb.
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General Experimental Procedure NMR spectra were recorded on a Bruker Avance 400 spectrometer
(400 MHz 1H, 100 MHz 13C). Column chromatography was performed
with silica gel 60 (70-230 mesh, Merck). Thin layer chromatography was
performed with plastic backed plates coated with silica gel F254 and the
plates were visualized by spraying with vanillin/H2SO4 solution followed
by warming. Sample Collection Healthy cuttings of H. paziae were collected from propagated plants at
the Philippine Nuclear Research Institute Hoya Germplasm Collection
under MTA No. 2015-004 dated March 20, 2015. Accession PNRI-H.30
was obtained from the Institute of Plant Breeding, University of the
Philippines-Los Baños (UPLB) in 2008 when it was authenticated by
Simeona V. Siar of the UPLB, Laguna, Philippines. The plant orginated
from Quezon province. This study is part of our research on the chemical constituents of Philip
pine endemic and indigenous hoyas. We earlier reported the isolation
of lupenone and lupeol from the roots; lupeol, squalene and β-sitosterol
from the leaves, and betulin from the stems of H. mindorensis Schlechter.2
In another study, we reported the isolation of lupeol, α-amyrin,
β-amyrin, lupeol acetate, α-amyrin acetate, and β-amyrin acetate from
the stems; and α-amyrin, bauerenol, squalene, lutein, β-sitosterol, and
stigmasterol from the leaves of H. multiflora Blume.3 Recently, the isola
tion of β-amyrin cinnamate and taraxerol from the stems and taraxerol,
triglycerides, chlorophyll a, and a mixture of β-sitosterol and stigmaster
ol from the leaves of H. wayetii Kloppenb. has been reported.4 Further
more, the isolation of taraxerol, taraxerone, a mixture of β-sitosterol and
stigmasterol, and a mixture of α-amyrin cinnamate and β-amyrin cin
namate from the stems; taraxerol, taraxerone, and β-sitosterol from the
roots; a mixture of α-amyrin cinnamate and β-amyrin cinnamate from
the flowers and squalene, β-sitosterol, and saturated hydrocarbons from
the leaves of H. buotii has been reported.5 Most recently, we reported
the isolation of β-amyrin cinnamate, squalene, β-sitosterol, a mixture
of β-amyrin, α-amyrin and lupeol and saturated hydrocarbons from
the leaves and squalene, taraxerol, lupeol cinnamate, and a mixture of
β-sitosterol and stigmasterol from the stems of H. diversifolia.6 Melissa Borlagdan1,2, Fernando B. Aurigue3, Ian A. Van Altena4, Consolacion Y. Ragasa1,5* 1Department of Chemistry, De La Salle University, 2401 Taft Avenue, Manila 1004, PHILIPPINES. 2Department of Science and Technology-Food and Nutrition Research Institute, Bicutan,Taguig, Metro Manila, PHILIPPINES. 3Department of Science and Technology- Philippine Nuclear Research Institute, Commonwealth Avenue, Diliman, Quezon City 1101, PHILIPPINES. 4School of Environmental and Life Sciences, Faculty of Science and Information Technology, The University of Newcastle-Australia, Callaghan, NSW, 2308, AUSTRALIA. 5De La Salle University Science & Technology Complex, Leandro V. Locsin Campus, Biñan City, Laguna 4024, PHILIPPINES. ABSTRACT Corresponding author: Consolacion Y. Ragasa, Department of Chemistry,
De La Salle University, 2401 Taft Avenue, Manila 1004, PHILIPPINES. Tel./Fax: +632 5360230
Email: consolacion.ragasa@dlsu.edu.ph
DOI : 10.5530/pj.2016.5.13 Corresponding author: Consolacion Y. Ragasa, Department of Chemistry,
De La Salle University, 2401 Taft Avenue, Manila 1004, PHILIPPINES. Tel./Fax: +632 5360230
Email: consolacion.ragasa@dlsu.edu.ph
DOI : 10.5530/pj.2016.5.13 Corresponding author: Consolacion Y. Ragasa, Department of Chemistry,
De La Salle University, 2401 Taft Avenue, Manila 1004, PHILIPPINES. Chemical investigation of the dichloromethane extracts of the stems of
Hoya paziae Kloppenb. led to the isolation of taraxerol (1), taraxeryl acetate
(2), and a mixture α-amyrin acetate (3), and β-amyrin acetate (4) in about
2.5:1 ratio. The structures of 1–4 were identified by comparison of their
NMR data with those reported in the literature. Email: consolacion.ragasa@dlsu.edu.ph
DOI : 10.5530/pj.2016.5.13 Email: consolacion.ragasa@dlsu.edu.ph
DOI : 10.5530/pj.2016.5.13 Keywords: Hoya paziae, Apocynaceae, taraxerol, taraxeryl acetate,
α-amyrin acetate, β-amyrin acetate Triterpenes from Hoya paziae Kloppenb. Triterpenes from Hoya paziae Kloppenb. Pharmacogn. J. 2016;8(5):487-489i Pharmacogn. J. 2016;8(5):487-489
A multifaceted peer reviewed journal in the field of Pharmacognosy and Natural Products
www.phcogj.com | www.journalonweb.com/pj Pharmacogn. J. 2016;8(5):487 489
A multifaceted peer reviewed journal in the field of Pharmacognosy and Natural Products
www.phcogj.com | www.journalonweb.com/pj g
; ( )
A multifaceted peer reviewed journal in the field of Pharmacognosy and Natural Products
www.phcogj.com | www.journalonweb.com/pj Pharmacognosy Journal, Vol 8, Issue 5, Sep-Oct, 2016 Original Article Original Article INTRODUCTION We report herein the isolation of taraxerol (1), taraxeryl acetate (2), and
a mixture α-amyrin acetate (3) and β-amyrin acetate (4) in about 2.5:1
ratio from the stems of H. paziae. The chemical structures of compounds
1–4 are presented in Figure 1. Hoya is one of the genera of the family Apocynaceae that has been used
as traditional medicine but it has become more useful as a source for
ornamental plants due to its waxy foliage and attractive flowers. There
are at least 109 species of Hoya found in the Philippines, 88 of these are
endemic to the country.1 Unlike most other native hoyas, Hoya paziae
is not a vine but an epiphytic shrub with scandent stems that tend to be
pendent due to their own weights, especially when there are plenty of
branches and foliage. The leaves are decussate instead of being opposite,
while the flowers have white corolla with a purplish red and white corona. It was originally found in Antique, Oriental Mindoro, and Quezon
provinces and is now commercialized locally and abroad as a hanging
ornamental plant with blooms that last up to six days and emit a pleas
antly sweet scent during the first day after opening.1 No report of the
chemical constituents or ethnobotanical uses of the plant have been
found. REFERENCES 1. Aurigue FB. 2013. A Collection of Philippine Hoyas and Their Culture, Philippine
Council for Agriculture, Aquatic and Natural Resources Research and Development
(PCAARRD). Department of Science and Technology (DOST). 195 pages. 2. Ebajo Jr VD, Shen CC, Ragasa CY. Triterpenes and sterol from Hoya mindorensis. Der Pharma Chemica. 2014; 6(4):321-5. 3. Ebajo Jr VD, Shen CC, Ragasa CY. Terpenoids and sterols from Hoya multiflora
Blume. J Appl Pharm Sci. 2015;5(3):33-9. Isolation of Chemical Constituentsh A research grant from the De La Salle University Science Foundation
through the University Research Coordination Office is gratefully
acknowledged. The air-dried stems (169.9 g) of H. paziae were ground in an Osterizer
blender, soaked in CH2Cl2 for three days and then filtered. The filtrate
was concentrated under vacuum to afford a crude extract (7 g) which
was chromatographed by gradient elution with CH2Cl2, followed by
increasing amounts of acetone by 10% increments by volume. The CH2Cl2
fraction was rechromatographed (2 ×) using 2.5% EtOAc in petroleum
ether to yield a mixture of 3 and 4 (4 mg) after washing with petroleum
ether. The 20% acetone in CH2Cl2 fraction was rechromatographed
(3 ×) using 2.5% EtOAc in petroleum ether to afford 2 (2 mg). The
30% acetone in CH2Cl2 fraction was rechromatographed (3 ×) using 5%
EtOAc in petroleum ether to yield 1 (3 mg) after washing with petroleum
ether. The authors declare no conflict of interest. The authors declare no conflict of interest. The authors declare no conflict of interest. RESULTS AND DISCUSSION 4. Ebajo Jr VD, Aurigue FB, Brkljaca R, Urban S, Ragasa CY. Chemical constituents
of Hoya wayetii Kloppenb. Int J Pharmacog Phytochem Res. 2015; 7(5):1041-5. Silica gel chromatography of the dichloromethane extract of H. paziae
yielded compounds 1–4. Their NMR spectra are in accord with data
reported in the literature for taraxerol5 (1), taraxeryl acetate7 (2),
α-amyrin acetate8 (3), and for β-amyrin acetate8 (4). The 2.5:1 ratio of the
mixture of α-amyrin acetate (3) and β-amyrin acetate (4) was deduced
from the intensities and integrations of the 1H NMR resonances for the
olefinic protons of 3 at δ 5.11 (t, J = 3.6 Hz) and δ 5.16 (t, J = 3.6 Hz) of 4 . 5. Ebajo Jr VD, Brkljaca R, Urban S, Ragasa CY. Chemical constituents of Hoya
buotii Kloppenb. J Appl Pharm Sci. 2015; 5(11):69-72. 6. Panajon NM, Aurigue FB, Shen CC, Ragasa CY. Triterpenes and sterols from
Hoya diversifolia Blume. J Appl Pharm Sci. 2016;6(6):79-82. 7. Thuy TT, Sung TV, Frank K, Wessjohann L. Triterpenes from the roots of
Codonopsis pilosula. J Chem. 2008;46(4):515- 20. 8. Feleke S, Brehane A. Triterpene compounds from the latex of Ficus sur I. Bull
Chem Soc Ethiop. 2005;19(2):307-10. PICTORIAL ABSTRACT
Consolacion Y. Ragasa: Obtai
Ragasa is a Full Professor of th
nila. Her research focuses on t
plants. She has published exte
Ragasa was awarded the Pillar
the Philippines Achievement Aw
Achievement Award in Chemic
2001). She is a member of the PICTORIAL ABSTRACT PICTORIAL ABSTRACT PICTORIAL ABSTRACT General Isolation Procedure A glass column 18 inches in height and 1 inch internal diameter was used
for the fractionation of the crude extracts. Twenty (20) mL fractions Figure 1: Chemical structures of taraxerol (1), taraxeryl acetate (2), α-amyrin
acetate (3), and β-amyrin acetate (4) from Hoya paziae. Figure 1: Chemical structures of taraxerol (1), taraxeryl acetate (2), α-amyrin
acetate (3), and β-amyrin acetate (4) from Hoya paziae. Pharmacognosy Journal, Vol 8, Issue 5, Sep-Oct, 2016 487 Borlagdan et al.: Hoya paziae Borlagdan et al.: Hoya paziae were collected. A glass column 12 inches in height and 0.5 inch internal
diameter was used for the rechromatography of fractions from the crude
extracts. Two (2) mL fractions were collected. Fractions with spots of
the same Rf value were combined and rechromatographed in appropriate
solvent systems until TLC pure isolates were obtained. Rechromatog
raphy and final purifications were conducted using Pasteur pipettes as
columns. One (1) mL fractions were collected. These results indicate that H. paziae shares similar chemical characteristics
with other members of the genus Hoya: H. wayetii,4 H. buotii5 and
H. diversifolia6 which yielded taraxerol (1); and H.multiflora3 which
afforded α-amyrin acetate (3) and β-amyrin acetate (4). This is the first
report on the isolation of taraxeryl acetate from Philippine native hoyas. SUMMARY • This is the first study on the chemical constituents of Hoya paziae, an
ornamental plant native to the Philippines. • The dichloromethane extracts of the stems of H. paziae yielded the tri
terpenes taraxerol, taraxeryl acetate, α-amyrin acetate, and β-amyrin
acetate. • H. paziae shares similar chemical characteristics with other members of
the genus Hoya: H. wayetii, H. buotii and H. diversifolia which yielded
taraxerol; and H. multiflora which afforded α-amyrin acetate and β-amyrin
acetate. • This is the first report on the isolation of taraxeryl acetate from Philippine
native hoyas. ABOUT AUTHORS Consolacion Y. Ragasa: Obtained her Ph. D. in Chemistry from the University of the Philippines - Diliman. Dr. Ragasa is a Full Professor of the Chemistry Department and a University Fellow of De La Salle University - Ma
nila. Her research focuses on the isolation and structure elucidation of the chemical constituents of Philippine
plants. She has published extensively in natural products, phytochemistry and pharmacognosy journals. Dr. Ragasa was awarded the Pillar of Lasallian Excellence Award in Research (2013), National Research Council of
the Philippines Achievement Award in Chemical Research (2003), Philippine Federation of Chemistry Societies
Achievement Award in Chemical Research (2002), and St. Miguel Febres Cordero Research Award (SY 2000-
2001). She is a member of the editorial board of several international and national journals. 488 Pharmacognosy Journal, Vol 8, Issue 5, Sep-Oct, 2016 Borlagdan et al.: Hoya paziae Fernando B. Aurigue: Obtained his Master of Science in Plant Breeding (Horticulture) from the University of
the Philippines - Los Baños, College, Laguna, Philippines where he also obtained his Bachelor of Science in Ag
riculture, Major in Horticulture, Minor in Plant Tissue Culture and Embryology. He is a Professional Regulations
Commission-Registered Agriculturist, an accredited researcher of the National Research Council of the Philip
pines, a member of the Philippine Council for Agriculture, Aquatic and Natural Resources Research and Devel
opment Pool of Experts in Medicinal and Ornamental Crops, and a Senior Science Research Specialist at the
Philippine Nuclear Research Institute-Department of Science and Technology. His research works on the genus
*Hoya* have been recognized by the Flora Malesiana Foundation and his contributions to the advancement
of Horticulture in the Philippines made him an Honorary Member of the Philippine Horticultural Society, Inc. Ian van Altena: Is a lecturer in chemistry in the School of Environmental and Life Sciences, The University
of Newcastle–Australia. He lectures courses in organic and environmental chemistry and conducts research
programs in the area of natural products chemistry. He has a particularly interest in medicinal plants, algal
chemotaxonomy and marine chemical ecology. Day-to-day research is driven by the isolation of compounds
from plants and marine invertebrate and their structure elucidation mainly by nuclear magnetic resonance
spectroscopy and mass spectrometry. Melissa Sausa Borlagdan: Is a graduate student of De La Salle University taking Master of Science in Chem
istry. She graduated Bachelor of Science in Biochemistry at the University of the Philippines –Manila. Pharmacognosy Journal, Vol 8, Issue 5, Sep-Oct, 2016 ABOUT AUTHORS She is
currently employed as Science Research Specialist I at the Food and Nutrition Research Institute Department
of Science and Technology (FNRI-DOST). She works on projects studying nutrient availability, dietary fiber com
position, phytochemical and glycemic index of different food sources. 489 Pharmacognosy Journal, Vol 8, Issue 5, Sep-Oct, 2016
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Application of dry olive residue-based biochar in combination with arbuscular mycorrhizal fungi enhances the microbial status of metal contaminated soils
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Application of dry olive
residue‑based biochar
in combination with arbuscular
mycorrhizal fungi enhances
the microbial status of metal
contaminated soils José A. Siles1,7, Inmaculada García‑Romera2,7, Tomas Cajthaml3,4, Jorge Belloc2,
Gloria Silva‑Castro2, Jirina Szaková5, Pavel Tlustos5 & Mercedes Garcia‑Sanchez6* Biochar made-up of dry olive residue (DOR), a biomass resulting from the olive oil extraction industry,
has been proposed to be used as a reclamation agent for the recovery of metal contaminated soils. The aim of the present study was to investigate whether the soil application of DOR-based biochar
alone or in combination with arbuscular mycorrhizal fungi (AMF) leads to an enhancement in the
functionality and abundance of microbial communities inhabiting metal contaminated soils. To study
that, a greenhouse microcosm experiment was carried out, where the effect of the factors (i) soil
application of DOR-based biochar, (ii) biochar pyrolysis temperature (considering the variants 350
and 500 °C), (iii) soil application dose of biochar (2 and 5%), (iv) soil contamination level (slightly,
moderately and highly polluted), (v) soil treatment time (30, 60 and 90 days) and (vi) soil inoculation
with Funneliformis mosseae (AM fungus) on β-glucosidase and dehydrogenase activities, FA (fatty
acid)-based abundance of soil microbial communities, soil glomalin content and AMF root colonization
rates of the wheat plants growing in each microcosm were evaluated. Biochar soil amendment did
not stimulate enzyme activities but increased microbial abundances. Dehydrogenase activity and
microbial abundances were found to be higher in less contaminated soils and at shorter treatment
times. Biochar pyrolysis temperature and application dose differently affected enzyme activities, but
while the first factor did not have a significant effect on glucosidase and dehydrogenase, a higher
biochar dose resulted in boosted microbial abundances. Soil inoculation with F. mosseae favored the
proliferation of soil AMF community and increased soil glomalin content as well as rates of AMF root
colonization. This factor also interacted with many of the others evaluated to significantly affect
soil enzyme activities, microbial abundances and AMF community. Our results indicate that the
application of DOR-based biochar along with AMF fungi is an appropriate approach to improve the
status of microbial communities in soils with a moderate metal contamination at short-term. Contamination by metals has generally chronic consequences for soils since most of these pollutants are not sub-
jected to microbial or chemical degradation and remain in these ecosystems for long-lasting periods, compromis-
ing the quality services provided by soils and negatively impacting soil life and its functions1. From a microbial 1Department of Plant & Microbial Biology, University of California at Berkeley, Berkeley, CA, USA. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports/ perspective, metals have shown to have a detrimental impact on soil microbial functionality2,3, abundance4,5 and
taxonomic diversity6. The extent of this impact is dependent on the type and level of metal contamination and
the type of soil considered4,5. Many soils chronically contaminated with metals are characterized by harboring
microbial communities with a poor microbial activity and taxonomically restricted to metal-resistant groups,
which limits their functional capabilities7,8. Soil microorganisms can be used as indicators of ecosystem quality
because of their capability to quickly respond to environmental shifts. Likewise, they play a pivotal role in the
functioning of the entire ecosystem as they are involved in soil organic matter (SOM) decomposition, nutrient
cycling, maintaining soil structure and stablishing symbiotic relationships with plants9,10. Therefore, effective
reclamation treatments of metal polluted soils should not only include strategies to reduce the bioavailability of
metals, but also to enhance the status of the autochthonous microbial communities. Biochar has attracted great attention to be used as a reclamation agent for metal contaminated soils in the last
few years11. This carbon-rich material is obtained through pyrolysis and is characterized by having an alkaline pH,
a porous structure, a large surface area, a high cation exchange capacity (CEC) and abundant surface functional
groups12,13. Biochar can directly (through complexation, cation exchange, electrostatic interactions, reduction and
precipitation processes) or indirectly (through affecting soil pH, CEC and SOM contents) reduce the mobility and
bioavailability of metals and their transfer to other ecosystem as well as their uptake by plants14,15. Biochars based
on municipal and animal wastes, wood, crop residues as well as biosolids have shown to reduce the bioavailability
of metals in contaminated soils, as reviewed by Hean et al.16 and Yuan et al.17. However, in most of the cases, it is
not well known whether the metal immobilization mediated by biochar is concomitant with an enhancement of
the soil microbial status. In this way, integrative studies such as that of Xu et al.18, showing that biochar made of
macadamia nutshell reduced soil Cd and Pb bioavailability and increased microbial respiration, abundance and
carbon use efficiency, should be performed more commonly. Likewise, it is important to assess how factors that
affect the reclamation effectiveness of biochar such as the inherent properties of the starting material, pyrolysis
conditions (temperature and time), amendment doses, mixing depth, soil properties and climate conditions13,14
impact soil microbial communities during reclamation processes. www.nature.com/scientificreports/ p
g
p
Arbuscular mycorrhizal fungi (AMF) establish symbiotic relationships with plants and expand the interface
between plants and soil environment19. AMF ameliorate plant metal stress and enhance plant abilities in terms of
nutrient and water uptake as well as pathogen resistance20,21 in different degrees depending on the AMF species
considered20. This microbial group also plays an important role in reducing soil metal bioavailability by binding
metals to the fungal structures and by complexing the pollutants through glomalin, an extracellular glycoprotein
produced by all the AMF22. Metals, especially at high levels, can negatively affect the germination of AMF spores,
the growth of extraradical mycelium and the mycorrhizal colonization19,23. The initial successful establishment
of AMF symbiosis may thus be challenging in contaminated soils. The application of organic amendments such
as biochar has shown to enhance AMF performance by increasing the availability of nutrients, changing soil
physicochemical properties and improving spore germination24. Additionally, a number of studies have reported
that AMF inoculation and biochar application have synergistic effects on promoting soil fertility and plant yield
in metal contaminated soils25. In the Mediterranean region, the olive oil industry produces huge amounts of an organic-matter-rich waste
called dry olive residue (DOR), which has been proposed to be used as reclamation agent for metal polluted
soils26. As it was demonstrated by Hovorka et al.27, DOR, especially after being transformed by saprotrophic fungi,
has capabilities to absorb Cd, Zn and Pb. However, desorption experiments showed that metals were weakly and
unstably immobilized. In this context, it has been hypothesized that the conversion of DOR into biochar may
improve the capabilities of DOR to keep metals bound to a higher extent. Additionally, soil application of fungal
transformed-DOR in combination with AMF is a viable practice to restore microbial functionality and biomass
in soils chronically contaminated with metals28. Integrative studies assessing the potential of DOR-based biochar
along with AMF application to reduce the bioavailability of metals and to enhance the activity and abundance of
microbial communities in chronically metal polluted soils are missing. It has been recently found that DOR-based
biochar decreased available contents of Cd and produced an ambiguous effect on the mobility of As, Pb and Zn
in three soils presenting a pollution gradient collected from the same location15. www.nature.com/scientificreports/ We aimed in the present work at
investigating whether the addition of DOR-based biochar alone or in combination with the AMF Funneliformis
mosseae leads to an enhancement in the functionality and abundance of microbial communities inhabiting metal
contaminated soils in concomitance with an alleviation of the metal mobility. Application of dry olive
residue‑based biochar
in combination with arbuscular
mycorrhizal fungi enhances
the microbial status of metal
contaminated soils 2Department
of Soil Microbiology and Symbiotic Systems, Estación Experimental del Zaidín, Consejo Superior de
Investigaciones Científica (EEZ-CSIC), Granada, Spain. 3Institute of Microbiology of the Academy of Sciences,
Prague, Czech Republic. 4Faculty of Science, Institute for Environmental Studies, Charles University, Prague,
Czech Republic. 5Department of Agro‑Environmental Chemistry and Plant Nutrition, Faculty of Agrobiology,
Food and Natural Resources, Prague, Czech Republic. 6Eco&Sols, CIRAD, INRAE, IRD, Institut Agro Montpellier,
Université Montpellier, Montpellier, France. 7These authors contributed equally: José A. Siles and Inmaculada
García-Romera. *email: garcia.sanchez.mercedes@gmail.com | https://doi.org/10.1038/s41598-022-17075-5 Scientific Reports | (2022) 12:12690 www.nature.com/scientificreports/ Results
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i Evaluation of the factors DOR‑based biochar application, soil contamination level, soil treat‑
ment time, F. mosseae inoculation and their interactions throughout the experiment. In a
first analysis, we evaluated the effect of the factors soil application of DOR-based biochar (B), soil contamina-
tion level (S), soil treatment-time (T), F. mosseae (M) inoculation and their interactions on the status of soil
microbial communities (soil enzyme activities, FA-based microbial abundance), EE-GRSPcontent and AMF
root colonization percentage (Table 1, Figs. 1, 2, 3). The soil enzyme activities assayed, namely β-glucosidase
and dehydrogenase, were differently influenced by the factors considered and their interactions. Specifically,
β-glucosidase activity was reduced after soil amendment with DOR-based biochar, while dehydrogenase was
not significantly affected. Soil contamination level was a significant factor for both activities, with the least con-
taminated soil showing significantly higher β-glucosidase and dehydrogenase activities throughout the experi-
ment. Interestingly, the interaction between biochar application and soil contamination level was significant
only for the β-glucosidase activity (B × S, p < 0.001). Longer treatment times led to a significant increase and
decrease in β-glucosidase and dehydrogenase activities, respectively. The inoculation of F. mosseae boosted the
soil β-glucosidase activity, which was also affected by the interaction between F. mosseae inoculation and biochar https://doi.org/10.1038/s41598-022-17075-5 Scientific Reports | (2022) 12:12690 | www.nature.com/scientificreports/ Table 1. Results of MANOVA and post-hoc analyses on the effect of the factors application of DOR-based
biochar, soil contamination level (“low”, “medium” and “high”), soil treatment time (30, 60 and 90 days),
inoculation of F. mosseae and their interactions on soil enzyme activities (β-glucosidase and dehydrogenase),
PLFA- and NLFA-based microbial abundance, easily extractable glomalin-related soil protein content
(EE-GRSP) and AMF-root colonization rate. Median values for each variable at each factor level are also
shown. a For post-hoc tests, the values are expressed as µPNP (g soil dm)−1 (1 h)−1. b For post-hoc tests, the
values are expressed as μmol INTF (g soil dm)−1 (1 h)−1. c For post-hoc tests, the values are expressed as µg
PLFA or NLFA (g soil dm)−1. d For post-hoc tests, the values are expressed as mg (g soil dm)−1. e For post-hoc
tests, the values are expressed as %. f F-valuesignificance (degrees of freedom). Results
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i For MANOVA analyses, F-values
in bold denote statistical significance (p ≤ 0.05), significance levels are shown at *p < 0.05, **p < 0.01 and
***p < 0.001; for post-hoc Tukey’s HSD tests, median values of followed by different letters are significantly
different (p < 0.05). Factors
β-glucosidasea
Dehydrogenaseb
PLFAtot
c
PLFAbac
c
PLFAGram+
c
PLFAGram-
c
PLFAact
c
PLFAfun
c
NLFAAMF
c
EE-GRSPd
AMF-root
colonizatione
Biochar application (B)
Fsignificance
(df)f
47.15*** (1)
0.19 (1)
36.04***
(1)
33.1*** (1)
11.44*** (1)
75.5*** (1)
8.74*** (1)
47.00***
(1)
13.48*** (1)
2.70 (1)
1.83 (1)
Post-hoc test
No appli-
cation
1283.79 b
0.39 a
10.54 a
7.02 a
2.67 a
2.87 a
1.07 a
0.17 a
0.63 a
0.16 a
5.00 a
Applica-
tion
1191.40 a
0.39 a
13.80 b
9.44 b
3.26 b
4.10 b
1.25 b
0.28 b
0.77 b
0.16 a
6.00 a
Soil contamination level (S)
Fsignificance
(df)f
18.65*** (2)
2251.15*** (2)
314.44***
(2)
495.05***
(2)
258.73***
(2)
340.47***
(2)
555.19***
(2)
125.60***
(2)
872.95***
(2)
2553.75*** (2)
29.00*** (2)
Post-hoc test
Low
1459.21 b
0.45 b
14.95 b
10.53 b
4.11 b
4.33 b
1.54 c
0.20 b
0.87 b
0.17 a
7.00 b
Medium
1127.80 a
0.46 b
15.25 b
10.95 c
3.85 b
4.96 c
1.31 b
0.38 c
1.36 c
0.16 a
9.00 c
High
1239.45 a
0.14 a
7.93 a
4.96 a
1.73 a
2.26 a
0.58 a
0.15 a
0.16 a
0.19 b
5.00 a
Soil treatment time (T)
Fsignificance
(df)f
485.17*** (2)
14.68*** (2)
52.40***
(2)
81.44*** (2)
14.00*** (2)
98.24***
(2)
19.41***
(2)
55.25***
(2)
64.06*** (2)
821.10*** (2)
362.20*** (2)
Post-hoc test
30 days
1119.52 a
0.45 b
14.96 c
10.13 b
3.29 b
5.20 c
1.30 c
0.32 b
0.58 a
0.17 a
14.00 c
60 days
1196.07 b
0.42 b
12.62 b
8.55 b
2.98 b
3.80 b
1.23 b
0.30 b
1.16 c
0.19 c
5.75 b
90 days
1341.95 c
0.38 a
12.54 a
8.58 a
2.50 a
3.34 a
1.17 a
0.20 a
0.70 b
0.15 b
3.55 a
F. Results
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i mosseae inoculation (M)
Fsignificance
(df)f
8.81*** (1)
2.62 (1)
0.74 (1)
23.97*** (1)
3.85 (1)
1.66 (1)
5.57* (1)
1.07 (1)
24.56*** (1)
3122.76*** (1)
24.29*** (1)
Post-hoc test
No inocu-
lation
1204.16 a
0.43 a
12.79 a
8.90 b
3.07 a
3.78 a
1.19 b
0.23 a
0.78 a
0.16 a
5.00 a
Inocula-
tion
1270.64 b
0.33 a
13.09 a
8.89 a
3.16 a
4.13 a
1.16 a
0.29 a
0.86 b
3.41 b
8.57 b
Significant
interac-
tions
B × S***, B × M*,
S × T***,
S × M***,
T × M***,
B × S × M***,
S × T × M*
S × T***,
S × M***, T × M*,
B × T × M***,
S × T × M***
B × T***,
B × M***,
S × T***,
B × S × T***
B × T***,
B × M***,
S × T***,
S × M***,
M × T***,
B × S × T***,
S × T × M***
B × T***,
S × T***,
S × M***,
B × S × T***,
S × T × M***
B × T***,
B × M***,
S × T***,
B × S × T***
B × S***,
B × T***,
B × M***,
S × T***,
S × M***,
B × S × T*
B × M***,
S × T***,
B × S × T*
S × T***,
S × M***,
B × S × T*,
S × T × M***
B × S*, S × T***,
S × M***,
T × M***,
S × T × M***,
B × S × T × M***
B × T***,
S × T***,
S × M***,
M × T***,
S × T × M* Table 1. Results of MANOVA and post-hoc analyses on the effect of the factors application of DOR-based
biochar, soil contamination level (“low”, “medium” and “high”), soil treatment time (30, 60 and 90 days),
inoculation of F. mosseae and their interactions on soil enzyme activities (β-glucosidase and dehydrogenase),
PLFA- and NLFA-based microbial abundance, easily extractable glomalin-related soil protein content
(EE-GRSP) and AMF-root colonization rate. Median values for each variable at each factor level are also
shown. a For post-hoc tests, the values are expressed as µPNP (g soil dm)−1 (1 h)−1. b For post-hoc tests, the
values are expressed as μmol INTF (g soil dm)−1 (1 h)−1. Results
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i c For post-hoc tests, the values are expressed as µg
PLFA or NLFA (g soil dm)−1. d For post-hoc tests, the values are expressed as mg (g soil dm)−1. e For post-hoc
tests, the values are expressed as %. f F-valuesignificance (degrees of freedom). For MANOVA analyses, F-values
in bold denote statistical significance (p ≤ 0.05), significance levels are shown at *p < 0.05, **p < 0.01 and
***p < 0.001; for post-hoc Tukey’s HSD tests, median values of followed by different letters are significantly
different (p < 0.05). Table 1. Results of MANOVA and post-hoc analyses on the effect of the factors application of DOR-based
biochar, soil contamination level (“low”, “medium” and “high”), soil treatment time (30, 60 and 90 days),
inoculation of F. mosseae and their interactions on soil enzyme activities (β-glucosidase and dehydrogenase),
PLFA- and NLFA-based microbial abundance, easily extractable glomalin-related soil protein content
(EE-GRSP) and AMF-root colonization rate. Median values for each variable at each factor level are also
shown. a For post-hoc tests, the values are expressed as µPNP (g soil dm)−1 (1 h)−1. b For post-hoc tests, the
values are expressed as μmol INTF (g soil dm)−1 (1 h)−1. c For post-hoc tests, the values are expressed as µg
PLFA or NLFA (g soil dm)−1. d For post-hoc tests, the values are expressed as mg (g soil dm)−1. e For post-hoc
tests, the values are expressed as %. f F-valuesignificance (degrees of freedom). For MANOVA analyses, F-values
in bold denote statistical significance (p ≤ 0.05), significance levels are shown at *p < 0.05, **p < 0.01 and
***p < 0.001; for post-hoc Tukey’s HSD tests, median values of followed by different letters are significantly
different (p < 0.05). Table 1. Results of MANOVA and post-hoc analyses on the effect of the factors application of DOR-based
biochar, soil contamination level (“low”, “medium” and “high”), soil treatment time (30, 60 and 90 days),
inoculation of F. mosseae and their interactions on soil enzyme activities (β-glucosidase and dehydrogenase),
PLFA- and NLFA-based microbial abundance, easily extractable glomalin-related soil protein content
(EE-GRSP) and AMF-root colonization rate. Median values for each variable at each factor level are also
shown. a For post-hoc tests, the values are expressed as µPNP (g soil dm)−1 (1 h)−1. b For post-hoc tests, the
values are expressed as μmol INTF (g soil dm)−1 (1 h)−1. Results
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i c For post-hoc tests, the values are expressed as µg
PLFA or NLFA (g soil dm)−1. d For post-hoc tests, the values are expressed as mg (g soil dm)−1. e For post-hoc
tests, the values are expressed as %. f F-valuesignificance (degrees of freedom). For MANOVA analyses, F-values
in bold denote statistical significance (p ≤ 0.05), significance levels are shown at *p < 0.05, **p < 0.01 and
***p < 0.001; for post-hoc Tukey’s HSD tests, median values of followed by different letters are significantly
different (p < 0.05). application (B × M, p < 0.05). On the other hand, although the AM fungus presence or absence did not signifi-
cantly modify the dehydrogenase activity, the interaction of this factor with the soil treatment time and the factor
biochar application or soil contamination level was significant (B × T × M, p < 0.001; S × T × M, p < 0.001, Table 1,
Fig. 1).i application (B × M, p < 0.05). On the other hand, although the AM fungus presence or absence did not signifi-
cantly modify the dehydrogenase activity, the interaction of this factor with the soil treatment time and the factor
biochar application or soil contamination level was significant (B × T × M, p < 0.001; S × T × M, p < 0.001, Table 1,
Fig. 1). g
All the PLFA-based microbial fractions were significantly boosted by the soil amendment with DOR-based
biochar (Table 1). Soil contamination level and treatment time were also significant factors for all the groups
of PLFA markers, with soils “low” and “medium” showing significantly higher microbial abundances than the
soil “high” and with the highest biomass contents being obtained after 30 and 60 days of the experiment. The
microbial communities inhabiting the three soils responded to biochar addition in terms of abundance in a
time-dependent manner as demonstrated by the significance of the interaction B × S × T for all the microbial
fractions (Table 1, Fig. 2). A decline in the population of bacteria and actinobacteria was found in response to F. mosseae inoculation and this factor resulted not to be significant for the rest of PLFA-based microbial groups. Yet, https://doi.org/10.1038/s41598-022-17075-5 Scientific Reports | (2022) 12:12690 | www.nature.com/scientificreports/ Figure 1. Results
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i Box plots showing levels of β-glucosidase and dehydrogenase activities in soils contaminated at low,
medium and high levels and amended at the doses of 2 and 5% with DOR-based biochar produced at 350 and
500 °C after 30, 60 and 90 days of experiment. Controls refers to unamended soils. Soils were inoculated with
the AMF F. mosseae. The boxes represent the interquartile range (IQR) between the first and third quartiles
(25th and 75th percentiles, respectively) and the vertical line inside the box defines the median. Whiskers
represent the lowest and highest values within 1.5 times the IQR from the first and third quartiles, respectively. Dots represent outliers. Figure 1. Box plots showing levels of β-glucosidase and dehydrogenase activities in soils contaminated at low,
medium and high levels and amended at the doses of 2 and 5% with DOR-based biochar produced at 350 and
500 °C after 30, 60 and 90 days of experiment. Controls refers to unamended soils. Soils were inoculated with
the AMF F. mosseae. The boxes represent the interquartile range (IQR) between the first and third quartiles
(25th and 75th percentiles, respectively) and the vertical line inside the box defines the median. Whiskers
represent the lowest and highest values within 1.5 times the IQR from the first and third quartiles, respectively. Dots represent outliers. the interaction between soil biochar addition and AMF application (B × M) had a significant effect on PLFAtot,
PLFAbac, PLFAGram-, PLFAact and PLFAfun. d
h d ff
d
l
h
f
l
d
h bac
Gram
act
fun
Regarding the different parameters measured in relation to the community of AMF, soil amendment with
DOR-based biochar significantly increased NLFAAMF in soil; nevertheless, soil contents of EE-GRSP and the
percentage of AMF root colonization did not significantly vary (Table 1, Fig. 3). Soil contamination level was a
significant factor for NLFAAMF, EE-GRSP and AMF root colonization. Soil NLFA-based abundance of AMF and
AMF root colonization were significantly higher in soil “medium” compared with the other two soils; instead,
the highest values for EE-GRSP were recorded in soil “high” throughout the experiment (Fig. 3). The highest
soil contents for NLFAAMF and EE-GRSP were obtained at time 60 days, while the percentage of AMF root
colonization was the highest after 30 days of treatment; i.e., the factor soil treatment was significant for the three
parameters. As expected, the inoculation of F. Results
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i mosseae resulted effective at boosting NLFAAMF, soil content of
EE-GRSP and AMF root colonization. However, the interaction between biochar and AMF inoculation was not
significant for any of them (Table 1, Fig. 3). The interaction B × S × T × M showed to be significant (p < 0.001) only
for EE-GRSP. Spearman correlation analyses showed that contents of NLFAAMF and EE-GRSP were significantly
positively correlated (ρ = 0.138, p = 0.009). Percentage of AMF root colonization did not significantly correlate
with NLFAAMF (ρ = 0.101, p = 0.058) nor with EE-GRSP contents (ρ = 0.027, p = 0.616). Evaluation of the factors pyrolysis temperature and application dose of the DOR‑based bio‑
char, soil contamination level, soil treatment time, F. mosseae inoculation and their interac‑
tions. In a second analysis, only the amended microcosms were considered in order to evaluate the effect of
the factors biochar pyrolysis temperature (P) and biochar amendment dose (D) along with the soil contamina-
tion level (S), soil treatment time (T), F. mosseae inoculation (M) and their interactions on the different param-
eters considered in the study (Tables 2, 3, 4, Figs. 1, 2, 3). Both production temperature and application dose
of DOR-based biochar significantly affected β-glucosidase and dehydrogenase activities, but opposite patterns
were observed. While β-glucosidase activity was significantly higher after soil application of BC500 (compared
with BC350) and at a dose of 2% (compared with a 5% dose), dehydrogenase was stimulated to a significantly
higher extent by BC350 and by a dose of 5% (Table 2, Fig. 1). The factors biochar production temperature and
application dose significantly interacted with soil contamination level for β-glucosidase (P × D × S, p < 0.001) and
with soil contamination and soil treatment time for dehydrogenase (P × D × S × T, p < 0.001). The inoculation of
F. mosseae did not significantly alter the β-glucosidase activity, but the interaction between AMF application
and biochar pyrolysis temperature and application dose was significant (P × D × M, p < 0.05, Table 2, Fig. 1). Significantly lower levels of dehydrogenase activity were obtained in the F. mosseae-inoculated microcosms. The https://doi.org/10.1038/s41598-022-17075-5 Scientific Reports | (2022) 12:12690 | www.nature.com/scientificreports/ Figure 2. Results
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i Box plots showing PLFA-based total, bacterial and fungal abundance in soils contaminated at low,
medium and high levels and amended at the doses of 2 and 5% with DOR-based biochar produced at 350 and
500 °C after 30, 60 and 90 days of experiment. Controls refers to unamended soils. Soils were inoculated with
the AMF F. mosseae. The boxes represent the interquartile range (IQR) between the first and third quartiles
(25th and 75th percentiles, respectively) and the vertical line inside the box defines the median. Whiskers
represent the lowest and highest values within 1.5 times the IQR from the first and third quartiles, respectively. Dots represent outliers. Figure 2. Box plots showing PLFA-based total, bacterial and fungal abundance in soils contaminated at low,
medium and high levels and amended at the doses of 2 and 5% with DOR-based biochar produced at 350 and
500 °C after 30, 60 and 90 days of experiment. Controls refers to unamended soils. Soils were inoculated with
the AMF F. mosseae. The boxes represent the interquartile range (IQR) between the first and third quartiles
(25th and 75th percentiles, respectively) and the vertical line inside the box defines the median. Whiskers
represent the lowest and highest values within 1.5 times the IQR from the first and third quartiles, respectively. Dots represent outliers. nteraction between the factor AMF inoculation and dose of application, soil contamination and treatment time
valuated was significant for dehydrogenase (D × S × T × M, p < 0.001).hiff i
The factor DOR pyrolysis temperature did not have a significant effect on the abundance of the different
PLFA-based microbial fractions, except for fungi (PLFAfun increased after soil BC350 application in compari-
son with BC500 addition, Table 3, Fig. 2). Instead, the factor dose was significant for all PLFA-based microbial
groups, except Actinobacteria, with the highest microbial abundances recorded at a dose of 5% (Table 3, Fig. 2). The factor dose showed to significantly affect the soil microbial abundance not only by itself, but also by its
interaction with the factors soil contamination level and soil treatment time for all the microbial groups except
fungi (D × S × T, p < 0.05, Table 3, Fig. 2). Soil inoculation with F. mosseae significantly increased total biomass,
Gram- and fungal PLFA contents, whilst bacterial contents decreased (Table 3, Fig. 2). Results
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i For PLFAtot, PLFAbac,
PLFAGram+, PLFAGram- and PLFAfun, the interactions P × D × S × M (p < 0.05) or P × D × T × M (p < 0.001) showed
to be significant (Table 3).il gi
DOR pyrolysis temperature did not significantly influence soil contents of mycorrhizal NLFA marker and
glomalin nor the percentages of AMF root colonization (Table 4, Fig. 3). However, increased values for all these
parameters were observed following DOR-based biochar application at a dose of 5% in comparison with the 2%
dose. The interaction of the factors P × D × T × M (p < 0.001) was significant only for AMF root colonization rate. The presence of F. mosseae in the rhizosphere of wheat plants significantly boosted NLFA markers for AMF, soil
glomalin contents and the AMF root colonization percentage (Table 4, Fig. 3). When only amended microcosms
are considered, soil EE-GRSP contents and NLFAAMF were significantly positively correlated (Spearman rank https://doi.org/10.1038/s41598-022-17075-5 Scientific Reports | (2022) 12:12690 | www.nature.com/scientificreports/ Figure 3. Box plots showing levels of NLFA-based abundance of AMF, easily extractable glomalin-related
soil content (EE-GRSP) and AMF-root colonization rate of wheat plants grown in soils contaminated at low,
medium and high levels and amended at the doses of 2 and 5% with DOR-based biochar produced at 350 and
500 °C after 30, 60 and 90 days of experiment. Control refers to unamended soils. Soils were inoculated with the
AMF F. mosseae. The boxes represent the interquartile range (IQR) between the first and third quartiles (25th
and 75th percentiles, respectively) and the vertical line inside the box defines the median. Whiskers represent
the lowest and highest values within 1.5 times the IQR from the first and third quartiles, respectively. Dots
represent outliers. Figure 3. Box plots showing levels of NLFA-based abundance of AMF, easily extractable glomalin-related
soil content (EE-GRSP) and AMF-root colonization rate of wheat plants grown in soils contaminated at low,
medium and high levels and amended at the doses of 2 and 5% with DOR-based biochar produced at 350 and
500 °C after 30, 60 and 90 days of experiment. Control refers to unamended soils. Soils were inoculated with the
AMF F. mosseae. The boxes represent the interquartile range (IQR) between the first and third quartiles (25th
and 75th percentiles, respectively) and the vertical line inside the box defines the median. Results
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i Whiskers represent
the lowest and highest values within 1.5 times the IQR from the first and third quartiles, respectively. Dots
represent outliers. correlation; ρ = 0.158, p = 0.008). However, mycorrhization rates were not significantly correlated with NLFAAMF
(ρ = 0.113, p = 0.058) nor with soil EE-GRSP contents (ρ = 0.0155, p = 0.795). www.nature.com/scientificreports/ www.nature.com/scientificreports/ Factors
β-glucosidasea
Dehydrogenaseb
DOR pyrolysis temperature (P)
Fsignificance (df)c
10.81*** (1)
7.52*** (1)
Post-hoc test
350 °C
1162.88 a
0.40 b
500 °C
1200.48 b
0.39 a
Biochar application dose (D)
Fsignificance (df)c
51.80*** (1)
15.86*** (1)
Post-hoc test
2%
1232.74 b
0.38 a
5%
1148.28 a
0.41 b
Soil contamination level (S)
Fsignificance (df)f
46.46*** (2)
5044.91*** (2)
Post-hoc test
Low
1436.14 b
0.45 b
Medium
1122.88 a
0.46 b
High
1229.23 a
0.14 a
Soil treatment time (T)
Fsignificance (df)f
936.05*** (2)
22.84*** (2)
Post-hoc test
30 days
1100.52 a
0.41 b
60 days
1191.51 b
0.42 b
90 days
1317.82 c
0.38 a
F. mosseae inoculation (M)
Fsignificance (df)f
0.51 (1)
18.17*** (1)
Post-hoc test
No inoculation
1196.07 a
0.44 b
Inoculation
1183.59 a
0.38 a
Significant interactions
P × D*, P × M*, D × M*, S × T***, T × M***, P × D × S***, P × S × T*,
P × D × M*, D × S × M***
D × S*, D × T***, D × M*, S × T***, S × M***, M × T*, P × D × T***,
D × S × T***, D × S × M***, D × T × M***, S × T × M***, P × D × S × T***,
D × S × T × M*** Table 2. Results of MANOVA and post-hoc analyses on the effect of the factors pyrolysis temperature of
DOR-based biochar (350 and 500 °C), application dose of DOR-based biochar (2 and 5%), soil contamination
level (“low”, “medium” and “high”), soil treatment time (30, 60 and 90 days), inoculation of F. mosseae (AMF)
and their interactions on soil enzyme activities (β-glucosidase and dehydrogenase) considering only amended
microcosms. Median values for each variable at each factor level are also shown. a For post-hoc tests, the values
are expressed as µPNP (g soil dm)−1 (1 h)−1. b For post-hoc tests, the values are expressed as μmol INTF (g soil
dm)−1 (1 h)−1. c F-valuesignificance (degrees of freedom). For MANOVA analyses, F-values in bold denote statistical
significance (p ≤ 0.05), significance levels are shown at *p < 0.05, **p < 0.01 and ***p < 0.001; for post-hoc
Tukey’s HSD tests, median values followed by different letters are significantly different (p < 0.05). Table 2. www.nature.com/scientificreports/ These findings are in line with other works stating that soil enzyme activities respond
to biochar application in various ways, depending on the type of enzyme and biochar, the biochar application
rate and soil properties32. In this way, biochars might affect soil microbial activities by means of the following
mechanisms, among others: (i) enriching soil with specific nutrients and substrates, which induces the microbial
production of enzymes; (ii) adsorbing extracellular enzymes and/or substrates on the surface or blocking the
reaction site of enzymes in a way that regulates their affinity for substrates; (iii) indirectly, by producing shifts nor dehydrogenase; in fact, β-glucosidase was inhibited. Although soil amendment with biochar tends to have a
positive effect on enzyme activities, decreased values in β-glucosidase activity followed soil application of biochar
derived from sewage sludge at rates of 4% and 8% (w/w) were reported33. In our experiment, the significance of
the interaction between biochar application and soil contamination level points out that the effect of biochar on
this enzyme is dependent on the pollution level of the soil. On the other hand, temperature of pyrolysis is a key
parameter in controlling the properties of biochar, especially to designate the short or long-term availabilities
of C in soil as well as the accumulation of mineral nutrients and/or OM combustion residue34. As demonstrated
by Vejvodová et al.15, BC500 was richer than BC350 in C and some other mineral nutrients (except N). In our
study, pyrolytic temperature of biochar significantly influenced both enzyme activities, although a clear pat-
tern was not observed. The highest values for β-glucosidase were found after the application of BC500, while
dehydrogenase was recorded at the highest extent in the microcosms amended with BC350. We also found that
β-glucosidase was inhibited to a higher extent after applying DOR-based biochar at a dose of 5% compared with
the 2% dose. Conversely, dehydrogenase activity was significantly higher after soil amendment at a dose of 5% in
comparison with the 2%. These findings are in line with other works stating that soil enzyme activities respond
to biochar application in various ways, depending on the type of enzyme and biochar, the biochar application
rate and soil properties32. www.nature.com/scientificreports/ Results of MANOVA and post-hoc analyses on the effect of the factors pyrolysis temperature of
DOR-based biochar (350 and 500 °C), application dose of DOR-based biochar (2 and 5%), soil contamination
level (“low”, “medium” and “high”), soil treatment time (30, 60 and 90 days), inoculation of F. mosseae (AMF)
and their interactions on soil enzyme activities (β-glucosidase and dehydrogenase) considering only amended
microcosms. Median values for each variable at each factor level are also shown. a For post-hoc tests, the values
are expressed as µPNP (g soil dm)−1 (1 h)−1. b For post-hoc tests, the values are expressed as μmol INTF (g soil
dm)−1 (1 h)−1. c F-valuesignificance (degrees of freedom). For MANOVA analyses, F-values in bold denote statistical
significance (p ≤ 0.05), significance levels are shown at *p < 0.05, **p < 0.01 and ***p < 0.001; for post-hoc
Tukey’s HSD tests, median values followed by different letters are significantly different (p < 0.05). nor dehydrogenase; in fact, β-glucosidase was inhibited. Although soil amendment with biochar tends to have a
positive effect on enzyme activities, decreased values in β-glucosidase activity followed soil application of biochar
derived from sewage sludge at rates of 4% and 8% (w/w) were reported33. In our experiment, the significance of
the interaction between biochar application and soil contamination level points out that the effect of biochar on
this enzyme is dependent on the pollution level of the soil. On the other hand, temperature of pyrolysis is a key
parameter in controlling the properties of biochar, especially to designate the short or long-term availabilities
of C in soil as well as the accumulation of mineral nutrients and/or OM combustion residue34. As demonstrated
by Vejvodová et al.15, BC500 was richer than BC350 in C and some other mineral nutrients (except N). In our
study, pyrolytic temperature of biochar significantly influenced both enzyme activities, although a clear pat-
tern was not observed. The highest values for β-glucosidase were found after the application of BC500, while
dehydrogenase was recorded at the highest extent in the microcosms amended with BC350. We also found that
β-glucosidase was inhibited to a higher extent after applying DOR-based biochar at a dose of 5% compared with
the 2% dose. Conversely, dehydrogenase activity was significantly higher after soil amendment at a dose of 5% in
comparison with the 2%. Discussion Studies assessing the effectiveness of treatments based on the soil application of organic amendments to recover
metal-contaminated soils should consider not only the effect of such treatments in the reduction of metal bio-
availability and/or mobility, but also their concomitant potential to improve the status of microbial communities
since microbes are indispensable in maintaining healthy and fertile soils. In this way, we proposed in the present
work to investigate the potential of DOR-based biochar application along with AMF inoculation (using the
fungus F. mosseae) in recovering the microbial communities of three soils presenting a metal-pollution gradient
as a complementary study to the previous insights about the DOR-based biochar impact on soil metal behavior
and plant nutrition status reported recently by Vejvodová et al.15. Our work was mainly focused on monitoring soil potential enzyme activities and community abundance
since these parameters have been regarded as useful indicators in assessing the impact of biochar on soil quality29. In fact, soil enzymatic activities have shown to respond faster than other soil variables to soil management and
disturbance processes30. In our experiment, the significantly highest values for β-glucosidase and dehydrogenase
were recorded in the least contaminated soil, which is in line with other works showing that increased metal pol-
lution negatively impacts microbial functionality31. Our preliminary expectations, based on studies such as that
of Pokharel et al.32, were to find an increase in microbial activities after soil amendment with biochar, especially
in the most contaminated soil. However, in our study, DOR-based biochar did not boost β-glucosidase activity https://doi.org/10.1038/s41598-022-17075-5 Scientific Reports | (2022) 12:12690 | www.nature.com/scientificreports/ In this way, biochars might affect soil microbial activities by means of the following
mechanisms, among others: (i) enriching soil with specific nutrients and substrates, which induces the microbial
production of enzymes; (ii) adsorbing extracellular enzymes and/or substrates on the surface or blocking the
reaction site of enzymes in a way that regulates their affinity for substrates; (iii) indirectly, by producing shifts https://doi.org/10.1038/s41598-022-17075-5 Scientific Reports | (2022) 12:12690 | www.nature.com/scientificreports/ Table 3. Results of MANOVA and post-hoc analyses on the effect of the factors pyrolysis temperature of the
DOR-based biochar (350 and 500 °C), application dose of DOR-based biochar (2 and 5%), soil contamination
level (“low”, “medium” and “high”), soil treatment time (30, 60 and 90 days) and inoculation of F. mosseae
(AMF) and their interactions on PLFA-based abundance of the different microbial groups considering only
amended microcosms. Median values for each variable at each factor level are also shown. a For post-hoc
tests, the values are expressed as µg PLFA (g soil dm)-1. b F-valuesignificance (degrees of freedom). For MANOVA
analyses, F-values in bold denote statistical significance (p ≤ 0.05), significance levels are shown at *p < 0.05,
**p < 0.01 and ***p < 0.001; for post-hoc Tukey’s HSD tests, median values followed by different letters are
significantly different (p < 0.05). www.nature.com/scientificreports/ Factors
PLFAtot
a
PLFAbac
a
PLFAGram+
a
PLFAGram-
a
PLFAact
a
PLFAfun
a
DOR pyrolysis temperature (P)
Fsignificance (df)b
0.32 (1)
0.10 (1)
2.64 (1)
0.87 (1)
1.29 (1)
16.03*** (1)
Post-hoc test
350 °C
13.55 a
9.22 a
3.32 a
4.10 a
1.23 a
0.29 b
500 °C
14.01 a
9.69 a
3.21 a
4.08 a
1.25 a
0.25 a
Biochar application dose (D)
Fsignificance (df)b
38.62*** (1)
38.93*** (1)
11.87*** (1)
47.26*** (1)
0.41 (1)
65.38*** (1)
Post-hoc
2%
13.57 a
9.28 a
3.09 a
4.01 a
1.23 a
0.23 a
5%
14.01 b
9.95 b
3.37 b
4.23 b
1.27 a
0.30 b
Soil contamination level (S)
Fsignificance (df)b
632.59*** (2)
908.16*** (2)
502.64*** (2)
524.27*** (2)
931.66*** (2)
181.50*** (2)
Post-hoc test
Low
15.44 b
10.78 b
4.21 c
4.42 b
1.56 c
0.20 b
Medium
15.41 b
11.16 c
3.92 b
4.99 c
1.32 b
0.38 c
High
8.00 a
5.04 a
1.73 a
2.41 a
0.58 a
0.16 a
Soil treatment time (T)
Fsignificance (df)b
35.34*** (2)
69.16*** (2)
0.20 (2)
101.52*** (2)
6.87*** (2)
88.32*** (2)
Post-hoc test
30 days
15.22 c
10.44 c
3.31 a
5.34 c
1.33 b
0.35 b
60 days
13.55 b
9.32 b
3.01 a
3.93 b
1.26 ab
0.31 b
90 days
12.65 a
8.95 a
4.02 a
3.71 a
1.23 a
0.20 a
F. www.nature.com/scientificreports/ b F-valuesignificance (degrees of freedom). For MANOVA
analyses, F-values in bold denote statistical significance (p ≤ 0.05), significance levels are shown at *p < 0.05,
**p < 0.01 and ***p < 0.001; for post-hoc Tukey’s HSD tests, median values followed by different letters are
significantly different (p < 0.05). Table 3. Results of MANOVA and post-hoc analyses on the effect of the factors pyrolysis temperature of the
DOR-based biochar (350 and 500 °C), application dose of DOR-based biochar (2 and 5%), soil contamination
level (“low”, “medium” and “high”), soil treatment time (30, 60 and 90 days) and inoculation of F. mosseae
(AMF) and their interactions on PLFA-based abundance of the different microbial groups considering only
amended microcosms. Median values for each variable at each factor level are also shown. a For post-hoc
tests, the values are expressed as µg PLFA (g soil dm)-1. b F-valuesignificance (degrees of freedom). For MANOVA
analyses, F-values in bold denote statistical significance (p ≤ 0.05), significance levels are shown at *p < 0.05,
**p < 0.01 and ***p < 0.001; for post-hoc Tukey’s HSD tests, median values followed by different letters are
significantly different (p < 0.05). in soil physicochemical properties; and (iv) releasing some small molecules that may act as allosteric regulators
or inhibitors of specific enzymes35. i
Interestingly, although soil amendment with DOR-based biochar decreased β-glucosidase activity, the inocu-
lation of F. mosseae had a significantly positive effect on this activity. Likewise, these two factors significantly
interacted to influence β-glucosidase, which points out the suitability of incorporating AMF in recovery strategies
of metal contaminated soils that involve the application of biochar21. Additionally, F. mosseae inoculation led to
a significant increase in AMF hyphal density (as demonstrated by increased EE-GRSP contents36), which would
have benefited the stability of soil aggregates and had a beneficial effect on β-glucosidase. Thereby, combining
DOR-based biochar and AMF inoculation may result in a synergistic effect based on the high C-substances input
supplied by adding DOR-based biochar along with the better stability of soil aggregates provided by AMF24,28,37. This result might also suggest that F. www.nature.com/scientificreports/ mosseae inoculation (M)
Fsignificance (df)b
13.15*** (1)
5.86*** (1)
0.29 (1)
27.47*** (1)
2.24 (1)
24.01*** (1)
Post-hoc test
No inoculation
13.03 a
9.93 b
3.18 a
3.90 a
1.22 a
0.25 a
Inoculation
14.13 b
9.09 a
3.33 a
4.41 b
1.31 a
0.30 b
Significant interactions
P × M***, D × S***,
D × T*, S × T***, T × M*,
P × D × S*, P × S × M***,
D × S × T***, D × S × M*,
S × T × M*, P × D × S × T*
P × D × T × M*,
P × T***,
P × M***,D × S***,
S × T***, S × M***,
T × M***, P × D × M*,
P × D × S*, D × S × T***,
D × S × M***,
S × T × M***,
P × D × S × T*,
P × D × T × M*
P × D ***, P × T*,
P × M*, D × S***,
D × M***, S × T***,
S × M***, T × M*,
P × D × S*, D × S × T***,
D × S × M***,
S × T × M***,
P × D × S × M*,
P × D × T × M*
P × T*, P × M***,
D × S *, D × T*,
S × T***, P × S × T*,
D × S × T*, S × T × M *,
P × D × T × M*
P × M***, D × S*,
S × T***, S × M*,
D × S × T*, P × S × M*
P × D*, P × M***,
D × S***, S × T***,
S × M*, D × S × M*,
P × D × T × M*** Table 3. Results of MANOVA and post-hoc analyses on the effect of the factors pyrolysis temperature of the
DOR-based biochar (350 and 500 °C), application dose of DOR-based biochar (2 and 5%), soil contamination
level (“low”, “medium” and “high”), soil treatment time (30, 60 and 90 days) and inoculation of F. mosseae
(AMF) and their interactions on PLFA-based abundance of the different microbial groups considering only
amended microcosms. Median values for each variable at each factor level are also shown. a For post-hoc
tests, the values are expressed as µg PLFA (g soil dm)-1. Scientific Reports | (2022) 12:12690 www.nature.com/scientificreports/ a For post-hoc tests, the values are expressed
as µg NLFA (g soil dm)−1. b For post-hoc tests, the values are expressed as g (g soil dm)−1. c For post-hoc tests,
the values are expressed as %. d F-valuesignificance (degrees of freedom). For MANOVA analyses, F-values in bold
denote statistical significance (p ≤ 0.05), significance levels are shown at *p < 0.05, **p < 0.01 and ***p < 0.001;
for post-hoc Tukey’s HSD tests, median values followed by different letters are significantly different (p < 0.05). Table 4. Results of MANOVA and post-hoc analyses on the effect of the factors pyrolysis temperature
of the DOR-based biochar (350 and 500 °C), application dose of the DOR-based biochar (2 and 5%), soil
contamination level (“low”, “medium” and “high”), soil treatment time (30, 60 and 90 days) and inoculation
of F. mosseae (AMF) and their interactions on NLFA-based abundance of AMF, easily extractable glomalin-
related soil content (EE-GRSP) and AMF-root colonization rate considering only amended microcosms. Median values for each variable at each factor level are also shown. a For post-hoc tests, the values are expressed
as µg NLFA (g soil dm)−1. b For post-hoc tests, the values are expressed as g (g soil dm)−1. c For post-hoc tests,
the values are expressed as %. d F-valuesignificance (degrees of freedom). For MANOVA analyses, F-values in bold
denote statistical significance (p ≤ 0.05), significance levels are shown at *p < 0.05, **p < 0.01 and ***p < 0.001;
for post-hoc Tukey’s HSD tests, median values followed by different letters are significantly different (p < 0.05). fractions (PLFAtot, PLFAbac, PLFAGram+, PLFAGram-, PLFAact and PLFAfun) was significantly positively affected by
DOR-based biochar. Furthermore, the 5% dose boosted microbial abundances to a significantly higher extent
than the 2% dose did. The better response of microbial populations to a higher dose of application of DOR-based
biochar could indicate an improvement of the physical parameters of soils (i.e., porosity, humidity and aeration)
and nutrient availability, which leads to a favorable habitat for microbial communities, as indicated by El-Naggar
et al.42. It has also been proposed that the highly porous structure of biochar could provide a congenial habitat
niche for the growth of soil microorganisms43. www.nature.com/scientificreports/ Likewise, the decrease found in Cd, Pb and Zn mobility after
the application of DOR-based biochar to soils “low” and “medium”15, clearly shows the beneficial impact of this
amendment on microbial communities by increasing pH and its feedback on nutrient availability and potential
reduction of metal toxicity44. Throughout our experiment, the highest PLFA values for the different microbial
groups were obtained in the “low” and “medium” soils, which, according to García-Sánchez et al.45, is indicative
of the better efficiency of DOR-based biochar in metal-stabilization in moderately contaminated soils in com-
parison with the extremely contaminated ones. Additionally, it is worth noting that abundances of the different
microbial groups were never the highest in the most contaminated soil, even after biochar amendment, which
shows the detrimental effect that metals exert on microbial abundances31. Although BC500 was richer in some
nutrients, microbial communities were not affected by these differences in terms of abundance. On the other fractions (PLFAtot, PLFAbac, PLFAGram+, PLFAGram-, PLFAact and PLFAfun) was significantly positively affected by
DOR-based biochar. Furthermore, the 5% dose boosted microbial abundances to a significantly higher extent
than the 2% dose did. The better response of microbial populations to a higher dose of application of DOR-based
biochar could indicate an improvement of the physical parameters of soils (i.e., porosity, humidity and aeration)
and nutrient availability, which leads to a favorable habitat for microbial communities, as indicated by El-Naggar
et al.42. It has also been proposed that the highly porous structure of biochar could provide a congenial habitat
niche for the growth of soil microorganisms43. Likewise, the decrease found in Cd, Pb and Zn mobility after
the application of DOR-based biochar to soils “low” and “medium”15, clearly shows the beneficial impact of this
amendment on microbial communities by increasing pH and its feedback on nutrient availability and potential
reduction of metal toxicity44. Throughout our experiment, the highest PLFA values for the different microbial
groups were obtained in the “low” and “medium” soils, which, according to García-Sánchez et al.45, is indicative
of the better efficiency of DOR-based biochar in metal-stabilization in moderately contaminated soils in com-
parison with the extremely contaminated ones. Additionally, it is worth noting that abundances of the different
microbial groups were never the highest in the most contaminated soil, even after biochar amendment, which
shows the detrimental effect that metals exert on microbial abundances31. www.nature.com/scientificreports/ mosseae stimulates indirectly the microbial activity by changing the patterns
of root exudates, as previously reported38.ii ,
p
y
p
FA have been broadly used to create a profile of fingerprints of the community structure using biomarkers
for specific groups of microorganisms39,40 and are useful indicators of soil attributes to evaluate the recovery of
the ecological soil functions, as Covino et al.41 suggested. In our study, the abundance of the different microbial https://doi.org/10.1038/s41598-022-17075-5 Scientific Reports | (2022) 12:12690 | www.nature.com/scientificreports/ DOR pyrolysis temperature (P)
Fsignificance (df)d
1.33 (1)
3.47 (1)
2.41 (1)
Post-hoc test
350 °C
0.83 a
0.160 a
6.00 a
500 °C
0.72 a
0.163 a
6.00 a
Biochar application dose (D)
Fsignificance (df)d
4.53* (1)
8.21*** (1)
42.41*** (1)
Post-hoc test
2%
0.72 a
0.160 a
5.50 a
5%
0.78 b
0.174 b
6.00 b
Soil contamination level (S)
Fsignificance (df)d
1268.44*** (2)
3810.96*** (2)
79.86*** (2)
Post-hoc test
Low
0.88 b
0.151 b
7.00 c
Medium
1.40 c
0.112 a
5.55 b
High
0.17 a
0.176 c
5.00 a
Soil treatment time (T)
Fsignificance (df)d
100.44*** (2)
1264.24*** (2)
971.85*** (2)
Post-hoc test
30 days
0.65 a
0.146 a
13.00 c
60 days
1.26 c
0.181 c
6.00 b
90 days
0.70 b
0.155 b
3.55 a
F. mosseae inoculation (M)
Fsignificance (df)d
26.54*** (1)
4259.38*** (1)
48.97*** (1)
Post-hoc test
No inoculation
0.73 a
0.159 a
5.00 a
Inoculation
0.81 b
0.164 b
7.00 b
Significant interactions
P × S***, S × T***, S × M***, T × M*, S × T × M***
S × M***, S × T***, T × M***, P × S × M*, P × T × M*,
S × T × M***, P × S × T × M***
P × D*, P × T*, D × S***, D × T***, D × M***,
S × T***, S × M***, T × M***, P × S × T*,
P × D × T***, P × T × M***, D × S × T***,
D × T × M***, S × T × M*, P × D × S × T***,
P × D × S × M*, P × S × T × M***, P × D × T × M***,
D × S × T × M*** Table 4. Results of MANOVA and post-hoc analyses on the effect of the factors pyrolysis temperature
of the DOR-based biochar (350 and 500 °C), application dose of the DOR-based biochar (2 and 5%), soil
contamination level (“low”, “medium” and “high”), soil treatment time (30, 60 and 90 days) and inoculation
of F. mosseae (AMF) and their interactions on NLFA-based abundance of AMF, easily extractable glomalin-
related soil content (EE-GRSP) and AMF-root colonization rate considering only amended microcosms. Median values for each variable at each factor level are also shown. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Factors
NLFAAMF
a
EE-GRSPb
AMF-root colonizationc
DOR pyrolysis temperature (P)
Fsignificance (df)d
1.33 (1)
3.47 (1)
2.41 (1)
Post-hoc test
350 °C
0.83 a
0.160 a
6.00 a
500 °C
0.72 a
0.163 a
6.00 a
Biochar application dose (D)
Fsignificance (df)d
4.53* (1)
8.21*** (1)
42.41*** (1)
Post-hoc test
2%
0.72 a
0.160 a
5.50 a
5%
0.78 b
0.174 b
6.00 b
Soil contamination level (S)
Fsignificance (df)d
1268.44*** (2)
3810.96*** (2)
79.86*** (2)
Post-hoc test
Low
0.88 b
0.151 b
7.00 c
Medium
1.40 c
0.112 a
5.55 b
High
0.17 a
0.176 c
5.00 a
Soil treatment time (T)
Fsignificance (df)d
100.44*** (2)
1264.24*** (2)
971.85*** (2)
Post-hoc test
30 days
0.65 a
0.146 a
13.00 c
60 days
1.26 c
0.181 c
6.00 b
90 days
0.70 b
0.155 b
3.55 a
F. mosseae inoculation (M)
Fsignificance (df)d
26.54*** (1)
4259.38*** (1)
48.97*** (1)
Post-hoc test
No inoculation
0.73 a
0.159 a
5.00 a
Inoculation
0.81 b
0.164 b
7.00 b
Significant interactions
P × S***, S × T***, S × M***, T × M*, S × T × M***
S × M***, S × T***, T × M***, P × S × M*, P × T × M*,
S × T × M***, P × S × T × M***
P × D*, P × T*, D × S***, D × T***, D × M***,
S × T***, S × M***, T × M***, P × S × T*,
P × D × T***, P × T × M***, D × S × T***,
D × T × M***, S × T × M*, P × D × S × T***,
P × D × S × M*, P × S × T × M***, P × D × T × M***,
D × S × T × M*** fractions (PLFAtot, PLFAbac, PLFAGram+, PLFAGram-, PLFAact and PLFAfun) was significantly positively affected by
DOR-based biochar. Furthermore, the 5% dose boosted microbial abundances to a significantly higher extent
than the 2% dose did. The better response of microbial populations to a higher dose of application of DOR-based
biochar could indicate an improvement of the physical parameters of soils (i.e., porosity, humidity and aeration)
and nutrient availability, which leads to a favorable habitat for microbial communities, as indicated by El-Naggar
et al.42. www.nature.com/scientificreports/ It has also been proposed that the highly porous structure of biochar could provide a congenial habitat
niche for the growth of soil microorganisms43. Likewise, the decrease found in Cd, Pb and Zn mobility after
the application of DOR-based biochar to soils “low” and “medium”15, clearly shows the beneficial impact of this
amendment on microbial communities by increasing pH and its feedback on nutrient availability and potential
reduction of metal toxicity44. Throughout our experiment, the highest PLFA values for the different microbial
groups were obtained in the “low” and “medium” soils, which, according to García-Sánchez et al.45, is indicative
of the better efficiency of DOR-based biochar in metal-stabilization in moderately contaminated soils in com-
parison with the extremely contaminated ones. Additionally, it is worth noting that abundances of the different
microbial groups were never the highest in the most contaminated soil, even after biochar amendment, which
shows the detrimental effect that metals exert on microbial abundances31. Although BC500 was richer in some
nutrients, microbial communities were not affected by these differences in terms of abundance. On the other
Table 4. Results of MANOVA and post-hoc analyses on the effect of the factors pyrolysis temperature
of the DOR-based biochar (350 and 500 °C), application dose of the DOR-based biochar (2 and 5%), soil
contamination level (“low”, “medium” and “high”), soil treatment time (30, 60 and 90 days) and inoculation
of F. mosseae (AMF) and their interactions on NLFA-based abundance of AMF, easily extractable glomalin-
related soil content (EE-GRSP) and AMF-root colonization rate considering only amended microcosms. Median values for each variable at each factor level are also shown. a For post-hoc tests, the values are expressed
as µg NLFA (g soil dm)−1. b For post-hoc tests, the values are expressed as g (g soil dm)−1. c For post-hoc tests,
the values are expressed as %. d F-valuesignificance (degrees of freedom). For MANOVA analyses, F-values in bold
denote statistical significance (p ≤ 0.05), significance levels are shown at *p < 0.05, **p < 0.01 and ***p < 0.001;
for post-hoc Tukey’s HSD tests, median values followed by different letters are significantly different (p < 0.05). www.nature.com/scientificreports/ hand, it is reasonable to argue that nutrients supplied by biochar decreased in soil microcosms over time, which
would explain the decreasing microbial abundances obtained with longer experiment times. The inoculation
of F. mosseae had an ambiguous impact on microbial abundances since Gram− bacteria and fungal populations
were positively affected, in contrast with the decline found in Gram+ bacteria and Actinobacteria groups. This
suppressive effect has been already reported by García-Sánchez et al.28. The reason for this effect may be the
production of antagonistic metabolites by AM fungal exudates, as it has been reported for Glomus sp.46.i p
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Previous studies have argued that biochar benefits the growth of AMF by increasing the availability of micro-
nutrients, changing soil physical and chemical properties, promoting spore germination and hyphal branching47. In our study, although NLFAAMF contents increased after the application of DOR-based biochar, neither levels
of EE-GRSP in soil nor percentages of AMF root colonization were higher after soil application of DOR-based
biochar. A possible explanation for this contradictory result may be that the susceptibility of plant roots to be
colonized depends on the plant species, as some authors have suggested20,28. This argument could also explain
the lack of a positive correlation between AMF root colonization and soil contents of NLFAAMF or EE-GRSP
in our experiment. The reduction in EE-GRSP contents could be also due to the presence of certain chelating
humic substances supplied by DOR-based biochar36. On the other hand, when only amended microcosms were
considered, the 5% dose significantly favored the NLFAAMF, EE-GRSP content and AMF root colonization in
comparison with the 2% dose. This finding points out that the key factor influencing the effect of DOR-based
biochar on soil AMF community seems to be its dose application. Despite the use of biochar, the lowest AMF root
colonization rates were obtained in the most contaminated soil, presenting tenfold the contents of some metals
of the soil “medium”, which highlights the negative effect that metals exert on AMF development23. As expected,
soil inoculation with F. mosseae led to significantly higher soil contents of NLFAAMF and EE-GRSP and higher
AMF root colonization values. It is worth noting that we found a significantly positive correlation between soil
contents of the NLFA 16:1ω5 and EE-GRSP throughout our experiment. www.nature.com/scientificreports/ But interestingly, these two biomarkers
have been related to different compartments of AMF community in soil; NLFA 16:1ω5 represents storage lipids
in AMF spores, while EE-GRSP is rather a marker of the mycorrhizal mycelium in soil48. Therefore, although
the quantification of the NLFA 16:1ω5 in soil may not represent the actual active mycorrhizal population, its
assessment has been considered as complementary to that of EE-GRSP to achieve an integrative analysis of soil
AMF community48. In any case, the NLFA 16:1ω5 has been shown to be a more reliable marker of soil AMF
population than the PLFA 16:1ω549. In our study, the highest values in soil NLFAAMF and EE-GRSP contents were
found after 60 days, while rates of root colonization were the highest at time 30 days. After that sampling time,
colonization rates decreased, probably as the result of a reduced physical growing space for AMF. Conclusionsh The feasibility of the soil application of DOR-based biochar alone or in combination with F. mosseae to enhance
the status of microbial communities (in terms of potential functionality and abundance) inhabiting three soils
with a gradient of metal contamination was evaluated to complement our previous study15. We showed that
microbial enzyme activities (β-glucosidase and dehydrogenase) were differently affected by the specific proper-
ties of DOR-based biochar, which resulted to be dependent on the temperature of production (350 vs. 500 °C). The application dose of DOR-based biochar induced significant changes in the soil microbial status, especially
at boosting the FA-based abundance of all microbial groups. The efficiency of DOR-based biochar in improving
soil microbial status was higher when it was applied to the moderately contaminated soil. Soil inoculation with
F. mosseae led to a proliferation of AMF community, increased mycorrhization rates of wheat plants and higher
levels of β-glucosidase activity. Interestingly, although the F. mosseae inoculation itself had a limited effect on
boosting PLFA-based microbial abundances, it significantly interacted with the soil amendment with DOR-based
biochar to favor the microbial proliferation. Therefore, our results postulates that the soil application of DOR-
based biochar in combination with AMF inoculation is a suitable approach to improve the microbial status of
moderately metal contaminated soils at the short term. We recognize that these conclusions are limited to the
soils assayed and the experimental conditions used. In this way, further studies evaluating this approach at the
long term are needed under different metal-contaminated environments. www.nature.com/scientificreports/ Although BC500 was richer in some
nutrients, microbial communities were not affected by these differences in terms of abundance. On the other https://doi.org/10.1038/s41598-022-17075-5 Scientific Reports | (2022) 12:12690 | www.nature.com/scientificreports/ Materials and methods Site description and tested soils. As explained by Vejvodová et al.15, the chronically contaminated soils
used in this experiment were collected in the vicinity of Trhové Dušníky village, which is located in the Příbam
district of the Czech Republic. A detailed description of the location, including a detailed map of the area, is
available elsewhere50. The intense regional mining and smelting activities in this area during the last centuries
have resulted in soil contamination at different rates with metals such as As, Cd, Pb and Zn as the result of poly-
metallic mineral depositions51. In our experiment, three sites differing in the levels of metal concentration were
selected15 and identified as “low” (slightly polluted), “medium” (moderately polluted) and “high” (highly pol-
luted). The locations chosen were the following: soil “low” (49° 43′ 15.730″ N; 13° 58′ 33.126″ E), soil “medium”
(49° 42′ 43.450″ N; 13° 59′ 7.615″ E), and soil “high” (49° 43′ 9.353″ N; 14° 0′49.828″ E). The three soils are at
the same location under the same environmental and climatic conditions and are thus comparable. Soil samples
from each site were collected at a depth of 0–20 cm, immediately homogenized, air-dried at room temperature
and finally passed through a 5 mm mesh sieve. Soils were stored in polythene bags and kept at room temperature
until their use. “Low” and “medium” soils were classified as Cambisol and the “high” soil as Fluvisol and they
differed on their their physicochemical characteristics which have previously been reported15. www.nature.com/scientificreports/ autoclaving it three times and stored at 4 °C prior to use. Its basic characteristics have previously been described
by Siles et al.10. The biochar was produced in laboratory conditions using a pyrolytic furnace Carbolite 301
(Carbolite Gero, Great Britain). The pyrolysis was performed in an electrically heated quartz tube for 25 min at
the target temperatures of 350 °C (from now on referred to as BC350) and 500 °C (BC500) in the presence of
nitrogen (N2 flow of 4.5 L per min). The DOR-based biochar was immediately homogenized after pyrolysis and
physicochemical composition was determined and published elsewhere15. autoclaving it three times and stored at 4 °C prior to use. Its basic characteristics have previously been described
by Siles et al.10. The biochar was produced in laboratory conditions using a pyrolytic furnace Carbolite 301
(Carbolite Gero, Great Britain). The pyrolysis was performed in an electrically heated quartz tube for 25 min at
the target temperatures of 350 °C (from now on referred to as BC350) and 500 °C (BC500) in the presence of
nitrogen (N2 flow of 4.5 L per min). The DOR-based biochar was immediately homogenized after pyrolysis and
physicochemical composition was determined and published elsewhere15. Inoculum of arbuscular mycorrhizal fungus. The AM fungus used in this experiment was Funneli-
formis mosseae (formerly named as Glomus mosseae) due to its resistance and adaptation to metal-contaminated
soils as our previous studies showed15,28. The mycorrhizal inoculum was a mixture of rhizosphere soil contain-
ing spores, hyphae and mycorrhizal root fragments. This material was obtained by using trap pot cultures of
Medicago sativa L., consisting of soil, spores, mycelia and colonized root fragments (10 sporocarps g−1, with 1–5
spores per sporocarp). Experimental design and set up. The experimental design consisted of a factorial system including the
following factors: (i) soil application rate of the DOR-based biochar, comprising the levels 0% (control), 2% and
5%; (ii) pyrolysis temperature of the DOR-based biochar, which includes the temperatures 350 °C and 500 °C;
(iii) soil metal contamination level, comprising the levels “low” (slightly contaminated), “medium” (moder-
ately contaminated) and “high” (highly contaminated); (iv) soil treatment time, including the levels 30, 60 and
90 days; and (v) AMF inoculation, comprising the levels application or not of F. mosseae. The experiment was set
up in identical 0.3 L polypropylene pots. Approximately, 300 g of “low”, “medium” or “high” multi-contaminated
soil was placed into each pot. www.nature.com/scientificreports/ BC350 and BC500 were applied and manually mixed with the soil to reach the
concentrations of 2% and 5% (w/w). One half of the amended pots were inoculated with F. mosseae by add-
ing 8 g of inoculum, as suggested by Vejvodová et al.15; meanwhile, the other half received the same weight of
inoculum filtrate (Whatman no. 1 filter paper) containing soil microbiota free of AM fungal propagules. An
initial irrigation was realized gravitationally in order to reach the given value of the soil water holding capacity
(WHC = 60%). Soil samples with or without application of DOR-based biochar and F. mosseae were also set up
and used as controls. Five replicates were established for each treatment. In total, the experiment consisted of 325
pot microcosms (5 factors × 13 factor levels × 5 replicates).h p
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The use of plants in the present study complies with international/institutional guidelines. Before planting,
wheat seeds (Triticum aestivum L.) were surface disinfected by immersion in 2% (v/v) hydrogen peroxide for
8 min52. Seed germination was carried out at 25 ºC in trays containing vermiculite as substrate for two weeks. Afterward, one 15-day old wheat plant was planted in each pot. The experiment was run in greenhouse condi-
tions (supplementary light 25/19 °C and 50% relative humidity), as previously described15, and microcosms
were regularly watered in order to maintain the initial moisture conditions. Soil microcosms were destructively
sampled after 30, 60 and 90 days. Soil from each pot at each time sampling was collected, homogenized, sieved
(2 mm mesh) and subdivided into two subsamples. One subsample was kept at 4 °C for biochemical analyses
and the other one was frozen at − 80 °C and then freeze-dried for FA analyses. Soil enzyme activities. Dehydrogenase activity (EC 1.1) was measured using 0.5% INT (2-p-iodofenil-3-p-
nitrofenil-5-feniltetrazolio) as substrate following the methodology of Camiñas et al.53. 1 g of soil was incubated
with 2 mL of the substrate 2-p-iodophenyl-3-p-nitrophenyl-5-phenyltetrazolium (INT) 0.5% and 1.5 mL of 1 M
Tris–HCl buffer pH 7.5 for 1 h at 40 °C. Next, the iodonitrotetrazolium formazan (INTF) formed was extracted
with a 1:1 (v:v) mixture of ethanol and dimethyl-formamide, and its absorbance at 490 nm was measured. β-glucosidase activity (EC 3.2.1.21) was determined using 0.05 M PNG (p-nitrophenyl-β-d-glucopyranoside)
as substrate following the procedure described by Eivazi et al.54. Site description and tested soils
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d Biochar preparation. The biochar used in this study was produced from dry olive residue (DOR), which
was supplied by the olive oil manufacturer Sierra Sur S.A. (Granada, Spain). The DOR was sieved, sterilized by https://doi.org/10.1038/s41598-022-17075-5 Scientific Reports | (2022) 12:12690 | www.nature.com/scientificreports/ www.nature.com/scientificreports/ 1 g of dry soil was mixed with 5 mL of a solu-
tion of p-nitrophenyl β-d-glucopyranoside dissolved at a concentration of 0.025 M in buffer MUB (0.1 M, pH
6), and incubated at 37 °C for 2 h. The substrate used was transformed into p-nitrophenol due to the action of
β-glucosidase, and the concentration of this compound was determined at 400 nm after the addition of 1 mL of
0.5 M CaCl2 and 4 mL of 0.1 M THAM buffer pH 12. Fatty acid analysis. Soil samples were firstly extracted using a mixture of chloroform–methanol-phosphate
buffer (1:2:0.8; v/v/v), according to Bligh and Dyer55. Thereafter, the lipids were fractioned into neutral lipids
(NLFAs), glycolipids and polar lipids (PLFAs), using solid-phase extraction cartridges (LiChrolut Si-60, Merck,
White-house Station, USA), by elution with chloroform, acetone and methanol, respectively. PLFAs and NLFAs
were then subjected to mild alkaline methanolysis, as described by Šnajdr et al.56, and free methyl esters of
NLFAs and PLFAs were analyzed by gas chromatography-mass spectrometry (450-GC, 240-MS ion trap detec-
tor, Varian, Walnut Creek, CA), following the procedure described by Ref.57.i )
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Bacterial biomass (PLFAbact) was quantified as a sum of i14:0, i15:0, a15:0, 16:1ω5, 16:1ω7, 16:1ω9, 10Me-16:0,
i16:0, i17:0, a17:0, cy17:0, 17:0, 10Me-17:0, 18:1ω7, 10Me-18:0 and cy19:00. Actinobacteria abundance (PLFAact)
was determined according to 10Me-16:0, 10Me-17:0 and 10Me-18:0. Fungal biomass (PLFAfun) was quantified
based on the content of 18:2ω6,956. The ∑ PLFAs was used to estimate the total microbial biomass (PLFAtot). The
NLFA 16:1ω5 was assigned as a marker for the quantification of AM fungal population40. Root mycorrhization rate and soil glomalin content. The percentage of mycorrhizal fungi root
length infected was performed using a microscope (20–40×) after clearing a portion of the roots with 3% H2O2
(v/v) and 10% KOH (w/v) and staining in a 0.05% solution of fuchsine blue in lactic acid (w/v). The gridline
intercept method58 was used for determining the percentage of AM root colonization. https://doi.org/10.1038/s41598-022-17075-5 Scientific Reports | (2022) 12:12690 | www.nature.com/scientificreports/ To determine contents of easily extractable glomalin-related soil protein (EE-GRSP) in soil, 1 g of soil was
firstly suspended in 8 mL of citrate buffer (20 mM, pH 7.0) and the suspension was then autoclaved at 121 °C
for 30 min Wright and Upadhyaya59, centrifuged at 10,000×g for 15 min and filtered through Whatman no. 1
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Authors thank for financial support of the European Regional Development Fund—Project No. CZ.02.1.01/0.0
/0.0/16_019/0000845CZ.02.1.01/0.0/0.0/16_019/0000845 and by the Center for Geosphere Dynamics (UNCE/
SCI/006) and part of the equipment was supported by the Operational Programme Prague—Competitiveness
(Project No. CZ.2.16/3.1.00/21516). This study was also co-funded by the Spanish Ministry of Science and
Innovation Project No. RTI2018-094327-B-I00. www.nature.com/scientificreports/ Soil biochemical activities and the geometric mean of enzyme activities
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37. Alguacil, M. M., Caravaca, F., Azcón, R. & Roldán, A. Changes in biological activity of a degraded Mediterranean soil after using
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microbially-treated dry olive cake as a biosolid amendment and arbuscular mycorrhizal fungi. Eur. J. Soil Biol. 44, 347–354 (2008). 37. Alguacil, M. M., Caravaca, F., Azcón, R. & Roldán, A. Changes in biological activity of a degraded Mediterranean soil after using
microbially-treated dry olive cake as a biosolid amendment and arbuscular mycorrhizal fungi. Eur. J. Soil Biol. 44, 347–354 (2008). 38. Rodríguez-Caballero, G. et al. Arbuscular mycorrhizal fungi inoculation mediated changes in rhizosphere bacterial community
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0. Olsson, P. A. Signature fatty acids provide tools for determination of the distribution and interactions of mycorrhizal fungi in soil
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1. Chen, J. et al. Heavy metal pollution decreases microbial abundance, diversity and activity within particle-size fractions of a paddy
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43–551 (2019). https://doi.org/10.1038/s41598-022-17075-5 Scientific Reports | (2022) 12:12690 | Competing interests h p
g
The authors declare no competing interests. www.nature.com/scientificreports/ (including greenhouse experiment and analysis) and provided the financial support. T.C. performed the FA
analysis. J.B. was involved in preparing the greenhouse experiment, collecting soil samples and analyzing micro-
bial enzymes, EE-GRSP contents and percentage of root colonization. G.S.C was involved in the discussion and
analysis of data. J.S. and P.T. provided soils and prepared biochar samples. M.G.S. supervised the analyses, pro-
vided important advices, discussed the results, wrote the initial version of the manuscript, reviewed and edited
the final version. All authors read and approved the final manuscript. Additional information Correspondence and requests for materials should be addressed to M.G.-S. Correspondence and requests for materials should be addressed to M.G.-S. Author contributions J.A.S. performed the statistical analysis of the data, discussed the results, wrote the initial version of the man-
uscript, reviewed and edited the final version. I.G.R. conceived the study, planned the experimental design https://doi.org/10.1038/s41598-022-17075-5 Scientific Reports | (2022) 12:12690 | www.nature.com/scientificreports/ © The Author(s) 2022 Correspondence and requests for materials should be addressed to M.G.-S. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and
institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International
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the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2022 https://doi.org/10.1038/s41598-022-17075-5 Scientific Reports | (2022) 12:12690 |
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Effects of a mat pilates program on cardiometabolicparameters in elderly women
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Pakistan journal of medical sciences
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ABSTRACT Fourie Marinda,
2. Gildenhuys Magda,
3. Shaw Ina,
Office of the Deputy Pro Vice-Chancellor: Research,
Monash South Africa, P.O. Box X60, Ruimsig, 1725, Republic of South Africa. 4. Shaw Brandon,
5. Toriola Abel,
6. Daniel Ter Goon,
Centre for Biokinetics, Recreation and Sports Science,
University of Venda, X5050, Thohoyandou, Limpopo, 0590, South Africa. 1-5: Department of Sport, Rehabilitation and Dental Sciences,
Tshwane University of Technology, Private Bag X680, Pretoria,
Gauteng, 0001, Republic of South Africa. Correspondence:
Daniel Ter Goon,
Centre for Biokinetics, Recreation and Sports Science,
University of Venda, Thohoyandou, South Africa. E-mail: daniel_goon2004@yahoo.com
* Received for Publication:
November 1, 2012
* Accepted:
January 21, 2013 ABSTRACT Objective: This study aimed to determine the effects of mat Pilates on resting heart rate, resting blood
pressure and fasting blood glucose, cholesterol and triglycerides in elderly women. Methodology: Fifty sedentary, apparently healthy females aged 60 and older were randomly assigned into
a control (CG, n = 25) or an intervention (IG, n = 25) group. The IG took part in an eight-week progressive
mat Pilates exercise program, three times weekly while the CG did not take part in any structured exercises
throughout the eight-week period. All subjects underwent pre- and post-tests in which cardiometabolic
parameters were assessed. p
Results: In the eight-week mat Pilates program, the IG only demonstrated a significant (p ≤ 0.05) decrease
in systolic BP (p = 0.040) from 135.84 ± 14.66mmHg to 128.80 ± 16.36mmHg and a significant increase in
blood glucose (p = 0.000) from 5.07 ± 0.46mmol.L-1 to 5.83 ± 0.57mmol.L-1, whereas resting HR (p = 0.148)
(from 68.80 ± 12.58beats.min-1 to 73.20 ± 11.46beats.min-1), resting diastolic BP (p = 0.342) (from 75.64 ±
10.10mmHg to 77.44 ± 9.32mmHg), blood TC (p = 0.073) (from 5.37 ± 0.99mmol.L-1 to 5.67 ± 1.04mmol.L-1)
and blood TG (p = 0.384) (from 1.77 ± 0.88mmol.L-1 to 1.92 ± 0.87mmol.L-1) did not produce any significant
changes. g
Conclusion: Due to the contradictory nature of the cardiometabolic variables (except systolic BP) with
the findings of previous studies, it is difficult to establish a case for using Pilates as a substitute for
more conventional forms of exercising when exclusively attempting to favourably alter cardiometabolic
parameters at least among the elderly women in our sample. KEY WORDS: Pilates program, Resting heart rate, Resting blood pressure, Fasting blood glucose, Cholesterol,
Triglycerides, Elderly women. doi: http://dx.doi.org/10.12669/pjms.292.3099 How to cite this:
Marinda F, Magda G, Ina S, Brandon S, Abel T, Goon DT. Effects of a mat pilates program on cardiometabolic parameters in elderly
women. Pak J Med Sci 2013;29(2):500-504. doi: http://dx.doi.org/10.12669/pjms.292.3099 How to cite this:
Marinda F, Magda G, Ina S, Brandon S, Abel T, Goon DT. Effects of a mat pilates program on cardiometabolic parameters in elderly
women. Pak J Med Sci 2013;29(2):500-504. doi: http://dx.doi.org/10.12669/pjms.292.3099 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 500 Pak J Med Sci 2013 Vol. 29 No. 2 www.pjms.com.pk
1. Effects of a mat pilates program on cardiometabolic
parameters in elderly women Fourie Marinda1, Gildenhuys Magda2, Shaw Ina3,
Shaw Brandon4, Toriola Abel5, Daniel Ter Goon6 Original Article Original Article Original Article Open Access Open Access y
g
3.
Shaw Ina,
Office of the Deputy Pro Vice-Chancellor: Research,
Monash South Africa, P.O. Box X60, Ruimsig, 1725, Republic of South Africa.
4.
Shaw Brandon, 1.
Fourie Marinda, INTRODUCTION 2. Gildenhuys Magda,
3. Shaw Ina,
Office of the Deputy Pro Vice-Chancellor: Research,
Monash South Africa, P.O. Box X60, Ruimsig, 1725, Republic of South Africa. 4
Shaw Brandon With ageing maximum HR decreases1 due to
the natural pacemaker (the sinoarterial (SA) node)
losing some of its cells which may result in cardiac
arrhythmias in the elderly, as well as incompetency
among the valves inside the heart, which control
the direction of blood flow.2 Ageing also results
in a reduced elasticity in the arteries and possible
reductions
in
left
ventricular
contractility.1
Another age-related finding was that with ageing,
a decrease occurs in both the sympathetic and
parasympathetic regulation of the heart3-5 and
a reduced sensitivity of the heart to the body’s
chemical catecholamine stimulation.1 The aorta also 6. Daniel Ter Goon,
Centre for Biokinetics, Recreation and Sports Science,
University of Venda, X5050, Thohoyandou, Limpopo, 0590, South Africa. becomes thicker, stiffer and less flexible, which then
results in an increase in BP and causes the heart
to work harder, which may lead to myocardial
hypertrophy.2 However, this gradual stiffening and
inability of the arteries to dilate and constrict, can
explain the increases found in BP with ageing.2,6
Ageing also appears to be associated with specific
and selective impairments in baroreflex function,
which includes a decreased ability to alter cardiac
period in response to acute alterations in BP and
a decreased ability of the baroreflexes to buffer
changes in systemic BP.7 Kidneys of the elderly also
secrete less rennin,2 resulting in a decreased ability
to respond to decreases in BP by eventually causing
vasoconstriction and thus, BP to increase. such as, increases in flexibility, bone density and
dynamic balance as well as favourable changes in
body mass index and have a positive effect on mus-
cular strength and endurance.14-17 Correct breathing
during Pilates can also prevent excessive stress on
the heart, result in total body relaxation and mental
calmness.16 This reduction in stress as a result of Pi-
lates can reduce other serious complications. It may
directly or indirectly result in an improvement in
cardiometabolic parameters, especially in the elder-
ly, resulting in reduced risk of developing coronary
artery disease (CAD).18 However, little literature is
available on the effects of Pilates on cardiometabilic
parameters, therefore creating a need for scientific
evidence on the beneficial effects of Pilates on car-
diometabolic parameters. METHODOLOGY A sample of 50 elderly female subjects (≥ 60 years
of age), selected from caring facilities within Preto-
ria, Gauteng Province, South Africa were randomly
assigned into one of two groups using a random
numbers table; with 25 subjects undergoing an
eight-week mat Pilates program (IG), while the
other 25 subjects participated as a non-exercising
control group (CG). The research protocol was ap-
proved by the Institutional Review Boards of the
Tshwane University of Technology, Pretoria, South
Africa and was endorsed by the International Phys-
ical Activity Projects (IPAP). Permission to conduct
the study at the caring facilities was obtained from
the relevant care facilities and all subjects signed a
written informed consent form indicating all the ad-
vantages and risks involved of participation in the
study. All subjects were required to obtain medi-
cal clearance prior to commencement of pre-testing
procedures. Both groups took part in identical pre-
and post-tests. Subject demographics at baseline are
shown in Table-I. With regards to lipids, Gostynski et al.10 found
that the prevalence of dyslipidemia increases
with age, however, there has been much debate
whether cholesterol increases are the result of a
natural process of intrinsic ageing or whether it
is due to age-associated anthropometrics and/or
lifestyle changes.10 Schubert et al.11 found that age
is an important independent predictor for LDL-C
and TC in men and TC in women, but it is not as
influential as body composition and lifestyle on
HDL-C and TG in men and women and LDL-C in
women. Regardless of the link between overweight
and cholesterol levels Nakagawa et al,12 found that
intramyocellular lipid content also increases with
age in lean elderly. This might be related to blood
lipid and lipoprotein profiles, with a decrease in
HDL-C and might affect health risks and muscle
attenuation with age in lean elderly. All
subjects
were
required
to
undergo
cardiometabolic testing prior to and at completion
of the eight-week treatment period. Each subject’s
resting heart rate was measured in a seated position Table-I: Subject demographic data. Table-I: Subject demographic data. INTRODUCTION Ageing can also be seen as one of the largest
factors affecting glucose homeostasis. According
to Elahi and Muller and Stout,8,9 several glucose
age-related responses occur, such as a decrease in
glucose tolerance, inappropriate insulin secretion
and insulin insensitivity occurs, where in turn,
a number of possible mechanisms for glucose
intolerance in the elderly exist. Glucose intolerance
can be caused by changes in hormones involved
in the increase uptake and suppression of glucose
usage such as glucagon, growth hormone and
pancreatic hormones.9 With ageing, there is also
a tendency to increased total body weight and
increased distribution of adipose tissue in the
abdominal area, which mainly influence glucose
tolerance by causing insulin resistance.8,9 RESULTS At pre-test, the CG and IG were heterogeneous
for resting HR (p = 0.047). At pre-test, the groups
were, however, homogenous for resting systolic
BP (p = 0.736), resting diastolic BP (p = 0.143),
fasting blood glucose levels (p = 0.319), blood TC
(p = 0.754) and blood TG (p = 0.474). Following
the eight-week mat Pilates program, the IG only
demonstrated a significant (p ≤ 0.05) decrease in
systolic BP (p = 0.040) from 135.84 ± 14.66mmHg to
128.80 ± 16.36mmHg and a significant increase in
blood glucose (p = 0.000) from 5.07 ± 0.46mmol.L-1
to 5.83 ± 0.57mmol.L-1, whereas resting HR (p =
0.148) (from 68.80 ± 12.58beats.min-1 to 73.20 ±
11.46beats.min-1), resting diastolic BP (p = 0.342)
(from 75.64 ± 10.10mmHg to 77.44 ± 9.32mmHg),
blood TC (p = 0.073) (from 5.37 ± 0.99mmol.L-1 to
5.67 ± 1.04mmol.L-1) and blood TG (p = 0.384) (from
1.77 ± 0.88mmol.L-1 to 1.92 ± 0.87mmol.L-1) did
not produce any significant changes following the
eight-week exercise programme. Despite the CG
demonstrating no significant difference in resting
systolic BP (p = 0.705), resting diastolic BP (p =
0.471), blood TC (p = 0.054), blood TG (p = 0.321),
the CG did demonstrate significant changes in
resting HR and blood glucose from pre- to post-test
(p = 0.000 and p = 0.001, respectively) (Table-II). g
y
p y
Mat Pilates exercises were derived from Worth19
in order to compile the mat Pilates exercise
program. A session was allocated to explain the
basics of mat Pilates, before commencement of the
program. This session included an explanation
of the neutral position of the spine and also the
correct breathing techniques used during Pilates. After the introductory session the eight-week mat
Pilates exercise program commenced. The program
consisted of three non-consecutive sessions a
week, 60 minutes in duration, for eight-weeks
with increasing intensity. All sessions started with
breathing, followed by a flowing system from Table-II: Pre- and post-test cardiometabolic changes
in the mat Pilates and non-exercising control groups. 502 Pak J Med Sci 2013 Vol. 29 No. 2 www.pjms.com.pk
Table-II: Pre- and post-test cardiometabolic changes
in the mat Pilates and non-exercising control groups. METHODOLOGY The stethoscope
was placed below the cuff and the cuff inflated to
approximately 180 millimeters mercury (mmHg). The pressure was then slowly released and
different heart sounds were heard. The first sound
heard was used as the systolic blood pressure
(heart contraction) value and the sound heard
when the tone changed or disappeared was used
for the diastolic blood pressure (heart relaxation)
reading.2 Fasting blood glucose, total cholesterol
(TC) and triglycerides (TG) were measured by
means of using the Reflotron system (F. Hoffmann-
La Roche, Grenzarcherstrausse 124, CH-4070 Basel,
Switzerland) after an eight-hour fast. A multiclix
lancing device was used to prick the subjects’ finger. A drop of blood was then placed on a Reflotron
testing strip and the test strip placed in the Reflotron. The glucose, cholesterol and triglycerides readings
were then digitally displayed. Statistical analysis consisted of basic statistics to
determine pre- and post-test means and standard
deviations. A paired samples t-test was used to
determine if a significant change took place in
the measurements at post-test. Differences in
measurements were compared using a one-way
analysis of variance (ANOVA) using a Dunnett
T3 post-hoc analysis. Data was analyzed using
commercial software (Statistical Package for Social
Sciences (SPSS) Version 17, Chicago, IL) and
statistical significance set at p ≤ 0.05. METHODOLOGY Non-exercising
Mat Pilates
control group (CG)
program group (IG)
n = 25
n = 25
Age (years)
65.32 ± 5.01
66.12 ± 4.77
Weight (kg)
75.19 ± 14.78
71.71 ± 14.92
BMI (kg.m-2)
29.32 ± 5.44
28.32 ± 6.77
Values are means ± standard deviation ± SD; kilograms
per square meter The weakening of the cardiovascular system asso-
ciated with ageing, could be countered by increasing
levels of physical activity and functional fitness.13
Pilates results in various physical improvements Daniel Ter Goon et al. (after five minutes rest) by means of manually
applying mild pressure. Pressure was applied to
the radial artery on the lateral side of the anterior
forearm just proximal to the wrist.2 Resting BP was
measured by means of a sphygomanometer and a
stethoscope (Jiangsu Dengguan Medical Treatment
Instrument Co., Ltd.). The subject was required
to be supine for five minutes and the tested arm
was supported and the cuff was wrapped securely
around the right arm at heart level. The stethoscope
was placed below the cuff and the cuff inflated to
approximately 180 millimeters mercury (mmHg). The pressure was then slowly released and
different heart sounds were heard. The first sound
heard was used as the systolic blood pressure
(heart contraction) value and the sound heard
when the tone changed or disappeared was used
for the diastolic blood pressure (heart relaxation)
reading.2 Fasting blood glucose, total cholesterol
(TC) and triglycerides (TG) were measured by
means of using the Reflotron system (F. Hoffmann-
La Roche, Grenzarcherstrausse 124, CH-4070 Basel,
Switzerland) after an eight-hour fast. A multiclix
lancing device was used to prick the subjects’ finger. A drop of blood was then placed on a Reflotron
testing strip and the test strip placed in the Reflotron. The glucose, cholesterol and triglycerides readings
were then digitally displayed. standing, to sitting, to lying down exercises and
ended with the rest position. (after five minutes rest) by means of manually
applying mild pressure. Pressure was applied to
the radial artery on the lateral side of the anterior
forearm just proximal to the wrist.2 Resting BP was
measured by means of a sphygomanometer and a
stethoscope (Jiangsu Dengguan Medical Treatment
Instrument Co., Ltd.). The subject was required
to be supine for five minutes and the tested arm
was supported and the cuff was wrapped securely
around the right arm at heart level. DISCUSSION some independent effect on the risk of elevated BP,
where waist and hip circumferences are most im-
portant variables in women for obesity and BP. As
such, it can be assumed that the decrease found in
BP after completion of the eight-week mat Pilates
programme in the present study can be as a result
of the decrease found in waist circumference (WC)
from 83.50 ± 12.79mm to 81.44 ± 9.90mm at post-
test. Another influential mechanism in the decrease
in systolic BP could have been due to total body re-
laxation and mental calmness as a result of correct
breathing during the mat Pilates intervention,16 as
mental or emotional stress activates nervous and
endocrine responses, such as an increase in epi-
nephrine and norepinephrine stimulation that re-
sults in an increase in BP, while preparing the body
for physical activity.2,25 As individuals age, physiological functioning
decreases and most of the elderly develop at least
one of the following chronic diseases: hypertension
(49%), arthritis (36%), heart disease (31%), cancer
(20%) and/or diabetes (15%).20 It was however,
stated that exercise has major benefits for the car-
diovascular system (CVD, hypertension, stroke,
arrhythmias and peripheral artery disease) and
metabolism (body fat, glucose homeostasis and in-
sulin levels and lipids).21 Results obtained from the
present study, however, indicate that eight-weeks
of mat Pilates did not produce improvements in
any of the cardiometabolic variables tested except
systolic BP, which is a desirable finding as hyper-
tension is one of the most significant risk factors for
cerebrovascular and CVD.22i p y
y
Skeletal muscle is the primary site of glucose dis-
posal, therefore by increasing skeletal mass might
be an effective way to improve insulin action.26
Based on this statement, a decrease in blood glu-
cose in the IG would have been expected as the pre-
sent study demonstrated a significant (p = 0.006)
increase in lean body mass (LBM) (from 46.67 ±
6.33kg to 48.04 ± 7.52kg) in the IG. However, this
was not the case, instead both the control group
(CG) and the IG demonstrated a significant increase
in blood glucose. The mean fasting glucose lev-
els are significantly higher during the colder than
warmer months.27 The present study was conduct-
ed during the transition from a warmer to a colder
season, hence the possibility of the increase in blood
glucose observed in the present study. DISCUSSION The signifi-
cant increase in glucose from pre- to post-test can
also possibly be due to different instead of fixed
nutritional consumption followed by each subject,
several days prior to testing.i The present study correlates with the findings
of Jago et al.14 regarding diastolic BP by indicating
that no significant changes occurred in diastolic
BP at post-test. In this regard, Jago et al.14 found
non-significant changes in systolic and diastolic
BP (from 108.2±9.4 mmHg to 102.5±5.6 mmHg and
from 62.7±6.2 mmHg to 58.9±6.9 mmHg, respec-
tively), following four-weeks of Pilates training in
girls. Even though no significant change occurred
in diastolic blood pressure (BP), the present study
demonstrated a significant reduction in systolic
BP. Brandaorondon et al.23 found that exercise sig-
nificantly decreases BP in hypertensive elderly and
provokes a decrease in BP that lasts for 22 hours, the
moderately hypertensive pre-test systolic BP value
should not be neglected. This reduction found in BP
after exercise in elderly hypertensive individuals
can be associated with a decrease in stroke volume
and left ventricular end-diastolic volume.23 The re-
duction in BP can also be attributed to a diet high in
cholesterol or fat that might result in fatty deposits
called plaques to clot the arteries, thus resulting in
narrowing of the arteries and increasing pressure. However, this cannot be confirmed in the present
study since no dietary analyses were conducted. Exercise also exerts beneficial effects on blood li-
pids, but since the separate measures of high-densi-
ty lipoprotein cholesterol (HDL-C) and low-density
lipoprotein cholesterol (LDL-C) were not assessed,
the vascular remodeling of existing arteries can also
not be confirmed in the present study. In general,
obesity and overweight are considered controlla-
ble risk factors for hypertension,18 but with a more
thorough investigation24 found that body mass
index (BMI), WHR, and skinfolds adiposity have y p
g
The present study found no significant changes
in TC and TG after completion of an eight-week
mat Pilates programme. RESULTS Variables
Non-exercising control group (CG) n = 25
Mat Pilates program group (IG) n = 25
Pre-test
Post-test
p-value
Pre-test
Post-test
p-value
Resting heart rate (beats.min-1)
62.48±8.98
74.92 ± 9.96
0.000*
68.80±12.58
73.20±11.46
0.148
Resting systolic blood pressure (mmHg)
134.48±13.65 136.00±17.83 0.705
135.84±14.66
128.80±16.36 0.040*
Resting diastolic blood pressure (mmHg) 81.36 ± 9.27
79.76 ± 9.31
0.471
75.64 ±10.10
77.44 ± 9.32
0.342
Glucose (mmol.L-1)
5.24 ± 0.71
5.75 ± 0.59
0.001*
5.07 ± 0.46
5.83 ± 0.57
0.000*
Total cholesterol (mmol.L-1)
5.27 ± 1.27
5.43 ± 1.27
0.054
5.37 ± 0.99
5.67 ± 1.04
0.073
Triglycerides (mmol.L-1)
1.62 ± 0.54
1.78 ± 0.71
0.321
1.77 ± 0.88
1.92 ± 0.87
0.384
Values are means ± standard deviation ± SD; *: Indicates significant difference from pre- to post-test (p ≤0.05) 502 Pak J Med Sci 2013 Vol. 29 No. 2 www.pjms.com.pk Pak J Med Sci 2013 Vol. 29 No. 2 www.pjms.com.pk 503 ACKNOWLEDGEMENTS 18. Wilmore JH, Costill DL, Kenney WL. Physiology of sport
and exercise. 4th ed: Champaign, IL: Human Kinetics; 2008. The authors would like to thank the Tshwane
University of Technology (TUT), South Africa for
their approval and financial support as well as the
International Physical Activity Projects (IPAP) for
their endorsement of the study and financial and
statistical support. 19. Worth Y. Needs to know Pilates? All the tips and techniques
you need to get a toned, flexible body. London: Collins; 2004. y
gl
y
20. Howley ET, Franks BD. Fitness professional’s handbook. 5th ed: Champaign, IL: Human Kinetics; 2007. 21. Simon H. Moderate exercise: no pain, big gains. Medscape
Internal Medicine. 2006;8(1) [Online]. Available from www. medscape.com/viewarticle/524377?src=mp. [Accessed:
08/10/2010]. CONCLUSIONS The present study demonstrated that eight-weeks
of Pilates is sufficient to produce a significant im-
provement in systolic BP, however, due to the
contradictory nature of the other cardiometabolic
variables with the findings of previous studies, it
is difficult to establish a case for using Pilates as a
substitute for more conventional forms of exercis-
ing when exclusively attempting to favourably alter
cardiometabolic parameters in elderly women. 14. Jago R, Jonker ML, Missaghian M, Baranowski T. Effect of 4
weeks of Pilates on the body composition of young girls. J
Prev Med. 2006;42:177–180. 15. Johnson EG, Larsen A, Ozawa H, Wilson CA, Kennedy
KL. The effects of Pilates-based exercise on dynamic
balance in healthy adults. J Bodywork Mov Therapies. 2007;11(3):238–242.i ( )
16. Robinson L, Fisher H, Knox J, Thomson G. The official body
control Pilates manual. London: Macmillan; 2000. 17. Sekendiz B, Altun O, Korkusuz F, Akýn S. Effects of Pilates
exercise on trunk strength, endurance and flexibility in
sedentary adult females. J Bodywork Mov Therapies. 2007;11:318–26. DISCUSSION The results found on blood
lipids in the present study was not attributable to
any of the anthropometric variables as the major-
ity of TG are stored in adipose cells28 and favour-
able changes from pre- to post-test was observed in
body fat percentage (BF%) (from 33.85 ± 6.67% to
32.23 ± 5.82%), WC (from 83.50 ± 12.79 millimeters
to 81.44 ± 9.90 millimeters), fat mass (FM) (from
25.03 ± 9.53 kilograms to 23.69 ± 8.06 kilograms)
and LBM (from 46.67 ± 6.33 kilograms to 48.04 ±
7.52 kilograms) (data not shown). A possible reason
might have been due to an increase in HDL-C with-
out a corresponding decrease in LDL-C, but as the
CG also showed an increase in TC, this could not Pak J Med Sci 2013 Vol. 29 No. 2 www.pjms.com.pk 503 503 Daniel Ter Goon et al. 10. Gostynski M, Gutzwiller F, Kuulasmaa K, Doring A,
Ferrario M, Grafnetter D, et al. Analysis of the relationship
between total cholesterol, age, body mass index among
males and females in the WHO MONICA Project. Int J Obes. 2004;28(8):1082–1090. be a reasonable assumption. Uncontrollable factors
affecting lipids are genetics, age and gender,29 but
as only elderly women were used in the study age
and gender could not have influenced the outcome. Boardley et al29 however, indicated that dietary in-
take also influences blood lipids. As such, the ob-
servation might also be, as in the case of glucose,
attributed to different instead of fixed nutritional
consumption followed by each subject several days
prior to testing. However, again this cannot be con-
firmed since no dietary analyses were conducted. 11. Schubert CM, Rogers NL, Remsberg KE, Sun SS, Chumlea
WC, Demerath EW, et al. Lipids, lipoproteins, lifestyle,
adiposity and fat-free mass during middle age: the Fels
Longitudinal Study. Int J Obes. 2006;30(2):251–260. 12. Nakagawa T, Hattori M, Harada K, Shirase R, Bando M,
Okano G. Age-related changes in intramyocellular lipid
in humans by in vivo 1 h-mr spectroscopy. Gerontol. 2007;53(4):218–223. ( )
13. Martins RA, Veríssimo MT, Coelho E Silva MJ, Cumming SP,
Teixeira AM. Effects of aerobic and strength-based training
on metabolic health indicators in older adults. Lipids Health
Dis. 2010;9:76. REFERENCES 22. Ogihara T, Rakugi H. Hypertension in the elderly a Japanese
perspective. Drugs Aging. 2005;22(4):297-314. 1. Nessel EH. The physiology of aging as it relates to sports. AMAA J. 2004;17:12-17. 1. Nessel EH. The physiology of aging as it relates to sports. AMAA J. 2004;17:12-17. 23. BrandaoRondon MUP, Alves MJNN, Braga AMFW, Teixeira
OTUN, Barretto ACP, Krieger EM, et al. Postexercise blood
pressure reduction in elderly hypertensive patients. J Am
Col Cardiol. 2002;39:676–682. 2. Seeley RR, Stephens TD, Tate P. Anatomy and physiology. 7th ed: New-York: McGraw-Hill; 2006. 2. Seeley RR, Stephens TD, Tate P. Anatomy and physiology. 7th ed: New-York: McGraw-Hill; 2006. 3. Carter JB, Banister EW, Blaber AP. Effect of endurance
exercise on autonomic control of heart rate. J Sports Med. 2003;33:33-46. 24. Badaruddoza Kaur N, Barna B. Inter-relationship of waist-to-
hip ratio (WHR), body mass index (BMI) and subcutaneous
fat with blood pressure among university-going Punjabi
Sikh and Hindu females. Int J Med Medical Sci. 2010;2:5-11. 4. Ergun U, Nurlu MDG, Komurcu F. Analysis of heart rate
variability: normal values of subjects over 60 years old. Int J
Neuroscience. 2008;118:165–173. 25. Prentice WE. Arnheim’s Principles of Athletic Training:
a Competency-based approach. (13th ed.): New-York:
McGraw-Hill; 2009. 5. Singh D, Deepak KK. Spectral evaluation of aging effects on
blood pressure and heart rate variations in healthy subjects. J Med Eng Technol. 2006;30(3):145–150. 26. Kitamura I, Takeshima N, Tokudome M, Yamanouchi K,
Oshida Y, Sato Y. Effects of aerobic and resistance exercise
training on insulin action in the elderly. Geriatr Gerontol
Int. 2003;3:50–55.i g
6. Lawrence A, Llinas RH. Hypertension and stroke. Hypertension and stroke: preceding p1-94. [Online]. Available
from:
http://web.ebscohost.com/ehost/
pdfviewer/pdfviewer?vid=4&hid=104&sid=0295c7b1-
a205-4205-a4bc-8889ac4695e2%40sessionmgr111 [Accessed:
08/07/2011] 2006.l 27. Gikas A, Sotiropoulos A, Pastromas V, Papazafiropoulou A,
Apostolou O, Pappas S. Seasonal variation in fasting glucose
and HbA1c in patients with type 2 diabetes. Primary Care
Diabetes. 2009;3(2):111–114. 7. Monahan KD. Effect of aging on baroreflex function in
humans. Am J Physio - Regulatory, Integrative Comp
Physiol. 2007;293:R3-R12. 28. Thompson DL. Fitness professional’s handbook. 5th ed:
Champaign, IL: Human Kinetics, 2007. y
8. Elahi D, Muller DC. Carbohydrate metabolism in the
elderly. Euro J Clin Nutr. 2000;54:S112-S120. p g
29. Boardley D, Fahlman M, Topp R, Morgan AL, Mcnevin
N. The impact of exercise training on blood lipids in older
adults. Am J Geriatr Cardiol. 2006;16(1):30-35. y
9. Stout RW. Glucose tolerance and ageing. J Royal Soc Med. 1994;87(10):608-609. REFERENCES 504 Pak J Med Sci 2013 Vol. 29 No. 2 www.pjms.com.pk 504 Pak J Med Sci 2013 Vol. 29 No. 2 www.pjms.com.pk
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https://openalex.org/W2038699893
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English
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Pharmacological Characterization of a 5-HT1-Type Serotonin Receptor in the Red Flour Beetle, Tribolium castaneum
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PloS one
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cc-by
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Abstract Serotonin (5-hydroxytryptamine, 5-HT) is known for its key role in modulating diverse physiological processes and behaviors
by binding various 5-HT receptors. However, a lack of pharmacological knowledge impedes studies on invertebrate 5-HT
receptors. Moreover, pharmacological information is urgently needed in order to establish a reliable classification system for
invertebrate 5-HT receptors. In this study we report on the molecular cloning and pharmacological characterization of a 5-
HT1 receptor from the red flour beetle, Tribolium castaneum (Trica5-HT1). The Trica5-HT1 receptor encoding cDNA shows
considerable sequence similarity with members of the 5-HT1 receptor class. Real time PCR showed high expression in the
brain (without optic lobes) and the optic lobes, consistent with the role of 5-HT as neurotransmitter. Activation of Trica5-HT1
in mammalian cells decreased NKH-477-stimulated cyclic AMP levels in a dose-dependent manner, but did not influence
intracellular Ca2+ signaling. We studied the pharmacological profile of the 5-HT1 receptor and demonstrated that a-
methylserotonin, 5-methoxytryptamine and 5-carboxamidotryptamine acted as agonists. Prazosin, methiothepin and
methysergide were the most potent antagonists and showed competitive inhibition in presence of 5-HT. This study offers
important information on a 5-HT1 receptor from T. castaneum facilitating functional research of 5-HT receptors in insects
and other invertebrates. The pharmacological profiles may contribute to establish a reliable classification scheme for
invertebrate 5-HT receptors. Citation: Vleugels R, Lenaerts C, Baumann A, Vanden Broeck J, Verlinden H (2013) Pharmacological Characterization of a 5-HT1-Type Serotonin Receptor in the
Red Flour Beetle, Tribolium castaneum. PLoS ONE 8(5): e65052. doi:10.1371/journal.pone.0065052 Editor: Hubert Vaudry, University of Rouen, France Editor: Hubert Vaudry, University of Rouen, France Received January 17, 2013; Accepted April 22, 2013; Published May 31, 2013 Copyright: 2013 Vleugels et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors thank the Interuniversity Attraction Poles program (Belgian Science Policy Grant P7/40) and the KU Leuven Research Foundation (GOA/11/
02) for financial support. RV and CL were supported by the Agency for Innovation by Science and Technology (IWT). HV was supported by the Research
Foundation of Flanders(FWO). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: jozef.vandenbroeck@bio.kuleuven.be Pharmacological Characterization of a 5-HT1-Type
Serotonin Receptor in the Red Flour Beetle, Tribolium
castaneum Rut Vleugels1, Cynthia Lenaerts1, Arnd Baumann2, Jozef Vanden Broeck1*, Heleen Verlinden1
1 Department of Animal Physiology and Neurobiology, Zoological Institute, KU Leuven, Leuven, Belgium, 2 Institute of Complex Systems (ICS4), Research Centre Ju¨lich,
Ju¨lich, Germany Introduction signaling pathways in all eukaryote organisms. The vertebrate 5-
HT GPCRs (5-HT1,2,4–7) were classified based on their sequence
similarities, gene organization, downstream signaling pathways
and pharmacological properties [17–20]. 5-HT1 and 5-HT5
receptors couple preferentially to Gi/o proteins and thus inhibit
cyclic AMP (cAMP) synthesis. 5-HT2 receptors couple preferen-
tially to Gq/11 proteins which cause an increase in cytosolic Ca2+
levels. 5-HT4, 5-HT6 and 5-HT7 receptors are all preferentially
linked to Gs proteins and promote cAMP production. Biogenic amines play an important role in very diverse
physiological processes and behaviors. In insects, the six major
biogenic amines are serotonin (5-hydroxytryptamine, 5-HT),
dopamine, tyramine, octopamine, acetylcholine and histamine. 5-HT is known to play a crucial role in the regulation of important
processes in most, if not all, animal phyla. Alterations in 5-HT
neurotransmission are associated with several human disorders,
such as migraine, depression, schizophrenia and anxiety [1]. Normal human processes, such as sleep, mood level, appetite,
sexual activity and learning abilities are also modulated by 5-HT. In insects, 5-HT signaling controls nutrition [2], modulation of
heart rate [3], secretory processes in the salivary gland [4–7],
development [8], circadian rhythms and sleep regulation [9,10],
aggression [11], behavioral gregarization in locusts [12,13],
phototactic behavior in honeybees [14] and learning and memory
in fruit flies [15,16]. The major classes, 5-HT1, 5-HT2, and 5-HT6, probably
evolved from a primordial 5-HT receptor over 750 million years
ago. The 5-HT5 and 5-HT7 receptor classes diverged from 5-HT1
650 to 700 million years ago [21,22]. Since these events even
predate the estimated divergence of protostomes and deutero-
stomes about 600 to 650 million years ago [23], the invertebrate
and vertebrate serotonergic systems are believed to possess roughly
the same main receptor classes [21,24]. However, evolution
allowed further differentiation in various subtypes within each
main class, and these subtypes are believed to have evolved
independently in vertebrates and invertebrates [21,25]. Thus far,
only four types of 5-HT receptors are characterized in insects,
namely 5-HT1A, 5-HT1B, 5-HT2, and 5-HT7 [8,14,26–29]. Classification of invertebrate 5-HT receptors according to the To mediate such a variety of processes, 5-HT acts through
multiple 5-HT receptor types. In vertebrates, 5-HT receptors are
divided in seven main classes. Six of these are G protein-coupled
receptors (GPCRs) and the sole exception, 5-HT3, is a ligand-
gated ion channel. qRT-PCR Study of Transcript Levels y
For determination of expression levels of the receptor, tissues
from sexually mature T. castaneum were dissected in phosphate
buffered saline (PBS) (NaCl 137 mM, KCl 2.7 mM, Na2HPO4
10 mM, KH2PO4 1.76 mM; pH 7.2) and snap-frozen in liquid
nitrogen. For all samples, tissues of at least fifteen animals were
pooled. Tissues were homogenized and RNA was extracted using
the RNAqueous Micro Kit (Ambion) according to the protocol
recommended by the kit. The protocol included an additional
DNase treatment to digest remaining DNA. Total RNA was
reverse transcribed into cDNA using SuperScriptIII reverse
transcriptase (Invitrogen) as recommended by the manufacturer,
and diluted ten-fold prior to use. Transcript levels were quantified
using the Fast Sybr Green assay kit (Applied Biosystems) in a
StepOne Plus detection system (ABI Prism, Applied Biosystems). Primers (sense primer 59-GCCCTCTGGCTGGGCTAT-39 and
antisense primer 59-CGGGTTGAAGATCGTGTAAATGA-39)
(Sigma-Aldrich) were used in final concentrations of 500 nM. Other conditions were as recommended by the manufacturer. Reactions were run in duplicate and incubated for 2 min at 50uC,
followed by 10 min at 95uC, followed by 40 cycles of [15 s at 95uC
and 1 min at 60uC]. The specificity of the PCR products was
assessed generating a dissociation curve (95uC for 15 s, 60uC for
1 min, and increase in temperature in 0.7uC increments from
60uC to 95uC). Agarose gel electrophoresis of the PCR products
confirmed the presence of a single band of the expected size and
sequencing confirmed their identity. The relative quantity of target
cDNA was quantified using the DDCT-method including normal-
ization to a calibrator on all PCR plates and an endogenous
control. From a list of seven housekeeping genes (Table S1; [34]),
the combination of genes for this endogenous control was
determined using GeNorm [35]. Expression was most stable for
RPs3 (ribosomal protein 3) and RPs18 with respect to sex and
tissue and these transcripts were thus selected for further use as
endogenous controls (results not shown). In the present study, we will discuss the characterization of a 5-
HT1 receptor from the red flour beetle, Tribolium castaneum (Trica5-
HT1). The genome of T. castaneum has been completely sequenced
(Tribolium Genome Sequencing Consortium) [30]. Use of annota-
tion software led to the discovery of twenty Tribolium genes that
code for putative biogenic amine GPCRs. All these proteins have
orthologues in Drosophila melanogaster and Apis mellifera [31]. Animal Rearing Conditions Beetles were reared in a dark incubator at 30uC on wheat flour
and brewer’s yeast in Petri dishes. Adult beetles were sexed based
on the presence of a small patch of short bristles on the inside of
the first pair of legs in males, according to the T. castaneum rearing
protocol (http://bru.gmprc.ksu.edu/proj/tribolium/wrangle.asp)
[32]. qRT-PCR Study of Transcript Levels Four
receptors could be assigned as putative 5-HT receptors based on
sequence similarity to 5-HT receptors of D. melanogaster and A. mellifera [8,14,26–28,31]. After cloning the Trica5-HT1 cDNA, we
analyzed its tissue distribution by quantitative real-time PCR
(qRT-PCR) and elucidated its downstream signaling pathway. In
cells expressing Trica5-HT1, application of 5-HT inhibited NKH-
477 (a water-soluble forskolin analog) stimulated cAMP synthesis. The pharmacological profile of the receptor was established after
application of several synthetic 5-HT receptor agonists and
antagonists. These results will facilitate future in vivo studies aiming
to unravel the contribution of individual 5-HT receptors to the
animals’ physiology and behavior. Cloning of Trica5-ht1 and Construction of pcTrica5-ht1
Expression Vector The full length sequence encoding the receptor was amplified
with PCR using whole body T. castaneum cDNA, Taq polymerase
(REDTaqHReadyMixTMPCR Reaction Mix, Sigma-Aldrich), and
10 mM
of
sense
primer
59-ATGGGGACAGTAAA-
TAATCCCTCCTG-39
and
antisense
primer
59-TTATC-
TAATTTTGCCCGAGCGG-39 (Sigma-Aldrich). Primers were
designed based on sequences available in Beetlebase (Tcas_3.0;
http://www.beetlebase.org/) [33] released by the Human Ge-
nome Sequencing Center. PCR started with initial denaturation
for 2 min at 95uC, followed by 35 cycles of [30 s at 94uC, 30 s at
62uC, 2 min at 68uC], followed by final elongation for 2 min at
68uC. Amplification products were run on a 1.2% agarose gel and
purified with the GenEluteTM Gel extraction Kit (Sigma–Aldrich). The DNA fragments were cloned into a pcDNA3.1/V5-His-
TOPOHTA expression vector via TA TOPO cloning (Invitrogen)
and transformed into One Shot TOP10 chemically competent
Escherichia coli cells (Invitrogen). Bacteria were grown according to
the protocol recommended by the kit. Plasmids were isolated via
the GenEluteTM HP Plasmid Miniprep kit (Sigma-Aldrich) and
DNA sequences were determined by means of the ABI PRISM
3130 Genetic Analyzer (Applied Biosystems) following the protocol
outlined in the ABI PRISM BigDye Terminator Ready Reaction Pharmacology of a Beetle 5-HT1 Receptor Pharmacology of a Beetle 5-HT1 Receptor Cycle Sequencing Kit (Applied Biosystems). Bacterial cells known
to contain the correct receptor insert were grown at large scale in
100 ml Luria–Bertani broth medium. The expression vectors were
subsequently isolated from these cells using the EndoFree Plasmid
Maxi Kit (Qiagen) according to the protocol recommended by the
kit. existing vertebrate classes is mainly based on well conserved amino
acid sequences and activated second-messenger systems for 5-HT
receptors across all species. On the other hand, pharmacological
profiles from insect (and other invertebrate) receptors seem to
differ significantly from those of vertebrates [25]. Since only very
few data about pharmacological properties of invertebrate 5-HT
receptors are available, there is no general classification system for
invertebrate 5-HT receptors based on pharmacological properties
yet. This explains the need for detailed pharmacological studies on
insect and other invertebrate 5-HT receptors. Materials and Methods Animal Rearing Conditions Introduction GPCRs play a vital role in many essential PLOS ONE | www.plosone.org May 2013 | Volume 8 | Issue 5 | e65052 1 Aequorin-luminescence Assay Cloning and Sequence Analysis of Trica5-ht1
A cDNA fragment encoding a 5-HT1 receptor from T. castaneum
was amplified by PCR. The open reading frame of Trica5-ht1
contains 1,644 nucleotides (Figure S1) encoding the Trica5-HT1
protein of 547 amino acids (Figure 1) with a calculated molecular
weight of 60.8 kDa. Transmembrane topology prediction revealed
the presence of seven putative transmembrane domains (TM1-7),
characteristic of all GPCRs. Consensus motifs for N-linked
glycosylation (N-x-[S/T]) are found in the extracellular N-
terminus, and consensus sites for phosphorylation by protein
kinase C (PKC) ([S/T-x-[R/K]) are located within the third
intracellular loop (Figure 1). The C7.69 residue (numbering
according to the Ballesteros-Weinstein system [40]) in the
intracellular C-terminus is a putative palmitoylation site. The
large third intracellular loop and the short intracellular C-terminal
region are consistent with other known 5-HT1 and biogenic amine
receptors that couple via Gi. Other typical biogenic amine and 5-
HT receptor characteristics are present as well. The DRY
tripeptide (D3.49R3.50Y3.51) located in the second intracellular loop
is the key to the conformational changes necessary for receptor
activation [41]. The combination of the D3.32 in TM3 with the
conserved W7.40 in TM7 is considered a unique fingerprint for
biogenic amine and trace amine GPCRs. The charged D3.32
residue is thought to interact with the protonated amine moiety of
amine ligands [42,43]. As in other 5-HT receptors, Trica5-HT1
typically has a conserved group of hydrophobic amino acids
(W3.28, F5.47, W6.48, F6.51, F6.52, W7.40, Y7.43) that form the
hydrophobic ligand-binding pocket within the tertiary structure. This binding pocket may be stabilized by a disulfide bridge formed
between C2.55 in TM2 and C3.25 in extracellular loop 1 [42,44,45]. In addition, the consensus sequence of non-peptide receptors in
TM6, F6.44-x-x-x-W6.48-x-P6.50, is followed by a pair of Phe
residues (F6.51 and F6.52) unique to aminergic receptors. Also the
N7.49P7.50-x-x-Y7.53 motif in TM7 is conserved, which may
participate
in
agonist
mediated
receptor
sequestration
and
resensitization [46]. Aequorin luminescence Assay
The transfected CHO cells were detached with phosphate
buffered saline (PBS) containing 0.2% EDTA and collected in
DMEM/F-12 (without phenol red, with L-glutamine and 10 mM
HEPES) (Gibco). The amount of viable cells was determined using
the NucleoCounter NC-100+TM (Chemometic). Cells were
pelleted
for 4 min at 800 rpm at room temperature and
resuspended in BSA-medium (DMEM/F12 without phenol red,
with L-glutamine and 10 mM HEPES, supplemented with 0.1%
bovine serum albumin) to a concentration of 56106 cells/ml. Aequorin-luminescence Assay Coelenterazine H (Invitrogen) was added to a final concentration
of 5 mM, and cells were gently shaken for 4 h at room temperature
in the dark. After a 10-fold dilution in BSA-medium, cells were
incubated another 30 min. The pharmacological ligands were
dissolved in BSA-medium. For agonists, 50 ml containing the final
ligand concentration was added to appropriate wells of a 96-well
plate. For antagonists, 25 ml of the antagonist solution was
supplemented with 25 ml of a 5-HT solution. Receptor activity
was measured as the light emission after adding 50 ml of the cell
suspension. Light emission was measured for 30 s using a Mithras
LB940 (Berthold Technologies). Subsequently, cells were lysed
with Triton X-100 (0.1% in BSA-medium) and light emission was
recorded for another 8 s. BSA-medium was used as a negative
control. Light emission from each well was calculated relative to
the total response (ligand+Triton X-100) using the output file of
Mikrowin2000 software (Mikrotek). Further analysis was done in
Graphpad Prism 5. Drugs The pharmacological ligands 3-hydroxytyramine (dopamine)
hydrochloride, 5-carboxamidotryptamine maleate (5-CT), 5-HT
hydrochloride (5-HT), 5-methoxytryptamine (5-MT), (6)-8-hy-
droxy-2-(dipropylamino)tetralin hydrobromide (8-OH-DPAT), a-
methylserotonin maleate (am-5-HT), (+)-butaclamol hydrochlo-
ride, ketanserin (+)-tartrate, methiothepin mesylate, methysergide
maleate, mianserin hydrochloride, prazosin hydrochloride, DL-
octopamine hydrochloride, SB-269970 hydrochloride, tyramine
hydrochloride, WAY-100635 maleate (WAY = N-{2-[4-(2-meth-
oxyphenyl)-1-piperazinyl]-ethyl}-N-(2-pyridinyl)
cyclohexanecar-
boxamide), and yohimbine hydrochloride were purchased from
Sigma-Aldrich. Pharmacology of a Beetle 5-HT1 Receptor Pharmacology of a Beetle 5-HT1 Receptor dispensed. After incubation for 3–4 h in a CO2 incubator at 37uC,
100 ml of SteadyLite Plus (Perkin-Elmer) was added to each well
and the plate was gently shaken for 15 min in the dark. Light
emission was measured for 5 s per well using a Mithras LB940
(Berthold Technologies). Medium containing IBMX was used as a
negative control. Data were analyzed as described for CHO cells. expressing apoaequorin (CHO-PAM28) [36], or both apoaequorin
and Ga16 (CHO-WTA11 cells) [37] and are thus still used as
additional antibiotics in the appropriate screens. For CHO cells,
the medium was supplemented with 10% fetal calf serum
(inactivated at 65uC) (Sigma-Aldrich). For HEK293 cells, the
medium was supplemented with 2% Ultroser G serum substitute
(Pall Life Sciences). expressing apoaequorin (CHO-PAM28) [36], or both apoaequorin
and Ga16 (CHO-WTA11 cells) [37] and are thus still used as
additional antibiotics in the appropriate screens. For CHO cells,
the medium was supplemented with 10% fetal calf serum
(inactivated at 65uC) (Sigma-Aldrich). For HEK293 cells, the
medium was supplemented with 2% Ultroser G serum substitute
(Pall Life Sciences). (
)
Cells were cultured in vitro as monolayers at 37uC, 5% CO2 and
high relative humidity, and subcultivated twice a week. For
transfection of CHO cells, 2.5 ml Opti-MEMHI (Invitrogen), 5 mg
plasmid DNA and 12.5 ml PlusTMreagent (Invitrogen) were mixed,
stored at room temperature for 5 min and next repleted with 30 ml
LipofectamineTMLTX (Invitrogen). After 30 min incubation at
room temperature, this transfection mixture was added dropwise
to the cells together with 5 ml medium. HEK293 cells were
cotransfected with receptor construct (4 mg) and CRE-luciferase
construct (2 mg), consisting of the open reading frame of the
reporter gene, luciferase, downstream of a multimerized cAMP-
response-element (CRE6x) [38,39]. All transfections were per-
formed in T75 flasks with a confluency of about 60 percent. Cells
were grown overnight, after which 15 ml of medium was added
for an additional overnight incubation period. Cell Culture and Transfection General binding studies were performed in Chinese hamster
ovary (CHO) WTA11 cells stably coexpressing apoaequorin and
the promiscuous Ga16. This allowed us to measure the pharma-
cology independent of the downstream signaling. CHO-PAM28
cells stably expressing apoaequorin, but not the promiscuous Ga16,
and human embryonic kidney cells (HEK) 293 cells were used to
measure downstream signaling via Ca2+ and cAMP, respectively. CHO cell lines were provided by Prof. Marc Parmentier
(University of Brussels, Belgium) and Dr. Michel Detheux
(Euroscreen S.A., Belgium). HEK293 cells were a gift from Prof. Arnd Baumann (Research Centre Ju¨lich, Germany. All cells were cultured in Dulbecco’s Modified Eagles Medium
nutrient mixture F12-Ham (DMEM/F12) (Invitrogen) supple-
mented with 1% penicillin/streptomycin (10000 units/ml penicil-
lin and 10 mg/ml streptomycin in 0.9% NaCl) (Invitrogen) to
prevent bacterial contamination of gram-positive and gram-
negative bacteria, respectively. For CHO-WTA11 cells, 250 mg/
ml zeocin (Invitrogen) was added and for CHO-PAM28 cells,
5 mg/ml puromycin dihydrochloride (Invitrogen) was added. Puromycin and zeocin were initially used to select for cells stably May 2013 | Volume 8 | Issue 5 | e65052 May 2013 | Volume 8 | Issue 5 | e65052 PLOS ONE | www.plosone.org 2 Luciferase Reporter-gene Assay Cotransfected HEK293 cells were detached and the amount of
viable cells was determined as described for CHO cells. Cells were
pelleted
for 4 min at 800 rpm at room temperature and
resuspended in DMEM/F12 medium (without phenol red, with
L-glutamine and 10 mM HEPES) containing 200 mM 3-isobutyl-
1-methylxanthine (IBMX, Sigma-Aldrich) to a concentration of
106 cells/ml. The pharmacological ligands were dissolved in
DMEM/F12 medium without phenol red containing 200 mM
IBMX. The water-soluble forskolin analog NKH-477 was added
to a concentration of 20 mM to measure Trica5-ht1-mediated
effects on cellular cAMP signaling. In each well of a 96-well plate,
50 ml of ligand suspension and 50 ml of cell suspension were BLASTx (http://blast.ncbi.nlm.nih.gov/blast/) searches indi-
cate similarities of the receptor to other insect 5-HT1 receptors
(Pea5-HT1, 60% identity; Am5-HT1A, 55% identity; Dm5-HT1A,
46% identity; Dm5-HT1B, 42% identity). When compared to the
computationally predicted sequence in Beetlebase [31], a stretch of May 2013 | Volume 8 | Issue 5 | e65052 PLOS ONE | www.plosone.org 3 Figure 1. Amino acid sequence alignment of T. castaneum 5-HT1 sequence (Trica5-HT1, GenBank accession no. KC196076). Alignment
against sequences of orthologous receptors from Apis mellifera (Am5-HT1A, no. CBI75449), Drosophila melanogaster (Dm5-HT1A, no. CAA77570 and
Dm5-HT1B, no. CAA77571), and Periplaneta americana (Pea5-HT1, no. CAX65666). Identical residues between the receptors are shown as white
characters against black background. Conservatively substituted residues are shaded. Putative transmembrane domains are indicated by grey bars
(TM1-7). Dots indicate putative phosphorylation sites for PKC, and stars indicate putative N-linked glycosylation sites. Inverted triangles indicate
differences between the current sequence derived from cloned cDNA and the annotated sequence from Beetlebase. doi:10.1371/journal.pone.0065052.g001
Pharmacology of a Beetle 5-HT1 Receptor Pharmacology of a Beetle 5-HT1 Receptor 75 bp is missing in our cloned receptor cDNA sequence. Since this
stretch is flanked by splice sites and not present in cDNA
sequences of other species, it is presumed to be an intron. After
multiple sequencing runs on different cDNA samples, also five
single base mismatches (three silent and two missense mutations)
were found between the amplified and the annotated sequence. Furthermore, two consecutive nucleotides were different, resulting
in an amino acid change (Figure S1). Figure 1. Amino acid sequence alignment of T. castaneum 5-HT1 sequence (Trica5-HT1, GenBank accession no. KC196076). Alignment
against sequences of orthologous receptors from Apis mellifera (Am5-HT1A, no. CBI75449), Drosophila melanogaster (Dm5-HT1A, no. CAA77570 and
Dm5-HT1B, no. CAA77571), and Periplaneta americana (Pea5-HT1, no. CAX65666). Transcript Level Study An initial tissue distribution screen of Trica5-HT1 mRNA in
sexually
mature
beetles
was
performed
using
qRT-PCR
(Figure 2A). Transcripts of the Trica5-ht1 gene were detected in
considerable amounts in the head and the gut, consistent with the
prevalence of 5-HT in central nervous system (CNS) and the
digestive tract of most animal species. However, expression in the
head appeared significantly more abundant. Differences between
males and females were not significant. In the fat body and the
reproductive tract of sexually mature beetles, only very low levels
of receptor transcript were observed. When transcript levels in the
brain (without the optic lobes) and the optic lobes were measured
separately, expression in both tissues seemed significantly higher
than in the gut (Figure 2B). Moreover, expression in the brain
(without the optic lobes) was 3.5 times higher compared to the
optic lobes. Although there are indications for 5-HT receptor
expression in the salivary gland of some insect species, we detected
almost no Trica5-HT1 transcripts in the salivary gland of adult
beetles. Potential antagonists were tested by simultaneously applying 5-
HT (100 nM) and a high dose of antagonist (100 mM) to Trica5-
HT1
expressing
cells
(Figure
4A). In
addition,
the
dose-
dependence of the antagonistic effects were measured with
antagonist
concentrations
ranging
from
10 nM
to
1 mM
(Figure 4B). From these experiments, we can conclude that
ketanserin and mianserin, two selective antagonists of mammalian
5-HT2,
and
butaclamol,
a
dopamine
receptor
antagonist,
displayed no detectable inhibition of 5-HT induced responses in
cells expressing Trica5-HT1. Mianserin and butaclamol even
seemed to have some agonistic effects at high concentration
(100 mM). On the other hand prazosin, a selective a1-adrenergic
receptor antagonist in mammals, was shown to be the most potent
antagonist. It decreased the effect of 5-HT on the receptor in a
dose-dependent manner with a half maximal inhibitory concen-
tration (IC50) of 1.39 mM (logIC50 = 25.8660.18, mean 6 SEM). The IC50 values for all antagonists are shown in Table 2. Methiothepin and methysergide, two non-selective antagonists of
mammalian 5-HT receptors also showed dose-dependent inhibi-
tion. Moderate inhibition was achieved with SB-269970, a
selective antagonist of mammalian 5-HT7. The level of inhibition
induced by WAY-100635 didn’t drop below 50% and was variable
which might be due to complex effects of the compound on the
receptor and/or other targets in the assay. Transcript Level Study Also yohimbine, known
to behave as both agonist and antagonist on some mammalian 5-
HT-receptors, showed only about 30% of inhibition, even at Pharmacology of a Beetle 5-HT1 Receptor Pharmacology of a Beetle 5-HT1 Receptor set at 100% (Figure 3B). Surprisingly, the most potent agonist was
the mammalian 5-HT2 receptor agonist, am-5-HT, with an EC50
value of 10.74 mM (logEC50 = 24.9760.18, mean 6 SEM). However, this is more than 100-fold less potent than 5-HT itself. Also the 5-HT analog, 5-CT, a selective agonist for mammalian 5-
HT1 and 5-HT7 receptors, and the non-selective 5-HT receptor
agonist, 5-MT, acted as partial agonists in a dose-dependent
manner. 5-CT was more potent but had a lower efficacy than 5-
MT. The EC50 values for all agonists are shown in Table 1. 8-OH-
DPAT, a partial and selective agonist for mammalian 5-HT1 and
5-HT7
receptors
evoked
responses
only
at
concentrations
$100 mM. The biogenic amines, dopamine, octopamine and
tyramine did not generate any detectable responses at concentra-
tions #100 mM (results not shown). Luciferase Reporter-gene Assay Identical residues between the receptors are shown as white
characters against black background. Conservatively substituted residues are shaded. Putative transmembrane domains are indicated by grey bars
(TM1-7). Dots indicate putative phosphorylation sites for PKC, and stars indicate putative N-linked glycosylation sites. Inverted triangles indicate
differences between the current sequence derived from cloned cDNA and the annotated sequence from Beetlebase. doi:10.1371/journal.pone.0065052.g001 Figure 1. Amino acid sequence alignment of T. castaneum 5-HT1 sequence (Trica5-HT1, GenBank accession no. KC196076). Alignment
against sequences of orthologous receptors from Apis mellifera (Am5-HT1A, no. CBI75449), Drosophila melanogaster (Dm5-HT1A, no. CAA77570 and
Dm5-HT1B, no. CAA77571), and Periplaneta americana (Pea5-HT1, no. CAX65666). Identical residues between the receptors are shown as white
characters against black background. Conservatively substituted residues are shaded. Putative transmembrane domains are indicated by grey bars
(TM1-7). Dots indicate putative phosphorylation sites for PKC, and stars indicate putative N-linked glycosylation sites. Inverted triangles indicate
differences between the current sequence derived from cloned cDNA and the annotated sequence from Beetlebase. doi:10.1371/journal.pone.0065052.g001 Figure 1. Amino acid sequence alignment of T. castaneum 5-HT1 sequence (Trica5-HT1, GenBank accession no. KC196076). Alignment
against sequences of orthologous receptors from Apis mellifera (Am5-HT1A, no. CBI75449), Drosophila melanogaster (Dm5-HT1A, no. CAA77570 and
Dm5-HT1B, no. CAA77571), and Periplaneta americana (Pea5-HT1, no. CAX65666). Identical residues between the receptors are shown as white
characters against black background. Conservatively substituted residues are shaded. Putative transmembrane domains are indicated by grey bars
(TM1-7). Dots indicate putative phosphorylation sites for PKC, and stars indicate putative N-linked glycosylation sites. Inverted triangles indicate
differences between the current sequence derived from cloned cDNA and the annotated sequence from Beetlebase. doi:10.1371/journal.pone.0065052.g001 75 bp is missing in our cloned receptor cDNA sequence. Since this
stretch is flanked by splice sites and not present in cDNA
sequences of other species, it is presumed to be an intron. After
multiple sequencing runs on different cDNA samples, also five single base mismatches (three silent and two missense mutations)
were found between the amplified and the annotated sequence. Furthermore, two consecutive nucleotides were different, resulting
in an amino acid change (Figure S1). May 2013 | Volume 8 | Issue 5 | e65052 May 2013 | Volume 8 | Issue 5 | e65052 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 4 Pharmacological Characterization and Downstream
Signaling Properties of Trica5-HT1 In order to pharmacologically characterize Trica5-HT1, we
used CHO-WTA11 cells stably expressing apoaequorin and the
promiscuous Ga16 subunit. No 5-HT evoked signal was observed
in non-transfected cells or in cells transfected with an empty vector
(results not shown). Significant responses were obtained when cells
expressing Trica5-HT1 were incubated with 100 mM 5-HT or the
5-HT receptor agonists am-5-HT, 5-CT, 5-MT and 8-OH-DPAT
(Figure 3A). The dose-response relationship for 5-HT and these
synthetic agonists was examined at concentrations ranging from
1 pM to 1 mM (Figure 3B). The resulting sigmoidal dose-response
curve of 5-HT shows receptor activation in a dose-dependent and
saturable manner. Half-maximal activation (EC50) was achieved at
5-HT
concentrations
of 95.15 nM (logEC50 = 27.0160.043,
mean 6 SEM). The maximal response was attained at 5-HT
concentrations of $10 mM. Since the efficacy achieved by any
agonist depends on the number of receptors expressed, we
measured a dose-response curve for 5-HT in every experiment
and normalized all agonist effects to the maximum 5-HT response, Figure 2. Expression profile of transcripts encoding Trica5-HT1 in sexually mature beetles. The data represent mean values of (A) three
independent samples of 306heads, 506guts, 206fat body and 506reproductive system; and (B) three independent samples of 15 beetles each; run
in duplicate 6 SEM, normalized relative to RPs3 (ribosomal protein 3) and RPs18 transcript levels. Statistically significant differences are indicated by
asterisks above the bars (p#0.05) (Kruskal-Wallis, IBM SPSS Statistics 20). Abbreviations: FB, fat body; RS, reproductive system; Brain, brain without the
optic lobes; OL, optic lobes; SalGl, salivary glands. doi:10.1371/journal.pone.0065052.g002 Figure 2. Expression profile of transcripts encoding Trica5-HT1 in sexually mature beetles. The data represent mean values of (A) three
independent samples of 306heads, 506guts, 206fat body and 506reproductive system; and (B) three independent samples of 15 beetles each; run
in duplicate 6 SEM, normalized relative to RPs3 (ribosomal protein 3) and RPs18 transcript levels. Statistically significant differences are indicated by
asterisks above the bars (p#0.05) (Kruskal-Wallis, IBM SPSS Statistics 20). Abbreviations: FB, fat body; RS, reproductive system; Brain, brain without the
optic lobes; OL, optic lobes; SalGl, salivary glands. doi:10.1371/journal.pone.0065052.g002 May 2013 | Volume 8 | Issue 5 | e65052 PLOS ONE | www.plosone.org 5 Pharmacology of a Beetle 5-HT1 Receptor Figure 3. Effect of various agonists on Trica5-HT1 in CHO-WTA11 cells. Pharmacological Characterization and Downstream
Signaling Properties of Trica5-HT1 The nature of inhibition obtained
with the three most potent antagonists: prazosin, methiothepin
and methysergide was further examined by studying the dose-
response relationship of 5-HT in presence of different concentra-
tions of antagonist (10 nM to 1 mM) (Figure 5). The higher the
concentration of antagonist, the higher was the resulting EC50
value for 5-HT activity. Since their efficacy didn’t change, these
compounds behaved as truly competitive antagonists. We used the
Gaddum/Schild plot to compare the inhibitory potencies of the
antagonists by their pA2 values (i.e. the logarithm of the
concentration of antagonist that doubles the amount of 5-HT
required for obtaining the same effect) [47]. The pA2 values (6
SEM) for prazosin, methiothepin and methysergide were, respec-
tively, 7.18 (60.13), 6.17 (60.11) and 5.96 (60.21), confirming
that prazosin has the highest affinity, followed by methiothepin
and methysergide. concentrations up to 100 mM. The nature of inhibition obtained
with the three most potent antagonists: prazosin, methiothepin
and methysergide was further examined by studying the dose-
response relationship of 5-HT in presence of different concentra-
tions of antagonist (10 nM to 1 mM) (Figure 5). The higher the
concentration of antagonist, the higher was the resulting EC50
value for 5-HT activity. Since their efficacy didn’t change, these
compounds behaved as truly competitive antagonists. We used the
Gaddum/Schild plot to compare the inhibitory potencies of the
antagonists by their pA2 values (i.e. the logarithm of the
concentration of antagonist that doubles the amount of 5-HT
required for obtaining the same effect) [47]. The pA2 values (6
SEM) for prazosin, methiothepin and methysergide were, respec-
tively, 7.18 (60.13), 6.17 (60.11) and 5.96 (60.21), confirming
that prazosin has the highest affinity, followed by methiothepin
and methysergide. Pharmacological Characterization and Downstream
Signaling Properties of Trica5-HT1 (A) Receptor activation after stimulation with 100 mM of agonist,
shown as the percentage of activation achieved with 100 mM of 5-HT (set at 100%). (B) Dose-dependent activation of Trica5-HT1 with synthetic 5-HT
receptor agonists, shown as the percentage of activation achieved with 1 mM 5-HT (maximum response = 100%). Cells treated with BSA-medium only
were used to define the basal level of 0%. Data represent the mean 6 SEM of (A) three independent measurements (each performed in duplicate)
and (B) seven independent measurements (each performed in duplicate) for 5-HT, or four independent measurements (three performed in triplicate,
one performed in duplicate) for the synthetic agonists am-5-HT, 5-MT, 5-CT and 8-OH-DPAT. doi:10.1371/journal.pone.0065052.g003 Figure 3. Effect of various agonists on Trica5-HT1 in CHO-WTA11 cells. (A) Receptor activation after stimulation with 100 mM of agonist,
shown as the percentage of activation achieved with 100 mM of 5-HT (set at 100%). (B) Dose-dependent activation of Trica5-HT1 with synthetic 5-HT
receptor agonists, shown as the percentage of activation achieved with 1 mM 5-HT (maximum response = 100%). Cells treated with BSA-medium only
were used to define the basal level of 0%. Data represent the mean 6 SEM of (A) three independent measurements (each performed in duplicate)
and (B) seven independent measurements (each performed in duplicate) for 5-HT, or four independent measurements (three performed in triplicate,
one performed in duplicate) for the synthetic agonists am-5-HT, 5-MT, 5-CT and 8-OH-DPAT. doi:10.1371/journal.pone.0065052.g003 concluded that Trica5-HT1 does not couple via Gq to the Ca2+
signaling pathway. In HEK293 cells, effects on the cAMP level
were examined for 5-HT concentrations ranging from 1 pM to
100 mM. Relative high variation in the data can be explained by
assay based variation since cells were not counted when dispensed. Basal levels of cAMP did not significantly change in cells
transfected with an empty vector. In Trica5-HT1 expressing cells,
a dose-dependent decrease in intracellular, NKH-477 stimulated
cAMP levels was registered (Figure 6). Half maximal reduction of
cAMP was observed at 82.7 nM 5-HT (logIC50 = 27.0860.30),
mean 6 SEM). Trica5-HT1 thus inhibits the cAMP production,
probably via the Gi protein. Maximal attenuation of cAMP
synthesis
(640%)
was
attained
with
5-HT
concentrations
$10 mM. concentrations up to 100 mM. doi:10.1371/journal.pone.0065052.t001 Discussion Additionaly, Am5-
HT1A was found in regions known to be important in visual
information processing, such as the optic lobes. Based on
behavioral tests 5-HT is assumed to reduce the positive phototactic
behavior of honeybees [14]. In P. americana, antibodies directed
against Pea5-HT1, stained some large somata in the pars
intercerebralis [29]. Thus, expression of Trica5-HT1 in beetle brains
may be due to expression in the mushroom bodies and/or cells of
the pars intercerebralis. Receptor expression in the gut may indicate a
role of 5-HT in the regulation of digestion or gut contraction. In
humans, for example, as much as 95% of the total 5-HT content
may reside in the intestine [56,57]. It is probable that a substantial
release of 5-HT in other animal species, such as Tribolium, takes place in the digestive tract as well. In several insects, 5-HT
immunoreactive nerve fibers have been localized in different parts
of the intestinal tract [58–60]. 5-HT was also shown to modulate
muscle contractions of the gut in several insects [61–66], although
it may also act as a paracrine factor and, for example, provoke the
release of other factors from neuroendocrine cells. Even then, high
5-HT levels in a given release organ do not necessarily co-incide
with the local level of receptor expression, which might explain the
relatively low 5-HT1 transcript levels in the gut compared to the
brain. Although only low transcript levels were observed in the
salivary glands of adult beetles, 5-HT also has been shown to be
important in salivation in several insect species [4–7,67]. In the
cockroach, 5-HT1 receptor expression was shown in the salivary
gland by RT-PCR and Western blotting [29]. Low expression
levels of Trica5-HT1 receptors in the salivary gland suggest that
possible 5-HT effects on salivation are regulated by other 5-HT
receptor subtypes. For example, in the blowfly, Calliphora vicina,
salivary glands express 5-HT2 and 5-HT7 receptors [67]. To examine the downstream signaling pathway of Trica5-HT1,
the receptor was expressed in CHO-PAM28 and HEK293 cells. Since no Ca2+ response was measured in CHO-PAM28 cells
expressing Trica5-HT1, the receptor does not engage Gq and the
PLC signaling pathway. In HEK293 cells, the receptor was
cotransfected with a CRE-luciferase construct to detect changes in
intracellular cAMP levels. 5-HT was found to decrease NKH-477-
stimulated cAMP synthesis in a dose-dependent manner. In
accordance with other 5-HT1 receptors, Trica5-HT1 couples to
Gi/o proteins that impair adenlylate cyclase activity. Discussion CHO-PAM28 and HEK293 cells were used to determine the
downstream signaling pathway of Trica5-HT1. No effect of 5-HT
was observed in CHO-PAM28 cells transfected with empty vector
or in cells transfected with the receptor. Therefore it can be In the present study, we have characterized Trica5-HT1, a 5-
HT1 receptor of the red flour beetle, T. castaneum. The obtained
sequence has considerable similarity with orthologous receptors
from other invertebrates [14,27,29,48–53] and mammals [20,54]. The sequence contains typical characteristics of 5-HT1 receptors,
such as a large third intracellular loop, a short C-terminal region, a
DRY motif in the second intracellular loop as well as other
conserved consensus sequences. Notably, differences between the
cloned and annotated nucleotide sequences were uncovered. These included single base mismatches and an intron in the
annotated open reading frame (Figure S1). So far, no introns have
been reported in the coding regions of vertebrate genes encoding
5-HT1 receptors [55]. However, introns were found in the D. melanogaster genes encoding Dm5-HT1A and Dm5-HT1B [27]. Table 1. EC50 values of agonists for Trica5-HT1 receptor
activation in CHO-WTA11 cells. agonist
EC50 (mM)
logEC50 (mean ± SEM)
5-HT
0.095
27.0160.043
am-5-HT
10.74
24.9760.18
5-CT
24.72
24.6160.17
5-MT
91.84
24.0460.12
8-OH-DPAT
551.0
23.2660.59
doi:10.1371/journal.pone.0065052.t001 Table 1. EC50 values of agonists for Trica5-HT1 receptor
activation in CHO-WTA11 cells. When studying the Trica5-HT1 transcript levels with qRT-
PCR, highest expression was observed in the brain (without optic
lobes), followed by the optic lobes. More detailed localization
studies in other insects also showed receptor expression in the optic May 2013 | Volume 8 | Issue 5 | e65052 May 2013 | Volume 8 | Issue 5 | e65052 6 Pharmacology of a Beetle 5-HT1 Receptor Figure 4. Effect of various antagonists on 5-HT mediated activation of Trica5-HT1 in CHO-WTA11 cells. (A) Effect of antagonists
(100 mM) on 5-HT (100 nM) mediated receptor activation. Receptor activation is shown as the percentage of activation achieved with 100 nM of 5-HT
(, EC50 value) (set at 100%). (B) Dose-dependent effect of 5-HT receptor antagonists on 5-HT (100 nM) mediated receptor activation. Receptor
activation is shown as the percentage of activation achieved with 10 nM of antagonist (set at 100%). Cells treated with BSA-medium only were used
to define the basal level of 0%. Discussion Data represent the mean 6 SEM of (A) three independent measurements (each performed in duplicate) and (B) two
(prazosin and methysergide) or three (methiothepin and SB-269970) independent measurements (each performed in triplicate). doi:10.1371/journal.pone.0065052.g004 Figure 4. Effect of various antagonists on 5-HT mediated activation of Trica5-HT1 in CHO-WTA11 cells. (A) Effect of antagonists
(100 mM) on 5-HT (100 nM) mediated receptor activation. Receptor activation is shown as the percentage of activation achieved with 100 nM of 5-HT
(, EC50 value) (set at 100%). (B) Dose-dependent effect of 5-HT receptor antagonists on 5-HT (100 nM) mediated receptor activation. Receptor
activation is shown as the percentage of activation achieved with 10 nM of antagonist (set at 100%). Cells treated with BSA-medium only were used
to define the basal level of 0%. Data represent the mean 6 SEM of (A) three independent measurements (each performed in duplicate) and (B) two
(prazosin and methysergide) or three (methiothepin and SB-269970) independent measurements (each performed in triplicate). doi:10.1371/journal.pone.0065052.g004 lobes as well as other parts of the brain [9,10,14,27,29]. In D. melanogaster, Dm5-HT1A and Dm5-HT1B receptors are expressed
in the mushroom bodies [9,10], the region of the brain involved in
learning and memory. Also in the mushroom bodies of A. mellifera,
high levels of Am5-HT1A were found [14]. Additionaly, Am5-
HT1A was found in regions known to be important in visual
information processing, such as the optic lobes. Based on
behavioral tests 5-HT is assumed to reduce the positive phototactic
behavior of honeybees [14]. In P. americana, antibodies directed
against Pea5-HT1, stained some large somata in the pars
intercerebralis [29]. Thus, expression of Trica5-HT1 in beetle brains
may be due to expression in the mushroom bodies and/or cells of
the pars intercerebralis. Receptor expression in the gut may indicate a
role of 5-HT in the regulation of digestion or gut contraction. In
humans, for example, as much as 95% of the total 5-HT content
may reside in the intestine [56,57]. It is probable that a substantial
release of 5-HT in other animal species, such as Tribolium, takes lobes as well as other parts of the brain [9,10,14,27,29]. In D. melanogaster, Dm5-HT1A and Dm5-HT1B receptors are expressed
in the mushroom bodies [9,10], the region of the brain involved in
learning and memory. Also in the mushroom bodies of A. mellifera,
high levels of Am5-HT1A were found [14]. doi:10.1371/journal.pone.0065052.t002 Discussion am-5-HT, 5-CT and 5-MT. The synthetic
agonists were more than a 100-fold less potent than 5-HT and
seemed to have a lower efficacy. However it is possible that the
agonists did not reach their maximum response although high
concentrations (1 mM) were already tested. Similar properties
have been reported for other insect 5-HT1 receptors [14,29]. Only
a very poor response was observed with 8-OH-DPAT, an agonist
for mammalian 5-HT1 and 5-HT7 receptors. Also on A. mellifera
and P. americana 5-HT1 receptors, 8-OH-DPAT acted as a poor
agonist [14,29]. For the antagonists, no inhibition was measured
upon application
of butaclamol,
ketanserin
and
mianserin. Although WAY-100635 is known as a potent and selective
antagonist of mammalian 5-HT1A receptors, no clear dose-
dependent effect was observed. In A. mellifera, however, WAY-
100635 acted as a partial antagonist of Am5-HT1 [14] and an
effective inhibition of agonist stimulated Pea5-HT1 was observed
[29]. A more effective inhibition was measured for the mammalian
5-HT7 receptor antagonist, SB-269970, but no information
regarding the possible effects of this antagonist is known from receptors from other arthropods [14,27,29,52,53]. Besides 5-HT,
three additional ligands caused dose-dependent activation of
Trica5-HT1, i.e. am-5-HT, 5-CT and 5-MT. The synthetic
agonists were more than a 100-fold less potent than 5-HT and
seemed to have a lower efficacy. However it is possible that the
agonists did not reach their maximum response although high
concentrations (1 mM) were already tested. Similar properties
have been reported for other insect 5-HT1 receptors [14,29]. Only
a very poor response was observed with 8-OH-DPAT, an agonist
for mammalian 5-HT1 and 5-HT7 receptors. Also on A. mellifera
and P. americana 5-HT1 receptors, 8-OH-DPAT acted as a poor
agonist [14,29]. For the antagonists, no inhibition was measured
upon application
of butaclamol,
ketanserin
and
mianserin. Although WAY-100635 is known as a potent and selective
antagonist of mammalian 5-HT1A receptors, no clear dose-
dependent effect was observed. In A. mellifera, however, WAY-
100635 acted as a partial antagonist of Am5-HT1 [14] and an
effective inhibition of agonist stimulated Pea5-HT1 was observed
[29]. A more effective inhibition was measured for the mammalian
5-HT7 receptor antagonist, SB-269970, but no information
regarding the possible effects of this antagonist is known from other arthropods. An effective dose-dependent inhibition was
measured for prazosin, methiothepin and methysergide, although
their IC50 values were in the micromolar range. Discussion However,
when interpolating these experimental data to physiological
processes in Tribolium, one must be aware of possible discrepancies
between effects observed in cultured cell lines and intracellular
processes occurring within the in vivo context of the organism. Table 2. IC50 values of antagonists for Trica5-HT1 receptor
inhibition in CHO-WTA11 cells. antagonist
IC50 (mM)
logIC50 (mean ± SEM)
prazosin
1.39
25.8960.18
methiothepin
16.38
24.7960.13
methysergide
33.97
24.4760.094
WAY-100635
no clear dose-dependence of inhibition
butaclamol
204.1
23.6960.20
SB-269970
205.4
23.6960.25
ketanserin
no inhibition
yohimbin
no inhibition
mianserin
no inhibition
doi:10.1371/journal.pone.0065052.t002 Table 2. IC50 values of antagonists for Trica5-HT1 receptor
inhibition in CHO-WTA11 cells. CHO-WTA11 cells were used to investigate the pharmacolog-
ical characteristics of Trica5-HT1. Application of 5-HT to these
cells resulted in dose-dependent receptor activities. The EC50
value of 95.15 nM is similar to values reported for 5-HT1 May 2013 | Volume 8 | Issue 5 | e65052 PLOS ONE | www.plosone.org May 2013 | Volume 8 | Issue 5 | e65052 7 Pharmacology of a Beetle 5-HT1 Receptor Figure 5. Effect of 5-HT on Trica5-HT1 in presence of different concentrations of antagonist. Dose-dependent activation of Trica5-HT1
was measured in CHO-WTA11 cells with 1 nM –1 mM 5-HT in presence of 10 nM –1 mM antagonist: (A) prazosin, (B) methysergide and (C)
methiothepin. Receptor activity is shown as the percentage of activation achieved with 1 mM of 5-HT (set at 100%). Cells treated with BSA-medium
only were used to define the basal level of 0%. Data represent the mean 6 SEM of three independent measurements (each performed in duplicate)
for prazosin and methiothepin and two independent measurements (each performed in duplicate) for methysergide. NA, no antagonist. doi:10.1371/journal.pone.0065052.g005 Figure 5. Effect of 5-HT on Trica5-HT1 in presence of different concentrations of antagonist. Dose-dependent activation of Trica5-HT1
was measured in CHO-WTA11 cells with 1 nM –1 mM 5-HT in presence of 10 nM –1 mM antagonist: (A) prazosin, (B) methysergide and (C)
methiothepin. Receptor activity is shown as the percentage of activation achieved with 1 mM of 5-HT (set at 100%). Cells treated with BSA-medium
only were used to define the basal level of 0%. Data represent the mean 6 SEM of three independent measurements (each performed in duplicate)
for prazosin and methiothepin and two independent measurements (each performed in duplicate) for methysergide. NA, no antagonist. doi:10.1371/journal.pone.0065052.g005 receptors from other arthropods [14,27,29,52,53]. Besides 5-HT,
three additional ligands caused dose-dependent activation of
Trica5-HT1, i.e. References 15. Sitaraman D, Zars M, Laferriere H, Chen YC, Sable-Smith A, et al. (2008)
Serotonin is necessary for place memory in Drosophila. Proc Natl Acad Sci U S A
105: 5579–5584. 1. Jones BJ, Blackburn TP (2002) The medical benefit of 5-HT research. Pharmacol Biochem Behav 71: 555–568. 1. Jones BJ, Blackburn TP (2002) The medical benefit of 5-HT research. Pharmacol Biochem Behav 71: 555–568. 2. Cohen RW, Friedman S, Waldbauer GP (1988) Physiological control of nutrient
self selection in Heliothis zea larvae - the role of serotonin. J Insect Physiol 34:
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rewarded olfactory short-term memory in Drosophila. J Neurogenet 26: 238–244. 3. Chiang RG, Chiang JA, Davey KG (1992) A sensory input inhibiting heart-rate
in an insect, Rhodnius prolixus. Experientia 48: 1122–1125. 17. Hoyer D, Clarke DE, Fozard JR, Hartig PR, Martin GR, et al. (1994)
International Union of Pharmacology classification of receptors for 5-
hydroxytryptamine (Serotonin). Pharmacol Rev 46: 157–203. 4. Berridge MJ, Patel NG (1968) Insect salivary glands - Stimulation of fluid
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functional diversity of 5-HT receptors. Pharmacol Biochem Behav 71: 533–554. 5. Trimmer BA (1985) Serotonin and the control of salivation in the blowfly
Calliphora. J Exp Biol 114: 307–328. 19. Hannon J, Hoyer D (2008) Molecular biology of 5-HT receptors. Behav Brain
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salivary-glands of adult female Aedes aegypti and its role in regulation of salivation. J Exp Biol 198: 167–174. 20. Nichols DE, Nichols CD (2008) Serotonin receptors. Chem Rev 108: 1614–
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control of cockroach salivary glands. Arch Insect Biochem Physiol 62: 141–152. 22. Walker RJ, Brooks HL, Holden-Dye L (1996) Evolution and overview of
classical transmitter molecules and their receptors. Parasitology 113: S3–S33. 8. Colas JF, Launay JM, Kellermann O, Rosay P, Maroteaux L (1995) Drosophila 5-
HT2 serotonin receptor: coexpression with fushi-tarazu during segmentation. Proc Natl Acad Sci U S A 92: 5441–5445. 23. Acknowledgments The authors gratefully acknowledge Marc Parmentier (University of
Brussels, Belgium) and Michel Detheux (Euroscreen S.A., Belgium) for
providing both CHO cell lines. They also thank Dr. Kristel Vuerinckx for
her help with dissections and cDNA sample preparations. receptor subtypes with specific pharmacological properties have
evolved within the main classes of vertebrate receptors. Pharmacology of a Beetle 5-HT1 Receptor Pharmacology of a Beetle 5-HT1 Receptor Figure 6. Dose-dependent effect of 5-HT on intracellular cAMP
levels in HEK293 cells. The effect of 5-HT (1 pM - 100 mM) on the
luciferase bioluminescence in HEK293 cells expressing Trica5-HT1 due to
changes in intracellular cAMP levels. Receptor activity is shown as the
percentage of activation achieved with 10 mM of NKH-477 (set at 100%). Luciferase bioluminescence due to basal intracellular cAMP levels is set
at 0%. The data represent the mean 6 SEM of four independent
measurements (three performed in triplicate, one performed in
duplicate). doi:10.1371/journal.pone.0065052.g006 characterization of all members constituting the invertebrate 5-HT
receptor family are needed to establish a reliable classification
scheme. Detailed pharmacological information for each 5-HT
receptor subtype will also aid in functional in vivo studies and can
be very useful for insect pest control. Author Contributions Conceived and designed the experiments: RV JVB AB HV. Performed the
experiments: RV CL HV. Analyzed the data: RV CL HV. Contributed
reagents/materials/analysis tools: AB JVB. Wrote the paper: RV AB JVB
HV. During the last decades, knowledge about 5-HT (and other
biogenic amine) receptors has increased. However, comprehensive
data on the pharmacology of insect or other invertebrate 5-HT
receptors is still missing. Unequivocal identification and extensive Conceived and designed the experiments: RV JVB AB HV. Performed the
experiments: RV CL HV. Analyzed the data: RV CL HV. Contributed
reagents/materials/analysis tools: AB JVB. Wrote the paper: RV AB JVB
HV. (TIF) Table S1
Nucleotide sequences of primers for T. castaneum
housekeeping genes. (DOCX) Figure 6. Dose-dependent effect of 5-HT on intracellular cAMP
levels in HEK293 cells. The effect of 5-HT (1 pM - 100 mM) on the
luciferase bioluminescence in HEK293 cells expressing Trica5-HT1 due to
changes in intracellular cAMP levels. Receptor activity is shown as the
percentage of activation achieved with 10 mM of NKH-477 (set at 100%). Luciferase bioluminescence due to basal intracellular cAMP levels is set
at 0%. The data represent the mean 6 SEM of four independent
measurements (three performed in triplicate, one performed in
duplicate). d i 10 1371/j
l
0065052 006 Discussion All three are
known to be non-selective antagonists for mammalian 5-HT
receptors, including 5-HT1 receptors. At high concentrations
($1 mM), they completely inhibited activation of Trica5-HT1 by
100 nM of 5-HT. When comparing dose-response curves of 5-HT
in the presence of different concentrations of antagonist, all three
antagonists behaved as competitive antagonists. Both the IC50 and
the pA2 values indicated that prazosin was the most potent
antagonist, followed by methiothepin and methysergide. In conclusion, our data support previous findings that primary
structures and signaling properties are well conserved between
vertebrate and invertebrate receptors, yet pharmacological prop-
erties can differ significantly between both phyla, and even
between different invertebrate species. The differences in phar-
macological profiles of vertebrate and invertebrate receptors may
be due to their large evolutionary distance. Selection during
evolution most likely was based on receptor properties such as
ligand binding and G protein coupling, but not on conservation of
recognition sites for man-made, synthetic ligands. Furthermore May 2013 | Volume 8 | Issue 5 | e65052 PLOS ONE | www.plosone.org 8 Supporting Information Figure S1
Nucleotide sequence of the T. castaneum 5-
HT1
receptor
sequence
(Trica5-HT1,
accession
no. KC196076). Inverted triangles indicate differences resulting in
another amino acid between the current sequence derived from
cloned cDNA and the annotated sequence from Beetlebase. Diamonds indicate silent mutations. The arrow indicates the splice
site where a stretch of 75 residues is present in the Beetlebase
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Mechanism of sea-ice expansion in the Indian Ocean sector of Antarctica: Insights from satellite observation and model reanalysis
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RESEARCH ARTICLE a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 Babula JenaID1*, Avinash Kumar1, M. Ravichandran1, Stefan Kern2 1 ESSO - National Centre for Antarctic and Ocean Research, Ministry of Earth Science, Government of India,
Vasco-da-Gama, India, 2 Integrated Climate Data Center (ICDC), Center for Earth System Research and
Sustainability, University of Hamburg, Hamburg, Germany * bjena@ncaor.gov.in Editor: João Miguel Dias, Universidade de Aveiro,
PORTUGAL Received: May 23, 2018
Accepted: August 16, 2018
Published: October 3, 2018 Abstract a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 In the backdrop of global warming, Antarctic sea-ice variability showed an overall expansion
with the regional heterogeneity of increasing and decreasing patterns. Analysis of satellite
derived sea-ice extent, during 1979 to 2015, in the Indian Ocean sector of Antarctica (IOA)
revealed expansion of 2.4±1.2% decade-1. We find strengthening of westerly wind during
the austral summer (between 50˚S to 62˚S) facilitated northward advection of a cool and
fresh layer. Also, the strong westerly wind cools the upper ocean due to net heat loss from
the ocean surface. The combined effect of northward advection of cold fresh layer and net
heat loss from the surface, favours sea-ice expansion in the subsequent seasons, in the
IOA region, north of 62˚S. However, sea-ice retreat was observed near the Kerguelen Pla-
teau, due to upper ocean warming, and hence a non-annular pattern of sea-ice extent in the
IOA was observed. OPEN ACCESS Citation: Jena B, Kumar A, Ravichandran M, Kern
S (2018) Mechanism of sea-ice expansion in the
Indian Ocean sector of Antarctica: Insights from
satellite observation and model reanalysis. PLoS
ONE 13(10): e0203222. https://doi.org/10.1371/
journal.pone.0203222 Editor: João Miguel Dias, Universidade de Aveiro,
PORTUGAL Mechanism of sea-ice expansion in the Indian
Ocean sector of Antarctica: Insights from
satellite observation and model reanalysis Babula JenaID1*, Avinash Kumar1, M. Ravichandran1, Stefan Kern2 Materials and methods The data sets used in this study are: (i) Monthly sea-ice extent and concentration (1979–2015)
from the Scanning Multichannel Microwave Radiometer (SMMR), the Special Sensor Micro-
wave Imager (SSMI), and the Special Sensor Microwave Imager Sounder (SSMIS) in polar ste-
reographic projection at 25×25 km spatial resolution acquired from the National Snow and Ice
Data Center (NSIDC), Colorado; (ii) National Oceanic and Atmospheric Administration
(NOAA) monthly optimum interpolated sea surface temperature (OI SST) v2 (1982–2015) at a
spatial resolution of 1˚×1˚ constructed after blending of Advanced Very High Resolution Radi-
ometer (AVHRR) and in-situ observations [30]; (iii) Quality controlled monthly potential tem-
perature, salinity, and current vector (1979–2015) from the European Centre for Medium-
Range Weather Forecast (ECMWF)’s advanced operational ocean reanalysis system (ORAS4)
and objective analysis product from Met Office Hadley Centre EN4 (EN.4.2.0) at a spatial reso-
lution of 1˚×1˚ with up to ~10 m vertical resolution; (iv) ECMWF’s ERA-Interim monthly
reanalysis data (1979–2015) on wind vector (10 m), and total column ozone at a spatial resolu-
tion of 25×25 km; (v) SAM monthly index (1979–2015) from National Center for Atmospheric
Research (NCAR) [13]; (vi) Gravity Recovery and Climate Experiment (GRACE) estimated
monthly mass balance data (2002–2015) for Antarctic drainage basins from Technische Uni-
versita¨t Dresden, Germany [31]; and (vii) Locations of various Southern Ocean climatological
fronts from the Australian Antarctic Data Centre [32]. In this article, we demarcated five dif-
ferent regional sectors of the Southern Ocean: Weddell Sea (60˚W–20˚E), Indian Ocean
(20˚E–90˚E), western Pacific Ocean (90–160˚E), Ross Sea (160˚E–130˚W), and the combined
Bellingshausen and Amundsen Seas (BAS, 130˚W –60˚W), following Parkinson and Cavalieri,
(2012) [33]. The sea-ice extent and concentration are derived from passive microwave brightness tem-
perature using the National Aeronautics and Space Administration (NASA) algorithm for the
period of 1979–2015. The sea-ice extent is computed by summing-up the areas of all pixels
having at least 15% of sea-ice concentration in the IOA between 20˚E to 90˚E [5]. We com-
puted seasonal averages for sea-ice extent, sea-ice concentration, sea surface temperature
(SST), wind, net heat flux, ocean current, potential temperature, and salinity from monthly
data corresponding to four different austral seasons (Summer—January to March; Autumn—
April to June; Winter—July to September; Spring—October to December). The seasonal cycle
for each year is removed by subtracting 37-year seasonal mean values [34]. Sea-ice expansion in the Indian Ocean sector of Antarctica due to enhanced sea-ice production and subsequent brine rejection [27], influencing the global
climate through meridional redistribution of heat, and polar ecosystem [28]. Considering its
importance, regional studies on sea-ice dynamics in the Indian Ocean Sector of Antarctica
(IOA) are essential from a global weather and climate perspective [29]. In this article, we first
show the satellite derived sea-ice variability in the IOA (20˚E to 90˚E), followed by a mecha-
nism on the role of westerly wind and the ocean current for driving the theromhaline structure
to cause the sea-ice growth are examined. Further, the observed growth in sea-ice that masks
the significant regional variations (sea-ice retreat) in some places near to the coast and south
Kerguelen Plateau, are investigated. PLOS ONE | https://doi.org/10.1371/journal.pone.0203222
October 3, 2018 Introduction The Southern Ocean plays a crucial role in the Earth’s climate system and its sea-ice coverage
is a key indicator of climate change that modulates the albedo, air-sea exchange of heat, fresh
water, carbon, ocean-atmospheric circulation, Antarctic ecosystems, and biogeochemical cycle
[1,2]. The influence of global warming on the Arctic sea-ice shows a profound declining trend
as expected, in contrast to the Antarctic sea-ice expansion. Several authors reported a signifi-
cant increase in Antarctic sea-ice extents [3–6] which has been linked to westerly wind forcing
[7–11]; southern annular mode (SAM) [7,12–14]; El Niño–Southern Oscillation (ENSO) [15];
stratospheric ozone depletion [16–21]; precipitation [22]; and ocean temperature [23,24];
however, the overall sea-ice expansion masks large regional variations [25]. At present there is
no consensus on the underlying mechanism to explain the observed sea-ice expansion. Regional scale processes and unidentified large-scale processes should be investigated to
explain the observed sea-ice expansion [26]. The interpretations of regional sea-ice variability
around Antarctica have been focussed on the most part of the Atlantic and the Pacific Ocean. Importance of the Indian Ocean sector of Antarctica is known for the bottom water formation Copyright: © 2018 Jena et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files. Funding: The author(s) received no specific
funding for this work. Competing interests: The authors have declared
that no competing interests exist. 1 / 16 PLOS ONE | https://doi.org/10.1371/journal.pone.0203222
October 3, 2018 Sea-ice expansion in the Indian Ocean sector of Antarctica EN4 [37]. In order to experiment whether the sea surface cooling has occurred as an effect of
heat transfer from the ocean, the net heat flux trend was computed during 1979–2011 from
ECMWF’s ORAS3 [38]. Ocean current trends from ORAS4 were calculated separately for the
zonal and meridional components and plotted the vector trends. Only the statistically signifi-
cant trend values were considered with p-values less than 0.05 (at 95% confidence level)
according to a two-tailed t-test. The water masses such as Antarctic surface water (AASW),
Antarctic intermediate water (AAIW), and circumpolar deep water (CDW) are characterised
after following the standard methodology [39,40]. In order to verify whether the observed increase in sea-ice extent and concentration are
compensated by the loss of sea-ice volume, we compute the sea-ice freeboard flux, i.e., sea-ice
freeboard ‘F’ times sea-ice area ‘A’ times the relevant component of the sea-ice motion vector
‘v’ for the period 2002 through 2017 for the gates denoted in S1 Fig. We use the sea-ice free-
board flux instead of the sea-ice (thickness) volume flux because the sea-ice thickness obtained
from the sea-ice freeboard is less good evaluated than the sea-ice freeboard itself. In addition,
it contains more uncertainties due to assumptions required for the sea-ice freeboard to thick-
ness conversion. In particular, the usage of a snow-depth climatology challenges the interpre-
tation of these sea-ice thickness values when using a time series as is done in this article. Sea-
ice freeboard ‘F’ as computed from Envisat (until March 2012) and CryoSat-2 (since Novem-
ber 2010) satellite altimetry [41,42] within the ESA-CCI sea-ice ecv project (see http://esa-cci. nersc.no) is available, e.g. from the Copernicus data portal for Envisat: http://catalogue.ceda. ac.uk/uuid/b1f1ac03077b4aa784c5a413a2210bf5 and CS2: http://catalogue.ceda.ac.uk/uuid/ In order to verify whether the observed increase in sea-ice extent and concentration are
compensated by the loss of sea-ice volume, we compute the sea-ice freeboard flux, i.e., sea-ice
freeboard ‘F’ times sea-ice area ‘A’ times the relevant component of the sea-ice motion vector
‘v’ for the period 2002 through 2017 for the gates denoted in S1 Fig. We use the sea-ice free-
board flux instead of the sea-ice (thickness) volume flux because the sea-ice thickness obtained
from the sea-ice freeboard is less good evaluated than the sea-ice freeboard itself. In addition,
it contains more uncertainties due to assumptions required for the sea-ice freeboard to thick-
ness conversion. SICCI-50km SIC data are used throughout the
entire AMSR-E–AMSR2 period; OSI-450 SIC data are used to fill the gap with AMSR-E /
AMSR2 observations from October 2011 through July 2012. OSI-450 SIC data, which have 25
km grid resolution, were re-sampled to the 50 km grid resolution of the SICCI-50km data set. From these daily SIC data we computed monthly averages. For sea-ice motion we use the
NSIDC sea-ice motion data set v03 [44] (http://nsidc.org/forms/nsidc-0116_or.html) with
monthly temporal resolution. For meridional fluxes at gates 1 and 2 we only use the v-compo-
nent of the ice motion; for zonal fluxes at gates 3 to 6 we only use the u-component of the ice
motion. We co-locate the sea-ice motion vectors, which are on the NSIDC polar-stereographic
grid with tangential plane at 70˚S, onto the EASE2.0 grid used for the SIC and sea-ice thickness
data. We only use cases with SIC > 70% in accordance with the settings of the sea-ice free-
board retrieval [41]. We used the uncertainty information provided with the SIC and sea-ice
thickness data sets to compute an uncertainty of our sea-ice freeboard fluxes. In S2–S4 Figs, we
show time series of the total sea-ice freeboard flux at the gates shown in S1 Fig together with
the uncertainty (error bars). Envisat and CS-2 based estimates are shown with different sym-
bols. We did not carry our any inter-sensor bias correction but we point out that the sea-ice
freeboard retrieval was optimised to mitigate the inter-sensor bias [41]. In particular, the usage of a snow-depth climatology challenges the interpre-
tation of these sea-ice thickness values when using a time series as is done in this article. Sea-
ice freeboard ‘F’ as computed from Envisat (until March 2012) and CryoSat-2 (since Novem-
ber 2010) satellite altimetry [41,42] within the ESA-CCI sea-ice ecv project (see http://esa-cci. nersc.no) is available, e.g. from the Copernicus data portal for Envisat: http://catalogue.ceda. ac.uk/uuid/b1f1ac03077b4aa784c5a413a2210bf5 and CS2: http://catalogue.ceda.ac.uk/uuid/
48fc3d1e8ada405c8486ada522dae9e8. The sea-ice area ‘A’ derived from the combined Eumet-
sat OSI-SAF / ESA-SICCI-2 AMSR-E / AMSR2 sea-ice concentration (SIC) data set (OSI-450:
http://dx.doi.org/10.15770/EUM_SAF_OSI_0008, SICCI-50km: http://dx.doi.org/10.5285/
5f75fcb0c58740d99b07953797bc041e) [43]. SICCI-50km SIC data are used throughout the
entire AMSR-E–AMSR2 period; OSI-450 SIC data are used to fill the gap with AMSR-E /
AMSR2 observations from October 2011 through July 2012. OSI-450 SIC data, which have 25
km grid resolution, were re-sampled to the 50 km grid resolution of the SICCI-50km data set. From these daily SIC data we computed monthly averages. For sea-ice motion we use the
NSIDC sea-ice motion data set v03 [44] (http://nsidc.org/forms/nsidc-0116_or.html) with
monthly temporal resolution. For meridional fluxes at gates 1 and 2 we only use the v-compo-
nent of the ice motion; for zonal fluxes at gates 3 to 6 we only use the u-component of the ice
motion. We co-locate the sea-ice motion vectors, which are on the NSIDC polar-stereographic
grid with tangential plane at 70˚S, onto the EASE2.0 grid used for the SIC and sea-ice thickness
data. We only use cases with SIC > 70% in accordance with the settings of the sea-ice free-
board retrieval [41]. We used the uncertainty information provided with the SIC and sea-ice
thickness data sets to compute an uncertainty of our sea-ice freeboard fluxes. In S2–S4 Figs, we
show time series of the total sea-ice freeboard flux at the gates shown in S1 Fig together with
the uncertainty (error bars). Envisat and CS-2 based estimates are shown with different sym-
bols. We did not carry our any inter-sensor bias correction but we point out that the sea-ice
freeboard retrieval was optimised to mitigate the inter-sensor bias [41]. ac.uk/uuid/b1f1ac03077b4aa784c5a413a2210bf5 and CS2: http://catalogue.ceda.ac.uk/uuid/
48fc3d1e8ada405c8486ada522dae9e8. The sea-ice area ‘A’ derived from the combined Eumet-
sat OSI-SAF / ESA-SICCI-2 AMSR-E / AMSR2 sea-ice concentration (SIC) data set (OSI-450:
http://dx.doi.org/10.15770/EUM_SAF_OSI_0008, SICCI-50km: http://dx.doi.org/10.5285/
5f75fcb0c58740d99b07953797bc041e) [43]. Materials and methods The time series of
data are fitted with a linear function to model the trend by using a least square method. Simi-
larly, the zonal average (20˚E to 90˚E) trends of potential temperature, salinity, and ocean cur-
rent are computed for different seasons. ORAS4 is an ocean reanalysis system where in both
in-situ and satellite data are being assimilated. EN4 is quality controlled subsurface ocean tem-
perature and salinity profiles and objectively analysed data. The detailed methodology of prod-
uct generation, and quality control approaches were given elsewhere for ORAS4 [35,36] and PLOS ONE | https://doi.org/10.1371/journal.pone.0203222
October 3, 2018 2 / 16 Sea-ice expansion in the Indian Ocean sector of Antarctica Table 1. Annual and seasonal trends (with standard errors) of sea-ice extent computed for the Antarctica and individual sectors over the period 1979–2015. Region
Annual
Summer (JFM)
Autumn (AMJ)
Winter (JAS)
Spring (OND)
103 km2 yr–1
% decade–1
103 km2 yr–1
% decade–1
103 km2 yr–1
% decade–1
103 km2 yr–1
% decade–1
103 km2 yr–1
% decade–1
SH
24.9±4.4
2.1±0.4
23.6±7.5
5.5±1.7
29.5±7.4
2.7±0.7
19.6±4.3
1.1±0.2
26.8±7.5
1.7±0.5
WS
5.2±1.8
2.6±0.9
20.4±5.4
5.4±3.2
-12.4±9.5
-3.3±2.5
-2.5±4.9
-0.4±0.8
1.9±5.3
0.3±0.9
IO
3.0±1.5
2.4±1.2
3.7±1.4
10.6±4.1
5.8±2.3
4.0±1.6
6.3±3.1
1.9±0.9
5.0±3.4
1.7±1.2
WPO
2.3±1.7
1.9±1.3
3.8±1.8
7.6±3.6
4.8±1.7
4.1±1.5
0.1±2.6
0.0±1.4
2.2±2.2
1.5±1.5
RS
11.1±2.8
3.8±1.0
7.2±4.2
6.3±3.6
13.3±4.0
4.5±1.3
10.2±3.5
2.5±0.9
13.7±4.0
3.8±1.1
BAS
-3.8±-2.0
-2.5±1.4
-11.6±2.0
-17.8±3.1
-6.8±2.8
-5.2±2.1
4.3±3.4
1.9±1.5
-0.9±3.7
-0.5±2.1
Southern Hemisphere (SH), Weddell Sea (WS), Indian Ocean (IO), Western Pacific Ocean (WPO), Ross Sea (RS), Bellingshausen and Amundsen Seas (BAS) tandard errors) of sea-ice extent computed for the Antarctica and individual sectors over the period 1979–2015. Table 1. Annual and seasonal trends (with standard errors) of sea-ice extent computed for the Antarctica a rate of 2.4%±1.2 per decade. The trends in IOA sea-ice extent have a substantial seasonal vari-
ability which peaks in summer (10.6%±4.1 decade-1), followed by autumn (4.0%±1.6 decade-
1), winter (1.9%±0.9 decade-1) and spring (1.7%±1.2 decade-1). We note that the absolute trend
values for IOA are relatively small in relation to the size of one grid-cell: ~25 km x ~25
km = 625 km2; hence, the annual trend of 3.0x103 km2 corresponds to 4–5 grid cells. Overall,
we find an increasing trend of sea-ice concentration and extent in the IOA, which is associated
with sea surface cooling (Figs 1 and 2). Analysis of sea-ice freeboard flux data during 2002–2017 suggest a slight increase in meridi-
onal northward flux from, at gate 1, around zero before 2010 to ~0.5 km3/day afterwards, and
at gate 2 from ~0.1 km3/day to ~0.4 km3/day (S2 Fig). At the two southernmost gates (3 and 5)
we find a general import of sea ice from the East at gate 5 and a general export of sea ice
towards the west at gate 3 –in accordance with the circum-Antarctic easterly winds (S3 and S4
Figs). We cannot find a change in this import and export over the time period considered. Observed sea-ice expansion In accordance with recent studies [45,46] we find an overall expansion of Antarctic sea-ice
extent over the 37-year period (1979–2015) by 24.9×103 ± 4.4×103 km2 yr-1 (or 2.1% decade-1,
p = 2.14×10−6) with regional heterogeneity that comprises of increasing and decreasing pattern
in different sectors (Table 1). For the IOA we find a significant expansion of sea-ice extent at a 3 / 16 PLOS ONE | https://doi.org/10.1371/journal.pone.0203222
October 3, 2018 PLOS ONE | https://doi.org/10.1371/journal.pone.0203222
October 3, 2018 https://doi.org/10.1371/journal.pone.0203222.t001 The
two northernmost gates (4 and 6) do not reveal any trends either (S3 and S4 Figs). At gate 6
flux values are too sparse and suggest a sea-ice important. At gate 4, import and export seem to
be in balance–except in the year 2010 where sea ice was imported into the IOA from the Wed-
dell Sea (positive = eastward sea-ice freeboard flux). What causes the marked increase in regional sea-ice with the background of global warm-
ing? Antarctic sea-ice expansion has been attributed to the positive trends in the SAM, associ-
ated with the poleward shifting of westerly wind [16] that reduces poleward heat transport and
induces cooling of the Antarctic atmosphere [12,14]. However, the magnitude of the Antarctic
sea-ice changes related to the SAM and El Niño–Southern Oscillation (ENSO) were found to
be smaller than the regional sea-ice trends [7]. Our result shows SAM index (1979–2015) has a
significant trend towards high-index polarity during the austral summer and autumn that
explains about 14% to 33% variance of the IOA sea-ice extent (S5 Fig and S1 Table). The
observed high-index polarity in SAM is influenced largely by the stratospheric ozone depletion
over the Antarctica [17,19]; possibly leads to Antarctic sea-ice increase [18,20]. An experiment
using atmospheric climate model revealed that the deepening of the Amundsen–Bellingshau-
sen Sea low in response to the stratospheric ozone depletion is accountable to the regional sea-
ice variability [18]. It is explained that the development of strong cyclonic circulation induced
by stratospheric ozone depletion intensifies windspeeds in austral autumn, deepens the
Amundsen Sea Low, which plays a key role in regional sea-ice expansion. In contrast, studies
using coupled climate models showed that ozone depletion over the Antarctica supposed to
result the Southern Ocean warming, and sea-ice loss, did not contribute significantly to the
observed sea-ice expansion [21,28,47–49]. We find the ozone depletion explains only about 4 / 16 PLOS ONE | https://doi.org/10.1371/journal.pone.0203222
October 3, 2018 Sea-ice expansion in the Indian Ocean sector of Antarctica Fig 1. Satellite derived sea-ice concentration trend contours (% decade-1) overlaid on the optimum interpolated sea surface temperature trend (˚C decade-1) at p<0.05,
for (a) summer, (b) autumn, (c) winter, and (d) spring. Blue and red colours indicate sea surface cooling and warming trend respectively. Overall, the increasing pattern
of sea-ice concentration is evident (associated with cooling) in the Indian Ocean sector of Antarctica (IOA) except the reduction in sea-ice near the south Kerguelen
Plateau (SKP), and coastal regions of the Prydz Bay (PB), Mawson (MS), Prince Harald (PH). Fig 1. Satellite derived sea-ice concentration trend contours (% decade-1) overlaid on the optimum interpolated sea surface temperature trend (˚C decade-1) at p<0.05,
for (a) summer, (b) autumn, (c) winter, and (d) spring. Blue and red colours indicate sea surface cooling and warming trend respectively. Overall, the increasing pattern
of sea-ice concentration is evident (associated with cooling) in the Indian Ocean sector of Antarctica (IOA) except the reduction in sea-ice near the south Kerguelen
Plateau (SKP), and coastal regions of the Prydz Bay (PB), Mawson (MS), Prince Harald (PH). https://doi.org/10.1371/journal.pone.0203222.g001 https://doi.org/10.1371/journal.pone.0203222.g001 Possible mechanism of sea-ice expansion The basal and upper portions of the Antarctica ice shelves are vulnerable to changes in ocean
and atmospheric forcing, respectively. Indeed, the melting rates are faster owing to the subsur-
face warming of the Southern Ocean [23,25,53–57]. The melt water accumulates as a cool and
fresh layer, thereby enhances the vertical stability and promoting sea-ice expansion [23]. In the
Eastern Antarctica, the ice-sheets are gaining mass due to the enhanced precipitation [58] (S6
Fig) and eventually the ice shelves melt either locally through basal melting or remotely by
drifting of icebergs [56]. In either case of mass gain or loss, the drainage networks are expected
to remain active and discharges more fresh water from the ice shelves into the ocean during
each austral summer, in addition to contribution of freshwater input from sea-ice melting. The persistent drainage network, interconnected streams, ponds and rivers of the Antarctica
export large amount of the ice shelves melt water (fresh water) into the ocean through water-
falls and dolines [59]. The melt water spreads and stratifies as low saline, cold water at the
upper ocean due to its lower density because the salinity dominates the density structure in the
Southern Ocean [60]. Our analysis reveals significant strengthening of westerly wind (up to 0.33±0.07 m s-1
decade-1) and thereby increasing the surface current (up to 0.35±0.08 cm s-1 decade-1) in the
austral summer (between 50˚S to 62˚S) suggesting northward transport of these comparably
fresh, cold water from the southern boundary of the Antarctic circumpolar current (Figs 3 and
4A–4D). The strengthening of westerly wind is driven by the tropospheric pressure variability,
which has oscillated southward (south of 60˚S) and northward between 30˚S to 50˚S [13,20]. The strengthening of westerly wind facilitates an anomalous sea surface cooling and freshening
north of 62˚S, due to northward movement of cold fresh water and net heat loss, during the
austral summer that persists in the successive seasons and favours for sea-ice expansion in the
IOA (Fig 4). A slight increase in northward flux of sea-ice freeboard is also evident in S2 Fig. This finding is supported by the ORAS4 and EN4, showing widespread freshening rate up to
-0.02 ±0.004 psu decade-1 in the surface and intermediate waters north of 62˚S (Fig 4E–4H, S7
Fig), which is consistent with the earlier observations on freshening of comparable magnitude
[61–63]. Sea-ice expansion in the Indian Ocean sector of Antarctica carry out due to the scarcity of in-situ data [18]. Regional studies over the Antarctica have
shown accelerated freshening due to the increased precipitation and melting of the west Ant-
arctic ice-sheet that enhances the thermohaline stratification and weakens the convective over-
turning, in turn reduces the ocean heat flux to, on the one hand, melt sea-ice and other hand
promote more sea-ice formation [50,51]. The oceanic heat flux available for sea-ice melting
decreases faster than the sea-ice growth in the weakly stratified Southern Ocean that favours
sea-ice expansion [4]. Another mechanism suggested that increased freshwater input by and
mass loss of the Antarctic ice sheet are caused by subsurface ocean warming which can drive
enhanced basal melting of ice-shelves [23,52]. However, the gravimetric mass balance analysis
of the ice-sheets adjacent to the IOA revealed a gain of about 6.83±0.5 Gt of mass annually (S6
Fig). With this background, the following possible mechanism is proposed to explain the
observed sea-ice expansion in the IOA. PLOS ONE | https://doi.org/10.1371/journal.pone.0203222
October 3, 2018 17% of variance in the IOA sea-ice extent during the austral summer and no significant corre-
lation found in other seasons (S1 Table). 17% of variance in the IOA sea-ice extent during the austral summer and no significant corre-
lation found in other seasons (S1 Table). 17% of variance in the IOA sea-ice extent during the austral summer and no significant corre-
lation found in other seasons (S1 Table). Although numerous studies were carried out to understand the atmospheric forcing on
recent Antarctic sea-ice trend, the role of oceanic influence on sea-ice has become difficult to Although numerous studies were carried out to understand the atmospheric forcing on
recent Antarctic sea-ice trend, the role of oceanic influence on sea-ice has become difficult to Fig 2. Anomalies of (a) sea-ice extent, (b) sea-ice concentration, (c) sea surface temperature, and (d) net heat flux, during the austral
autumn (1979–2015) over the IOA (averaged from 20˚E to 90˚E and 73˚S to 55˚S). Right panel figures (Figs e to h) are same as that of
left panel, but for the austral winter. The pink lines are the running mean of 10-year data. https://doi.org/10.1371/journal.pone.0203222.g002 Fig 2. Anomalies of (a) sea-ice extent, (b) sea-ice concentration, (c) sea surface temperature, and (d) net heat flux, during the austral
autumn (1979–2015) over the IOA (averaged from 20˚E to 90˚E and 73˚S to 55˚S). Right panel figures (Figs e to h) are same as that of
left panel, but for the austral winter. The pink lines are the running mean of 10-year data. https://doi.org/10.1371/journal.pone.0203222.g002 Fig 2. Anomalies of (a) sea-ice extent, (b) sea-ice concentration, (c) sea surface temperature, and (d) net heat flux, during the austral
autumn (1979–2015) over the IOA (averaged from 20˚E to 90˚E and 73˚S to 55˚S). Right panel figures (Figs e to h) are same as that of
left panel, but for the austral winter. The pink lines are the running mean of 10-year data. PLOS ONE | https://doi.org/10.1371/journal.pone.0203222
October 3, 2018 5 / 16 Possible mechanism of sea-ice expansion The joint impact of these processes produces anomalous
upper ocean cooling and freshening north of 62˚S, persists in the successive seasons and possi-
bly explains the observed sea-ice expansion (autumn and winter) while the sea-ice forms and
grows (Fig 6). However, salinification is observed near the coast (south of ~62˚S) due to
enhanced rejection of salt associated with high rate of sea-ice production and its export
[23,63,65]. This salinification across the coast in coastal polynyas by sea-ice growth has inter-
esting implications—partly for the sea-ice cover itself, via ice production and export of sea-ice
out of, e.g. the Cape Darnley polynya and Prydz Bay polynyas as well as smaller polynyas fur-
ther west, but in particular also in terms of water mass modification and anticipated dense
water mass production [68]. except few contrasting features at some places near to the coast and south Kerguelen Plateau
(Fig 1). The cooling trend prevails in the upper layer, whereas the subsurface warming is
observed below ~100 m depth (Fig 4A–4D). The subsurface warming is possibly due to the
advection of circumpolar deep water (CDW) which delivers warm water onto the ice shelves
through submarine troughs and leads to basal melting [24,57,66,67]. The subsurface warming
and its effect on ice shelves basal melting in turn helps accumulation of low saline and cold
water at the sea surface [23]. The observed strengthening of the westerly wind in austral sum-
mer induces the northward transport of these low saline, cool water that freezes readily into a
crystalline structure of sea-ice compared to the displaced water of relatively high saline and
warm waters. Concurrently, the westerly wind in austral summer generates the robust feature
of upper ocean cooling in the successive seasons through an overall loss of net heat flux from
the sea surface (Figs 2 and 5, S8 Fig). The joint impact of these processes produces anomalous
upper ocean cooling and freshening north of 62˚S, persists in the successive seasons and possi-
bly explains the observed sea-ice expansion (autumn and winter) while the sea-ice forms and
grows (Fig 6). However, salinification is observed near the coast (south of ~62˚S) due to
enhanced rejection of salt associated with high rate of sea-ice production and its export
[23,63,65]. This salinification across the coast in coastal polynyas by sea-ice growth has inter-
esting implications—partly for the sea-ice cover itself, via ice production and export of sea-ice
out of, e.g. Possible mechanism of sea-ice expansion The observed freshening has been reported in different sector of the Southern Ocean,
attributed to the accelerated freshwater fluxes from the Antarctic glacial melt [50,64], atmo-
spheric fluxes by excess precipitation over evaporation [62], and the northward transport of
sea-ice [9,63,65]. The accelerated freshening of sea surface can lower the SST by weakening
convection and vertical mixing through enhanced thermohaline stratification in the water col-
umn, which favours the sea-ice expansion [4]. The widespread sea surface cooling up to -0.2
±0.01˚C decade-1 observed in all seasons in synchronise with sea-ice expansion in the IOA, PLOS ONE | https://doi.org/10.1371/journal.pone.0203222
October 3, 2018 6 / 16 Sea-ice expansion in the Indian Ocean sector of Antarctica Fig 3. Spatial distribution of ERA-Interim 10m-wind speed trend (m s-1 decade-1) at p<0.05, with the vectors showing
the seasonal climatology for (a) summer, (b) autumn, (c) winter, and (d) spring, over the period of 1979–2015. https://doi.org/10.1371/journal.pone.0203222.g003 Fig 3. Spatial distribution of ERA-Interim 10m-wind speed trend (m s-1 decade-1) at p<0.05, with the vectors showing
the seasonal climatology for (a) summer, (b) autumn, (c) winter, and (d) spring, over the period of 1979–2015. https://doi.org/10.1371/journal.pone.0203222.g003 https://doi.org/10.1371/journal.pone.0203222.g003 except few contrasting features at some places near to the coast and south Kerguelen Plateau
(Fig 1). The cooling trend prevails in the upper layer, whereas the subsurface warming is
observed below ~100 m depth (Fig 4A–4D). The subsurface warming is possibly due to the
advection of circumpolar deep water (CDW) which delivers warm water onto the ice shelves
through submarine troughs and leads to basal melting [24,57,66,67]. The subsurface warming
and its effect on ice shelves basal melting in turn helps accumulation of low saline and cold
water at the sea surface [23]. The observed strengthening of the westerly wind in austral sum-
mer induces the northward transport of these low saline, cool water that freezes readily into a
crystalline structure of sea-ice compared to the displaced water of relatively high saline and
warm waters. Concurrently, the westerly wind in austral summer generates the robust feature
of upper ocean cooling in the successive seasons through an overall loss of net heat flux from
the sea surface (Figs 2 and 5, S8 Fig). PLOS ONE | https://doi.org/10.1371/journal.pone.0203222
October 3, 2018 Possible mechanism of sea-ice expansion the Cape Darnley polynya and Prydz Bay polynyas as well as smaller polynyas fur-
ther west, but in particular also in terms of water mass modification and anticipated dense
water mass production [68]. The dominance of increasing sea-ice concentration and decreasing SST observed near the
marginal ice zone, where sea-ice growth is evident (Fig 1 and S9 Fig). Interestingly, an excep-
tion is observed on the reduction of sea-ice concentration near the south Kerguelen Plateau
and coastal regions of the Prydz Bay, Mawson and Prince Harald, triggered by enhanced
warming throughout the water column both at the surface and subsurface (Fig 1 and S10 Fig). In particular, the waters above the southern Kerguelen Plateau (55˚S to 60˚S and 80˚E to 90˚E)
exhibit a consistent warming trend up to 0.26±0.01˚C decade-1 with a concurrent reduction in
sea-ice concentration up to 2% decade-1. Retreat in sea-ice extent is recorded on the south Ker-
guelen Plateau, showing a non-annular pattern of sea-ice variability in the IOA (Fig 6). 7 / 16 PLOS ONE | https://doi.org/10.1371/journal.pone.0203222
October 3, 2018 Fig 4. Depth-Latitude cross section of zonally averaged (20˚E to 90˚E) trends of potential temperature for (a) summer, (b) autumn, (c) winter, and (d) spring. The
trends of ocean current vectors are overlaid as magenta arrows (in figures a-d). Right panel figures indicate the trends of salinity for (e) summer, (f) autumn, (g) winter,
and (h) spring. Only the significant trend values (p < 0.05) are considered to plot the figures according to a two-tailed t-test and the dots are marked to show the
observations with the significant trends. The contour shows the climatology of temperature (left panel) and salinity (right panel). The data are obtained from the
European Centre for Medium-Range Weather Forecast (ECMWF)’s Ocean Reanalysis System 4 (ORAS4) for the period of 1979–2015. https://doi.org/10.1371/journal.pone.0203222.g004
Sea-ice expansion in the Indian Ocean sector of Antarctica Sea-ice expansion in the Indian Ocean sector of Antarctica Fig 4. Depth-Latitude cross section of zonally averaged (20˚E to 90˚E) trends of potential temperature for (a) summer, (b) autumn, (c) winter, and (d) spring. The
trends of ocean current vectors are overlaid as magenta arrows (in figures a-d). Right panel figures indicate the trends of salinity for (e) summer, (f) autumn, (g) winter,
and (h) spring. Possible mechanism of sea-ice expansion Only the significant trend values (p < 0.05) are considered to plot the figures according to a two-tailed t-test and the dots are marked to show the
observations with the significant trends. The contour shows the climatology of temperature (left panel) and salinity (right panel). The data are obtained from the
European Centre for Medium-Range Weather Forecast (ECMWF)’s Ocean Reanalysis System 4 (ORAS4) for the period of 1979–2015. Fig 4. Depth-Latitude cross section of zonally averaged (20˚E to 90˚E) trends of potential temperature for (a) summer, (b) autumn, (c) winter, and (d) spring. The
trends of ocean current vectors are overlaid as magenta arrows (in figures a-d). Right panel figures indicate the trends of salinity for (e) summer, (f) autumn, (g) winter,
and (h) spring. Only the significant trend values (p < 0.05) are considered to plot the figures according to a two-tailed t-test and the dots are marked to show the
observations with the significant trends. The contour shows the climatology of temperature (left panel) and salinity (right panel). The data are obtained from the
European Centre for Medium-Range Weather Forecast (ECMWF)’s Ocean Reanalysis System 4 (ORAS4) for the period of 1979–2015. https://doi.org/10.1371/journal.pone.0203222.g004 PLOS ONE | https://doi.org/10.1371/journal.pone.0203222
October 3, 2018 8 / 16 Sea-ice expansion in the Indian Ocean sector of Antarctica Fig 5. Spatial distribution of net heat flux trend (W m-2 decade-1) at p<0.05, over the period of 1979–2011, for (a) summer, (b) autumn, (c) winter, and (d) spring. Positive values indicate heat gain by the sea surface and negative values indicate heat loss from the sea surface. Significant heat loss trend observed in larger portion of
the Indian Ocean sector of Antarctica (IOA) particularly in austral autumn, winter and spring. Fig 5. Spatial distribution of net heat flux trend (W m-2 decade-1) at p<0.05, over the period of 1979–2011, for (a) summer, (b) autumn, (c) winter, and (d) spring. Positive values indicate heat gain by the sea surface and negative values indicate heat loss from the sea surface. Significant heat loss trend observed in larger portion of
the Indian Ocean sector of Antarctica (IOA) particularly in austral autumn, winter and spring. https://doi.org/10.1371/journal.pone.0203222.g005 https://doi.org/10.1371/journal.pone.0203222.g005 PLOS ONE | https://doi.org/10.1371/journal.pone.0203222
October 3, 2018 Conclusions The background colour shows the
mean state of sea surface temperature along with the cross section of zonally averaged (20˚E to 90˚E) temperature and salinity (contours) up to 500m depth,
utilizing Ocean Reanalysis System 4 (ORAS4) for the period of 1979–2015. Dashed rectangle (magenta) shows the zone of westerly wind strengthening in
austral summer suggests northward advection (blue-red blended arrow) of low saline and cool water from the southern boundary of the Antarctic circumpolar
current (sbACC), accompanied with the upper ocean cooling through an overall loss of net heat flux (black spiral arrow), thus favours overall sea-ice expansion. Dashed circle (red) shows the region of consistent warming trend in the entire water column on the south Kerguelen Plateau, lead to the localized sea-ice
retreat. Solid lines (colour) show the sea-ice extent in September with an increasing pattern over the larger portion of IOA, except on the south Kerguelen
Plateau. A notable episodic event on sea-ice retreat during 2017 is linked to the enhanced poleward advection of warm and moist air that led to strong melting
[69]. Locations of climatological Southern Ocean fronts are represented as black dashed lines [32]. White arrows show salinification near the coast (south of
~62˚S) due to enhanced rejection of salt associated with high rate of sea-ice production and its export [23,65]. https://doi.org/10.1371/journal.pone.0203222.g006
Sea-ice expansion in the Indian Ocean sector of Antarctica Fig 6. Schematic diagram illustrates the mechanism of sea-ice expansion in the Indian Ocean sector of Antarctica (IOA). The background colour shows the
mean state of sea surface temperature along with the cross section of zonally averaged (20˚E to 90˚E) temperature and salinity (contours) up to 500m depth,
utilizing Ocean Reanalysis System 4 (ORAS4) for the period of 1979–2015. Dashed rectangle (magenta) shows the zone of westerly wind strengthening in
austral summer suggests northward advection (blue-red blended arrow) of low saline and cool water from the southern boundary of the Antarctic circumpolar
current (sbACC), accompanied with the upper ocean cooling through an overall loss of net heat flux (black spiral arrow), thus favours overall sea-ice expansion. Dashed circle (red) shows the region of consistent warming trend in the entire water column on the south Kerguelen Plateau, lead to the localized sea-ice
retreat. Solid lines (colour) show the sea-ice extent in September with an increasing pattern over the larger portion of IOA, except on the south Kerguelen
Plateau. Conclusions This study reveals significant sea-ice expansion in the IOA associated with the robust feature
of sea surface cooling trend. SAM and stratospheric ozone depletion over the Antarctica par-
tially explained the variability in the IOA sea-ice extent. We suggest a mechanism which shows
significant strengthening of the westerly wind in austral summer that induces northward
transport of the low saline and cold water, accompanied with the upper ocean cooling through
an overall loss of net heat flux, conducive for sea-ice expansion. Apparently, an increasing pat-
tern of sea-ice concentration and a decrease in SST, are maximum near the marginal ice zone. Global ocean reanalysis data support our conclusion, showing widespread freshening at the
surface and intermediate waters. The upper ocean (~100 m) cooled significantly, except the
warming and corresponding sea-ice reduction near the south Kerguelen Plateau, and coastal
regions of the Prydz Bay, Mawson, Prince Harald. The remainder water column below ~100 m
warmed significantly, which has been detected earlier and linked to the advection of CDW
that delivers warm water onto the ice shelves [24,57,66]. In the coming decades, a subsurface
warming of the Southern Ocean may be favourable for sea-ice expansion through ice shelves
basal melting through accumulation of the low saline and cool water at the sea surface [23]. Despite the fact of Antarctic sea-ice expansion during 1979–2015, the record rate of springtime
sea-ice reduction in 2016–2017 is a further challenge to our understanding on the Antarctic
sea ice variability in a moderately short satellite observational record [69] (Fig 6). This episodic
sea-ice reduction has been ascribed to the persistence of the negative index polarity of SAM in
springtime led to westerly wind weakening and an overall surface warming of the coastal Ant-
arctica. The overall warming is linked to the enhanced poleward advection of warm and moist
air led to strong sea-ice melting. Regional atmosphere-ocean-sea-ice modelling is imperative
to characterise the sea-ice dynamics and quantify the individual contribution of various PLOS ONE | https://doi.org/10.1371/journal.pone.0203222
October 3, 2018 9 / 16 Sea-ice expansion in the Indian Ocean sector of Antarctica Fig 6. Schematic diagram illustrates the mechanism of sea-ice expansion in the Indian Ocean sector of Antarctica (IOA). https://doi.org/10.1371/journal.pone.0203222.g006 Conclusions The European Centre for Medium range Weather Forecast (ECMWF)’s EN4 data
shows the cross section of the zonally averaged (20˚E to 90˚E) trends of potential temperature
for (a) summer, (b) autumn, (c) winter, and (d) spring, over the period of 1979–2015. Right
panel figures indicates the trends of salinity for (e) summer, (f) autumn, (g) winter, and (h)
spring. Only the significant trend values (p < 0.05) are considered to plot the figures according
to a two tailed t-test and the dots are marked to show the observations with the significant
trends. The contours indicate the climatology of temperature and salinity overlaid on the color
shaded trend maps. (TIF)
S8 Fig. Sea surface temperature (SST) anomaly computed from multiple data showed robust
feature of sea surface cooling over the Indian Ocean sector of Antarctica (averaged from 20˚E
to 90˚E and 73˚S to 55˚S), for (a) summer, (b) autumn, (c) winter, and (d) spring. The opti-
mum interpolated SST (OI SST), Interim SST, Ocean Reanalysis System 4 (ORAS4) SST and physical processes. This study describes one of the realistic possible mechanisms that explain
the overall increasing trend of satellite derived sea-ice in the IOA. S3 Fig. Monthly total sea-ice freeboard flux across gates 3 and 4, i.e. the western entry to
our region of interest (see S1 Fig).
(TIF) S4 Fig. Monthly total sea-ice freeboard flux across gates 5 and 6, i.e. the eastern entry to
our region of interest (see S1 Fig). (TIF) S5 Fig. Time series of seasonal mean SAM index (Marshall., 2003) over the period of 1979–
2015. The dashed lines are the running mean of 10-year data for different seasons; indicates
significant trend towards high-index polarity during the austral summer (dashed red line) and
autumn (dashed black line). (TIF) S6 Fig. Annual mass variability of the Antarctic ice-sheets adjacent to the Indian Ocean
sector of Antarctica, showed a gain of about 6.83±0.5 Gt yr-1. The data are obtained from
the Technische Universita¨t Dresden, Germany, derived from the Gravity Recovery and Cli-
mate Experiment (GRACE) satellite. (TIF) S6 Fig. Annual mass variability of the Antarctic ice-sheets adjacent to the Indian Ocean
sector of Antarctica, showed a gain of about 6.83±0.5 Gt yr-1. The data are obtained from
the Technische Universita¨t Dresden, Germany, derived from the Gravity Recovery and Cli-
mate Experiment (GRACE) satellite. (TIF) S7 Fig. The European Centre for Medium range Weather Forecast (ECMWF)’s EN4 data
shows the cross section of the zonally averaged (20˚E to 90˚E) trends of potential temperature
for (a) summer, (b) autumn, (c) winter, and (d) spring, over the period of 1979–2015. Right
panel figures indicates the trends of salinity for (e) summer, (f) autumn, (g) winter, and (h)
spring. Only the significant trend values (p < 0.05) are considered to plot the figures according
to a two tailed t-test and the dots are marked to show the observations with the significant
trends. The contours indicate the climatology of temperature and salinity overlaid on the color
shaded trend maps. S7 Fig. The European Centre for Medium range Weather Forecast (ECMWF)’s EN4 data
shows the cross section of the zonally averaged (20˚E to 90˚E) trends of potential temperature
for (a) summer, (b) autumn, (c) winter, and (d) spring, over the period of 1979–2015. Right
panel figures indicates the trends of salinity for (e) summer, (f) autumn, (g) winter, and (h)
spring. Only the significant trend values (p < 0.05) are considered to plot the figures according
to a two tailed t-test and the dots are marked to show the observations with the significant
trends. The contours indicate the climatology of temperature and salinity overlaid on the color
shaded trend maps. S8 Fig. Conclusions A notable episodic event on sea-ice retreat during 2017 is linked to the enhanced poleward advection of warm and moist air that led to strong melting
[69]. Locations of climatological Southern Ocean fronts are represented as black dashed lines [32]. White arrows show salinification near the coast (south of
~62˚S) due to enhanced rejection of salt associated with high rate of sea-ice production and its export [23,65]. https://doi.org/10.1371/journal.pone.0203222.g006 PLOS ONE | https://doi.org/10.1371/journal.pone.0203222
October 3, 2018 10 / 16 Sea-ice expansion in the Indian Ocean sector of Antarctica physical processes. This study describes one of the realistic possible mechanisms that explain
the overall increasing trend of satellite derived sea-ice in the IOA. Supporting information
S1 Table. Cross-correlation (r) between sea-ice extent and ocean-atmospheric parameters
for different seasons with the significant relationship (p < 0.05) marked in bold. (DOCX)
S1 Fig. Location of flux gates superposed onto a map of the sea-ice freeboard distribution. Sea-ice freeboard fluxes are computed across 64˚S latitude (gates 1 and 2), at the western
(gates 3 and 4 at 20˚E) and eastern (gates 5 and 6 at 100˚E) entry to the Indian Ocean sector. The red line at ~53˚E separates between western IO = Weddell East and eastern IO = East Ant-
arctic West. (TIF)
S2 Fig. Monthly total sea-ice freeboard flux across gates 1 and 2 (see S1 Fig). Years and ver-
tical dashed lines denote the beginning of the respective calendar year. (TIF)
S3 Fig. Monthly total sea-ice freeboard flux across gates 3 and 4, i.e. the western entry to
our region of interest (see S1 Fig). (TIF)
S4 Fig. Monthly total sea-ice freeboard flux across gates 5 and 6, i.e. the eastern entry to
our region of interest (see S1 Fig). (TIF)
S5 Fig. Time series of seasonal mean SAM index (Marshall., 2003) over the period of 1979–
2015. The dashed lines are the running mean of 10-year data for different seasons; indicates
significant trend towards high-index polarity during the austral summer (dashed red line) and
autumn (dashed black line). (TIF)
S6 Fig. Annual mass variability of the Antarctic ice-sheets adjacent to the Indian Ocean
sector of Antarctica, showed a gain of about 6.83±0.5 Gt yr-1. The data are obtained from
the Technische Universita¨t Dresden, Germany, derived from the Gravity Recovery and Cli-
mate Experiment (GRACE) satellite. (TIF)
S7 Fig. Supporting information S1 Table. Cross-correlation (r) between sea-ice extent and ocean-atmospheric parameters
for different seasons with the significant relationship (p < 0.05) marked in bold. (DOCX) S1 Table. Cross-correlation (r) between sea-ice extent and ocean-atmospheric parameters
for different seasons with the significant relationship (p < 0.05) marked in bold. (DOCX) S1 Fig. Location of flux gates superposed onto a map of the sea-ice freeboard distribution. Sea-ice freeboard fluxes are computed across 64˚S latitude (gates 1 and 2), at the western
(gates 3 and 4 at 20˚E) and eastern (gates 5 and 6 at 100˚E) entry to the Indian Ocean sector. The red line at ~53˚E separates between western IO = Weddell East and eastern IO = East Ant-
arctic West. (TIF) S1 Fig. Location of flux gates superposed onto a map of the sea-ice freeboard distribution. Sea-ice freeboard fluxes are computed across 64˚S latitude (gates 1 and 2), at the western
(gates 3 and 4 at 20˚E) and eastern (gates 5 and 6 at 100˚E) entry to the Indian Ocean sector. The red line at ~53˚E separates between western IO = Weddell East and eastern IO = East Ant-
arctic West. (TIF) S1 Fig. Location of flux gates superposed onto a map of the sea-ice freeboard distribution. Sea-ice freeboard fluxes are computed across 64˚S latitude (gates 1 and 2), at the western
(gates 3 and 4 at 20˚E) and eastern (gates 5 and 6 at 100˚E) entry to the Indian Ocean sector. The red line at ~53˚E separates between western IO = Weddell East and eastern IO = East Ant-
arctic West. (TIF) S2 Fig. Monthly total sea-ice freeboard flux across gates 1 and 2 (see S1 Fig). Years and ver-
tical dashed lines denote the beginning of the respective calendar year. (TIF) S3 Fig. Monthly total sea-ice freeboard flux across gates 3 and 4, i.e. the western entry to
our region of interest (see S1 Fig). (TIF) S3 Fig. Monthly total sea-ice freeboard flux across gates 3 and 4, i.e. the western entry to
our region of interest (see S1 Fig). (TIF) S3 Fig. Monthly total sea-ice freeboard flux across gates 3 and 4, i.e. the western entry to
our region of interest (see S1 Fig).
(TIF) Sea surface temperature (SST) anomaly computed from multiple data showed robust
feature of sea surface cooling over the Indian Ocean sector of Antarctica (averaged from 20˚E
to 90˚E and 73˚S to 55˚S), for (a) summer, (b) autumn, (c) winter, and (d) spring. The opti-
mum interpolated SST (OI SST), Interim SST, Ocean Reanalysis System 4 (ORAS4) SST and PLOS ONE | https://doi.org/10.1371/journal.pone.0203222
October 3, 2018 11 / 16 Sea-ice expansion in the Indian Ocean sector of Antarctica EN4 SST data are used for the analysis. EN4 SST (pink line) shows large deviations (probably
uncertainties) compared to other datasets, particularly during austral autumn and winter. (TIF) S9 Fig. Sea-ice concentration trend contours (% decade-1) overlaid on the sea-ice concentra-
tion climatology (1979–2015), for (a) summer, (b) autumn, (c) winter, and (d) spring, gener-
ated using ERA-Interim reanalysis data. Only the significant trend values (p < 0.05) are
contoured according to a two tailed t-test. Overall, the increasing pattern of sea-ice concentra-
tion is evident in the Indian Ocean sector of the Southern Ocean except the reduction in sea-
ice near the south Kerguelen Plateau (SKP), and coastal regions of the Prydz Bay (PB), Maw-
son (MS), Prince Harald (PH). S10 Fig. The cross section of the zonal temperature trend at 85˚E (south Kerguelen Plateau)
for (a) summer, (b) autumn, (c) winter, and (d) spring, over the period of 1979–2015, com-
puted from the European Centre for Medium range Weather Forecast (ECMWF)’s Ocean
Reanalysis System 4 (ORAS4) data. Enhanced warming is observed throughout the water col-
umn both at the surface and subsurface with a concurrent reduction in sea-ice as shown in
Figs 2 and 6. (T
) Acknowledgments Authors are thankful to Dr. A. J. Luis, Dr. Rahul Mohan and Dr. Meloth Thamban of NCAOR
for their encouragement and support. The authors greatly acknowledge various organisations
such as National Snow and Ice Data Center (NSIDC), National Oceanic and Atmospheric
Administration (NOAA), European Centre for Medium Range Weather Forecast (ECMWF),
National Centre for Atmospheric Research (NCAR), Technische Universita¨t Dresden, and the
Australian Antarctic Data Centre for making various datasets available in their portals. The
authors thank the anonymous referees and the editor for their useful and constructive com-
ments and suggestions. This is NCAOR contribution no 40/2018. Author Contributions Conceptualization: Babula Jena. Data curation: Babula Jena, Stefan Kern. Data curation: Babula Jena, Stefan Kern. Formal analysis: Babula Jena, M. Ravichandran. Formal analysis: Babula Jena, M. Ravichandran. Methodology: Babula Jena. Software: Babula Jena, Avinash Kumar. Software: Babula Jena, Avinash Kumar. Software: Babula Jena, Avinash Kumar. Supervision: M. Ravichandran. Visualization: Babula Jena, Avinash Kumar. Visualization: Babula Jena, Avinash Kumar. Writing – original draft: Babula Jena. Writing – original draft: Babula Jena. Writing – review & editing: Babula Jena, Avinash Kumar, M. Ravichandran, Stefan Kern. References 1. Mayewski PA, Meredith MP, Summerhayes CP, Turner J, Worby A, Barrett PJ, et al. State of the antarc-
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The impact of life events and transitions on physical activity: A scoping review
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cc-by
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OPEN ACCESS Citation: Gropper H, John JM, Sudeck G, Thiel A
(2020) The impact of life events and transitions on
physical activity: A scoping review. PLoS ONE
15(6): e0234794. https://doi.org/10.1371/journal. pone.0234794 Background Physical activity (PA) is a fluctuating behavior and prone to change across the life course. Changes in PA may be particularly due to the experience of life events and transitions. For
well-timed and successful PA interventions, it is important to understand when and why indi-
viduals take up or terminate PA. Objectives This scoping review aims to examine the extent, range, and nature of research on the
impact of life events and transitions on PA and to summarize key findings. Editor: Stephanie Prince Ware, University of
Ottawa Heart Institute, CANADA
Received: November 8, 2019
Accepted: June 2, 2020
Published: June 22, 2020
Copyright: © 2020 Gropper et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. Editor: Stephanie Prince Ware, University of
Ottawa Heart Institute, CANADA
Received: November 8, 2019
Accepted: June 2, 2020
Published: June 22, 2020 Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Information files. Funding: The authors HG, GS, and AT
acknowledge financial support from the iReAct
graduate school, which is funded by the Ministry of
Research, Science, and the Arts of Baden-
Wu¨rttemberg; State Postgraduate Fellowship
Program, Germany (https://uni-tuebingen.de/en/
research/support/research-funding/research-
funding-baden-wuerttemberg/state-postgraduate-
fellowship-programme/). Moreover, we Hannes GropperID1,2*, Jannika M. JohnID1,2, Gorden Sudeck1,2, Ansgar Thiel1,2 1 Institute of Sports Science, Eberhard Karls University Tu¨bingen, Tu¨bingen, Germany, 2 Interfaculty
Research Institute for Sport and Physical Activity, Eberhard Karls University Tu¨bingen, Tu¨bingen, Germany * hannes.gropper@uni-tuebingen.de a1111111111
a1111111111
a1111111111
a1111111111
a1111111111 Results 107 studies that assessed 72 distinct life events and transitions were included and summa-
rized in ten categories. Events and transitions that are primarily associated with decreases
in PA were starting cohabitation, getting married, pregnancy, evolving parenthood, and the
transitions from kindergarten to primary school, from primary to secondary school, and from
high school to college or into the labor market. Retirement was associated with increases in
PA; yet, long-term trajectories across retirement indicated a subsequent drop in activity lev-
els. Divorce was associated with no changes in PA. No trends could be identified for chang-
ing work conditions, quitting or losing a job, starting a new relationship, widowhood, moving,
and diagnosis of illness. Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Information files. Methods A systematic literature search was conducted in PubMed, PsycINFO, PsycARTICLES,
SPORTDiscus, and Web of Science. Articles were included if they had been published in
peer-reviewed journals between 1998 and 2020 and assessed the impact of at least one life
event or transition on PA. Copyright: © 2020 Gropper et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited. PLOS ONE PLOS ONE RESEARCH ARTICLE Introduction Time and again, research has shown that regular physical activity (PA) is associated with phys-
ical [1, 2], as well as mental and social [3, 4] health benefits. Thus, PA is considered “a ‘best
buy’ for public health” [5]. However, PA is a highly complex and multidimensional behavioral
phenomenon [6], which is prone to change across the life course [7]. Despite being a signifi-
cant promoter of health and well-being, regular PA tends to decrease over the life course [8]. Total PA appears to decline particularly during adolescence and the transition to young adult-
hood [9]. Additionally, Shaw et al. [10] found “steady declines in leisure-time physical activity,
beginning in midlife and growing steeper at progressively older ages” (p.763). Contrary to
these findings, some longitudinal studies have also identified groups that remain at a given
activity level or even increase their amount of PA [11–13]. These results suggest that PA is a fluctuating behavior that is not stable over time [14],
marked by lapses and relapses [15], and shows a more pronounced trend towards declining
than towards stable or increasing levels. This is in line with research arguing that PA patterns
track at rather low to moderate levels across the life course [16, 17]. Considering both long-
term and short-term health benefits of regular PA [5], it is crucial to understand when and
why people take up or terminate PA. Discontinuity, and associated behavioral lapses and
relapses, might be triggered particularly by the experience of life events and transitions that
occur across the life course. Telama [17] summarizes that “because many transitions and life-
changing events experienced during the course of life influence physical activity, the level of
tracking of physical activity is likely to vary at different phases of life” (p. 187). Previously, two reviews have explicitly focused on the impact of various life-change events
on PA. Allender et al. [18] identified five general life changes that primarily account for drop
out from PA, namely, changes in employment status, residence, physical status, relationships,
and family structure. In addition, Engberg et al. [19] found evidence that major life events (e.g. the transition to university, beginning to work, cohabitation and marriage, pregnancy and
childbirth, divorce, widowhood, and retirement) may have both positive and negative effects
on PA, depending among others on age and gender. Conclusion Life events and transitions can be conceptualized as natural interventions that occur across
the life course and that are oftentimes associated with changes in PA behavior. Our study 1 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0234794
June 22, 2020 PLOS ONE The impact of life events and transitions on physical activity indicates that, despite some emerging trends, similar events do not necessarily have similar
impacts on PA across individuals. It also shows that the research landscape is characterized
by a lack of conceptual clarity and by disparate methodologies, making it difficult to synthe-
size results across studies. indicates that, despite some emerging trends, similar events do not necessarily have similar
impacts on PA across individuals. It also shows that the research landscape is characterized
by a lack of conceptual clarity and by disparate methodologies, making it difficult to synthe-
size results across studies. acknowledge support by Open Access Publishing
Fund of University of Tu¨bingen (https://uni-
tuebingen.de/en/facilities/university-library/
publishing-research/open-access/open-access-
publikationsfonds/). The funders had no role in
study design, data collection and analysis, decision
to publish, or preparation of the manuscript. acknowledge support by Open Access Publishing
Fund of University of Tu¨bingen (https://uni-
tuebingen.de/en/facilities/university-library/
publishing-research/open-access/open-access-
publikationsfonds/). The funders had no role in
study design, data collection and analysis, decision
to publish, or preparation of the manuscript. Competing interests: The authors have declared
that no competing interests exist. PLOS ONE | https://doi.org/10.1371/journal.pone.0234794
June 22, 2020 Introduction Recently, an umbrella review on the
behavioral determinants of PA across the life course has shown negative associations between
PA and the transition to university or emerging parenthood [20], while another review has
argued that major life events, life transitions, and the experience of trauma can trigger stress,
which in turn impairs efforts to be physically active [21]. In addition, several reviews have
focused on particular life events and transitions. These previous reviews have suggested that
the transition from primary to secondary school is associated with decreasing levels of total PA
[22]. Moreover, leaving high school [23] and evolving parenthood [24–26] are linked to
declines in PA, while retirement is linked to increases especially in leisure-time PA and exer-
cise [27]. These reviews have contributed to the understanding of the impact of life events on PA and
have shown that research on this topic is characterized by fragmentation and great heterogene-
ity of study designs and methods. Despite these achievements, we see three shortcomings in 2 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0234794
June 22, 2020 PLOS ONE The impact of life events and transitions on physical activity the recent review activity urging for a more comprehensive and up-to-date approach: First, the
last review dealing explicitly with the question as to how various life events and PA relate to
each other has been conducted in January 2011 [19] leaving the last nine years of research
unmapped. Second, both Allender et al. [18] and Engberg et al. [19] searched only one
(PubMed) and two databases (PubMed/MEDLINE and PsycINFO), respectively. Third, a the-
oretical underpinning of the search strategies and conceptual clarity in terminology is often
missing. Our aim is to examine the extent, range, and nature of previous research activity on the
impact of life events and transitions on PA, to compile key findings, to identify research gaps,
and, therefore, to provide an extension to the previous reviews [28, 29]. In particular, we aim
to address the following research question that guided our process: How do life events and
transitions impact PA behavior across the life course? We chose a scoping approach (i) to map
the landscape of previous research activity in a field that is quite heterogeneous and frag-
mented and (ii) to systematically summarize key findings. Methods In order to increase methodological rigor, the procedure of this scoping review adhered to the
Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping
Reviews (PRISMA-ScR) Checklist [30]. Due to a lack of conceptual and terminological clarity,
in the following section we first explicate some theoretical considerations that have built the
basis for our search strategy before we describe our methodological procedure in more detail. PLOS ONE | https://doi.org/10.1371/journal.pone.0234794
June 22, 2020 Theoretical and terminological considerations We conceptualize life events from a developmental perspective arguing that they can be con-
strued as natural interventions that occur across the life course and that may account for onto-
genetic change, growth, and development [31]. According to the working definition put
forward by Luhmann et al. [32], life events “mark the beginning or the end of a specific status. A status is a nominal variable with at least two levels” (p. 4). For example, marital status (i.e. single, married, divorced, or widowed) might change due to the life events of meeting a part-
ner, marriage, divorce, or the death of a spouse. Life events are thus singular occurrences that lead to a shift from one status to another. In
this regard, they may trigger periods of biological, psychological, and/or social adaptation and
readjustment and lead to behavioral changes. In the following, we term such adjustment peri-
ods transitions as they incorporate the processual character and temporality of life events and
describe their anticipation (depending on the foreseeability of an event), history, and aftermath
[33]. Both life events and transitions are interconnected and therefore two sides of the same
experience, yet different in their temporal nature. Minor every-day events (e.g. daily hassles),
slow transitions without clearly identifiable life events (e.g. aging, puberty), and non-events
(e.g. not having a child) are excluded from our conceptualization of the terms life event and
transition. Life events and transitions may mark turning points in (habitual) behavioral patterns [31],
such as PA. For our review, we employ a broad concept of PA defining it as any bodily move-
ment produced by skeletal muscle resulting in energy expenditure [34]. PA therefore occurs
across various domains and contexts such as leisure-time PA (including sport participation
and exercise), occupational PA, active commuting, daily activities, or domestic activities and at
different intensities such as light, moderate, vigorous, or moderate-to-vigorous. Table 1 sum-
marizes the key concepts that underpin our review. 3 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0234794
June 22, 2020 PLOS ONE The impact of life events and transitions on physical activity Table 1. Theoretical concepts. Literature search A broad, yet systematic, search strategy was developed to search the following databases:
PubMed (including MEDLINE), PsycINFO, PsycARTICLES, SPORTDiscus, and Web of Sci-
ence. Search terms were derived from our theoretical and terminological considerations and
in accordance with the terms employed in previous reviews [18, 19]. Table 2 displays the
search strategy, with #3 yielding the initial results from which studies where then selected. No
limits were used. The databases were searched on August 17, 2018. The search was updated on January, 27
2020. The updated information was combined with the results from the initial search. Results
were exported into EndNote Citation Manager and duplicates were removed. Then, two
researchers independently screened titles and abstracts for eligibility. Disagreements were
resolved through a process of critical debate. If consensus could not be reached, a third
researcher was consulted. Full-texts were assessed by a single researcher, who also extracted
data from the articles. When in doubt, the inclusion of full-texts was discussed with a second
researcher until consensus was reached. Moreover, included full-texts were used for cross-
referencing. Theoretical and terminological considerations Concept
Definition
Life event
In reference to the MeSH term definition for life-change events, yet with some adjustment, life
events are conceptualized as singular occurrences (including biological, psychological, social, and
environmental), which mark a change in status and therefore require (re)adjustment and effect a
change in an individual’s pattern of living [31, 32, 35]. Transition
Transitions are conceptualized as status passages that temporally exceed the duration of life
events. The duration of a transition varies as a function of the degree of (re)adjustment, which is
necessary to adapt to a new status. Generally, life events, which imply a change in status, may
trigger a transition or occur within the transitioning process itself (depending on an event’s
foreseeability) [36, 37]. Physical
activity
Physical activity is generally defined as any bodily movement produced by skeletal muscles
resulting in energy expenditure [34] and may occur across various domains and contexts and at
different intensities. https://doi.org/10.1371/journal.pone.0234794.t001 In reference to the MeSH term definition for life-change events, yet with some adjustment, life
events are conceptualized as singular occurrences (including biological, psychological, social, and
environmental), which mark a change in status and therefore require (re)adjustment and effect a
change in an individual’s pattern of living [31, 32, 35]. Transitions are conceptualized as status passages that temporally exceed the duration of life
events. The duration of a transition varies as a function of the degree of (re)adjustment, which is
necessary to adapt to a new status. Generally, life events, which imply a change in status, may
trigger a transition or occur within the transitioning process itself (depending on an event’s
foreseeability) [36, 37]. https://doi.org/10.1371/journal.pone.0234794.t001 https://doi.org/10.1371/journal.pone.0234794.t001 Inclusion and exclusion criteria Papers were included if they (i) focused on life events and/or transitions in accordance with
our theoretical conceptualizations; (ii) reported data on PA, which was assessed either objec-
tively (e.g. via accelerometer or pedometer) or through self-report (e.g. via questionnaires or
interviews); (iii) were either of prospective longitudinal or retrospective design in order to
compare PA behavior before and after a life event or transition; (iv) assessed healthy popula-
tions, except for when transitions into disease were analyzed; (v) were published in English or
German; (vi) were published in peer-reviewed journals between 1998 and 2020; and (vii) were
available as full-texts. Table 2. Search terms for the literature research. #1
“life event” OR “life events” OR “life change event” OR “life change events” OR “life-change event” OR “life-
change events” OR “life changing event” OR “life changing events” OR “life-changing event” OR “life-changing
events” OR “life experience” OR “life experiences” OR “life change” OR “life changes” OR “life-change” OR
“life-changes” OR transition
#2
“physical activity” OR exercis OR “sport” OR “sports”
#3
#1 AND #2 #1
“life event” OR “life events” OR “life change event” OR “life change events” OR “life-change event” OR “life-
change events” OR “life changing event” OR “life changing events” OR “life-changing event” OR “life-changing
events” OR “life experience” OR “life experiences” OR “life change” OR “life changes” OR “life-change” OR
“life-changes” OR transition PLOS ONE | https://doi.org/10.1371/journal.pone.0234794
June 22, 2020 Table 2. Search terms for the literature research. Table 2. Search terms for the literature research. Set Search Terms
#1
“life event” OR “life events” OR “life change event” OR “life change events” OR “life-change event” OR “life-
change events” OR “life changing event” OR “life changing events” OR “life-changing event” OR “life-changing
events” OR “life experience” OR “life experiences” OR “life change” OR “life changes” OR “life-change” OR
“life-changes” OR transition
#2
“physical activity” OR exercis OR “sport” OR “sports”
#3
#1 AND #2
https://doi.org/10.1371/journal.pone.0234794.t002 4 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0234794
June 22, 2020 PLOS ONE The impact of life events and transitions on physical activity Papers were excluded if they (i) were non-empirical (e.g. other reviews, editorials, com-
ments, essays, abstracts, conference submissions, etc.), dissertations, or intervention studies
(including randomized controlled trials); (ii) focused only on physical education or sedentary
behavior; (iii) did not examine changes in PA behavior itself but rather in psychological or
social variables associated with PA (e.g. motivational, intentional, or habitual variables); (iv)
used PA as a moderator or mediator for another dependent variable (e.g. weight gain); or (v)
dealt with daily hassles, non-events, or slow transitions. The study selection was an iterative
process and criteria for inclusion and exclusion were further refined and adapted as knowledge
on the topic grew, which is in line with the notion of a scoping review [28, 38]. Data extraction and synthesis Extracted data included the country in which the study was conducted, the life events and
transitions covered, study design, sample characteristics, the employed PA assessment tools,
and the PA domains and intensities that were examined (see S1 Table). If papers addressed
several research questions, we focused on the data relevant to our research question. Moreover,
if statistical models were adjusted (e.g. for sex, age, education, income, previous behavior,
etc.), we report the results from these adjusted analyses assuming that this is a more conserva-
tive, and therefore, a more cautious approach of interpreting results. As our primary objective
was to map the research landscape, we do not report on single mediation and interaction
effects. As a first synthesis step, we provide a summary of the research landscape in which we
also cluster the reported life events and transitions into distinct thematic categories. In a sec-
ond step, the identified categories are used as a basis for a narrative synthesis of the key find-
ings of the included studies (for both females and males if not otherwise specified). In order to
stay true to the scoping nature of the present review and to delineate general trends, we
refrained from summarizing results at the level of PA domains and intensities. Additionally, to
be included in the narrative summary, results for events and transitions had to be reported in
at least two studies. For the synthesis of the more general trends, results had to be presented in
at least four studies. PLOS ONE | https://doi.org/10.1371/journal.pone.0234794
June 22, 2020 Results The initial search yielded 22,869 articles with 15,935 remaining after duplicates were removed. After screening titles, abstracts, and full texts, 92 studies were included. Additionally, 15 rec-
ords were identified through cross-referencing resulting in a total of 107 articles (Fig 1). The included studies covered a total of 72 distinct life events and transitions, which we
grouped into 10 thematic categories. For the life event major personal achievement [40], a clear
allocation was not possible, since this event was considered to be rather broad and might
occur within various categorical domains. Forty-nine life events or transitions (68%) were cov-
ered in only one or two studies. Table 3 summarizes, which life event categories, and which
events and transitions in particular, were assessed across studies. Descriptive analyses of the publication landscape over the past twenty-two years revealed
an increasing research interest in the impact of life events and transitions on PA with 74 stud-
ies being published in the nine years since the last review on this topic has been conducted
[19]. More than half of the studies were conducted in the USA (n = 35; 32.7%), Canada (n =
13; 12.1%), and Australia (n = 12; 11.2%). Studies varied with regard to their design, with the
vast majority being prospective longitudinal studies (n = 88; 82.2%), followed by retrospective
assessments (n = 19; 17.8%). Follow-up time for the prospective longitudinal studies ranged
from three months [146] to 25 years [83]. Sample sizes varied from n = 7 [98] to n = 81,925
[123]. The age of participants ranged from three [44] to 91 years [109, 132]. Detailed study 5 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0234794
June 22, 2020 PLOS ONE The impact of life events and transitions on physical activity Fig 1. Flow chart of the systematic literature search. (adapted from the PRISMA flow diagram [39]). https://doi.org/10.1371/journal.pone.0234794.g001 Fig 1. Flow chart of the systematic literature search. (adapted from the PRISMA flow diagram [39]). https://doi.org/10.1371/journal.pone.0234794.g001 characteristics are summarized in S1 Table. Studies using the same samples are summarized in
S2 Table. A great variety of PA assessment tools was used. Ninety studies (84.1%) employed question-
naires to determine changes in PA. In total, 31 distinct PA questionnaires were used across
studies with the Godin Leisure-Time Exercise Questionnaire (n = 6) and the Active Australia
Survey (n = 4; three of which used the same sample) being the most recurring tools. PLOS ONE | https://doi.org/10.1371/journal.pone.0234794
June 22, 2020 Results Thirty-
four studies (31.8%) did not report, which questionnaires were employed. One study com-
bined the questionnaire assessment with interviews. Twenty-three studies (21.5%) used accel-
erometers or pedometers to assess PA. Of these, seven studies combined questionnaires and
accelerometers, eleven studies used only accelerometers (for four to ten consecutive days), and
five studies used pedometers usually in combination with either an accelerometer or a ques-
tionnaire. As a result, changes in PA behavior were assessed in different ways either taking
into account the amount of PA (e.g. frequency and duration, which was assessed either via
accelerometers, an open question, or a Likert scale) or by simply asking whether participants
engaged in PA or whether PA behavior has changed over time (e.g. yes/no). Studies assessed seven more or less distinct PA domains: leisure-time PA (n = 80; 74.8%),
occupational PA (n = 16; 15%), total PA (n = 22; 20.6%), walking and daily habitual PA
(n = 41; 38.3%), commuting and active transport (n = 20; 18.7%), domestic activities (n = 20; 6 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0234794
June 22, 2020 The impact of life events and transitions on physical activity PLOS ONE PLOS ONE PLOS ONE Table 3. Categories of life events and transitions covered across studies. Category
Life Event/Transition
n (%)
Study (Citation No.)
Education-related events/transitions (n = 38;
35.5%)
Transition to primary school
5 (4.7%)
[41–45]
Transition to secondary school
10 (9.3%)
[46–55]
Transition from middle to high school
2 (1.9%)
[56, 57]
Transition to post-secondary education/university/college
12
(11.2%)
[58–69]
Return to study
3 (2.8%)
[40, 70, 71]
(Post)graduation
8 (7.5%)
[59, 67, 70, 72–76]
Receiving more education
1 (0.9%)
[77]
Employment-related events/transitions (n = 41;
38.3%)
Entry into labor market
10 (9.3%)
[40, 58, 59, 67, 70–72, 76, 78, 79]
Changing jobs
6 (5.6%)
[40, 71, 72, 77, 80, 81]
Quitting/stopping/losing job
7 (6.5%)
[40, 72, 77, 79, 80, 82, 83]
Retirement
29
(27.1%)
[40, 80, 83–109]
Increased/decreased income
2 (1.9%)
[40, 77]
Going on welfare
1 (0.9%)
[72]
Difficulty finding a job
1 (0.9%)
[40]
Health-related events/transitions (n = 19;
17.8%)
Developing/recovering from a major personal illness (e.g. cancer)/injury
10 (9.3%)
[40, 72, 80, 109–115]
Developing/recovering from chronic diseases (e.g. overweight,
diabetes, etc.)
5 (4.7%)
[77, 80, 115–117]
Becoming normal weight
1 (0.9%)
[77]
Menopausal transition
4 (3.7%)
[40, 118–120]
Onset of menarche
1 (0.9%)
[121]
Major surgery
2 (1.9%)
[40, 80]
Involvement in a serious accident
2 (1.9%)
[40, 80]
Developing/recovering from a depression
2 (1.9%)
[77, 80]
Becoming (un)happy
1 (0.9%)
[77]
Developing/recovering from a long-term-disability
2 (1.9%)
[77, 109]
Developing/recovering from pain
2 (1.9%)
[77, 80]
Starting/stopping to smoke
1 (0.9%)
[77]
Starting/stopping to drink alcohol
1 (0.9%)
[77]
Becoming (un)healthy
1 (0.9%)
[77]
Becoming a caregiver
1 (0.9%)
[83]
Relationship-related events/transitions (n = 23;
21.5%)
Starting/stopping a relationship
5 (4.7%)
[40, 72, 76, 80, 122]
Starting cohabitation
6 (5.6%)
[40, 70, 76, 78, 123, 124]
Getting married
15
(14.0%)
[40, 59, 70–72, 76–78, 83, 109, 123–127]
Getting divorced/separated
11
(10.3%)
[40, 71, 72, 80, 83, 122, 123, 125–128]
Losing a spouse/partner/widowhood
9 (8.4%)
[40, 77, 80, 95, 123, 127–130]
Re-marriage
3 (2.8%)
[127, 128, 130]
Engagement
1 (0.9%)
[72]
Infidelity of spouse/partner
1 (0.9%)
[40]
Major decline in health of a spouse/partner
3 (2.8%)
[40, 114, 131]
Spouse/partner quitting/stopping/losing job
1 (0.9%)
[40]
Spouse’s/partner’s retirement
2 (1.9%)
[40, 132]
Spouse/partner moving into an institution
1 (0.9%)
[40]
(Continued) PLOS ONE | https://doi.org/10.1371/journal.pone.0234794
June 22, 2020 7 / 24 The impact of life events and transitions on physical activity PLOS ONE PLOS ONE Table 3. (Continued)
Category
Life Event/Transition
n (%)
Study (Citation No.)
Family-related events/transitions (n = 28;
26.2%)
Pregnancy/birth of a first/subsequent child
24
(22.4%)
[40, 58, 59, 70–72, 76, 78, 79, 109, 124,
133–145]
Becoming a single parent
2 (1.9%)
[40, 71]
Miscarriage
1 (0.9%)
[40]
Stillbirth
1 (0.9%)
[40]
Having a child with a disability/serious illness
1 (0.9%)
[40]
Death of a child
2 (1.9%)
[40, 83]
Major conflict with teenage/older children
1 (0.9%)
[40]
Increased hassles with parents
1 (0.9%)
[40]
Parents getting divorced/separated/re-married
1 (0.9%)
[40]
Serious conflict between family members
1 (0.9%)
[40]
Major decline in health of close friends/family
1 (0.9%)
[40]
Death of friend/family
5 (4.7%)
[40, 72, 80, 83, 109]
Family member being arrested
1 (0.9%)
[40]
Getting/losing social support
2 (1.9%)
[77, 115]
Child/other family member leaving home
1 (0.9%)
[40]
Birth of a grandchild
1 (0.9%)
[40]
Residence-related events/transitions (n = 10;
9.3%)
Moving
5 (4.7%)
[40, 72, 80, 83, 115]
Moving to an institution
2 (1.9%)
[40, 146]
Moving out of parents’ house/independence
6 (5.6%)
[40, 59, 67, 70, 72, 78]
Moving back to parents’ house
1 (0.9%)
[70]
Starting a mortgage
1 (0.9%)
[72]
Leisure-time related events/transitions (n = 1;
0.9%)
Starting/stopping a hobby
1 (0.9%)
[80]
Starting/stopping to go on holiday trips
1 (0.9%)
[80]
Victimization-related events/transitions (n = 2;
1.9%)
Distressing harassment at work
1 (0.9%)
[40]
Being pushed/grabbed/shoved/kicked/hit
1 (0.9%)
[40]
Being forced to take part in an unwanted sexual activity
1 (0.9%)
[40]
Being robbed
2 (1.9%)
[40, 80]
Criminal activity-related events/transitions
(n = 2; 1.9%)
Being arrested/going to jail
1 (0.9%)
[72]
Legal troubles/involvement in a court case
1 (0.9%)
[40]
Force majeure/material loss (n = 1; 0.9%)
Natural disaster
1 (0.9%)
[40]
Major loss/damage of personal property
1 (0.9%)
[40]
https://doi org/10 1371/journal pone 0234794 t003 18.7%), and school activities (n = 8; 7.5%). While some studies focused on PA domains, others
(additionally or instead) assessed PA intensities ranging from light (n = 18; 16.8%) to moderate
(n = 39; 36.4%) to vigorous (n = 34; 31.8%) to moderate-to-vigorous PA (n = 29; 27.1%), and
several combinations of the four intensity clusters (n = 38; 35.5%). As studies often merged
several PA domains and intensities, a wide variety of combinations was apparent (for a detailed
overview see S1 Table). PLOS ONE | https://doi.org/10.1371/journal.pone.0234794
June 22, 2020 PLOS ONE In this regard, an in-depth and differentiated analysis of findings
across all studies was not possible. Therefore, we abstracted from PA domains and intensities
and summarized trends. The following sub-chapters focus on the associations between specific
events and transitions and PA. Table 4 depicts, which of the included studies reported
increases, decreases, or no changes in PA for a respective event or transition. We only summa-
rized general trends for an event or transition when respective results were reported in at least
four studies. As single studies assessed different PA domains and intensities, they might be
reported multiple times (i.e. for increases, decreases, or no changes). Fig 2 summarizes trends
for events and transitions across the life course. 18.7%), and school activities (n = 8; 7.5%). While some studies focused on PA domains, others
(additionally or instead) assessed PA intensities ranging from light (n = 18; 16.8%) to moderate
(n = 39; 36.4%) to vigorous (n = 34; 31.8%) to moderate-to-vigorous PA (n = 29; 27.1%), and
several combinations of the four intensity clusters (n = 38; 35.5%). As studies often merged
several PA domains and intensities, a wide variety of combinations was apparent (for a detailed
overview see S1 Table). In this regard, an in-depth and differentiated analysis of findings
across all studies was not possible. Therefore, we abstracted from PA domains and intensities
and summarized trends. The following sub-chapters focus on the associations between specific
events and transitions and PA. Table 4 depicts, which of the included studies reported
increases, decreases, or no changes in PA for a respective event or transition. We only summa-
rized general trends for an event or transition when respective results were reported in at least
four studies. As single studies assessed different PA domains and intensities, they might be
reported multiple times (i.e. for increases, decreases, or no changes). Fig 2 summarizes trends
for events and transitions across the life course. 8 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0234794
June 22, 2020 PLOS ONE The impact of life events and transitions on physical activity Table 4. Trends and consistency of key findings across studies for events, when results were reported in 4
studies. PLOS ONE Life event/transition
Increase
Decrease
No change
Education-related events/
transitions
Kindergarten to primary
school
1 [42]
5 [41–45]
1 [41]
Primary to secondary school
4 [46–48, 50]
10 [47, 48, 50–57]
4 [47–49, 51]
High school to university/
college
14 [58, 60–69, 73–75]
2 [59, 65]
Family-related events/
transitions
Pregnancy, Parenthood1
5 [79, 136, 142, 144,
145]
20 [40, 59, 70, 71, 76, 78, 109, 124,
133, 134, 136–145]
5 [124, 135, 139,
142, 144]
Employment-related events/
transitions
Entry into the labor market
1 [72]
7 [40, 58, 71, 72, 76, 78, 81]
4 [59, 67, 77, 79]
Changing work conditions
2 [40, 72]
2 [71, 72]
Quitting, stopping, losing job
2 [40, 72]
2 [79, 83]
Retirement
25 [40, 84, 86–102,
104–109]
11 [83–86, 93, 98, 103–107]
8 [85–87, 92, 95, 96,
98, 103]
Relationship-related events/
transitions
Starting a new relationship
2 [40, 72]
3 [76, 122, 123]
2 [72, 122]
Starting cohabitation
3 [70, 76, 78]
1 [124]
Marriage
1 [125]
9 [40, 59, 70–72, 76–78, 109]
5 [72, 77, 83, 124,
126]
Divorce
2 [126, 127]
2 [40, 122]
5 [71, 83, 122, 125,
128]
Widowhood
3 [40, 129, 130]
4 [77, 95, 127, 128]
Health-related events/
transitions
Diagnosis of illness2
7 [77, 111–114, 117,
118]
7 [40, 77, 109, 110, 109, 114, 117]
2 [72, 114]
Residence-related events/
transitions
Moving
1 [79]
1 [109]
2 [72, 83]
1Pregnancy and parenthood were subsumed as one event as several studies assessed PA changes across pregnancy
including a postpartum period
2Diagnosis of illness also includes the diagnosis of a chronic condition Table 4. Trends and consistency of key findings across studies for events, when results were reported in 4
studies 1Pregnancy and parenthood were subsumed as one event as several studies assessed PA changes across pregnancy
including a postpartum period 2Diagnosis of illness also includes the diagnosis of a chronic condition Education-related events and transitions Educational transitions were covered from early childhood (e.g. from kindergarten to school)
to young adulthood (e.g. entry into post-secondary education or (post)graduation). Five stud-
ies assessed the transition from kindergarten to primary school and found mainly decreases
in PA [41–45]. Two of these studies reported a rebound effect to or beyond pre-school levels
[42, 44]. Similarly, findings on the transition from primary to secondary and from middle to high
school showed mainly declines in PA across this transition [47, 48, 50–57]. Yet, studies also
reported increases [46–48, 50] and no changes [47–49, 51] in PA, which was due to the assess-
ment of different PA domains and intensities and whether activities occurred during weekdays
or the weekend. 9 / 24 PLOS ONE | https://doi.org/10.1371/journal.pone.0234794
June 22, 2020 PLOS ONE The impact of life events and transitions on physical activity Fig 2. Summary of key findings of the impact of life events and transitions on PA across the life course. https://doi.org/10.1371/journal.pone.0234794.g002 Fig 2. Summary of key findings of the impact of life events and transitions on PA across the life course. https://doi.org/10.1371/journal.pone.0234794.g002 Studies, which focused on the transition from high school to post-secondary education (e.g. college or university) or leaving full-time education (e.g. started to work), showed that this is a
period during which emerging adults are vulnerable to decreasing PA levels [58, 60, 61, 63–65,
67–69, 73, 76]. A greater proportion of high school graduates decreased their PA levels as
opposed to those who increased PA [61, 62] and a shift from meeting PA guidelines to not
meeting them occurred [75]. Two studies indicated that changes in PA were dependent on the
environmental context, as high school graduates who started working were more likely to
engage in moderate-to-vigorous and occupational PA than those who transitioned to post-sec-
ondary education although, in general, both groups tended to decrease their PA levels [73, 74]. PLOS ONE | https://doi.org/10.1371/journal.pone.0234794
June 22, 2020 Employment-related events and transitions Beginning paid work or entry into the labor market was mainly associated with decreases in
PA. These declines were particularly pronounced in leisure-time PA [40, 58, 71, 72, 76, 78, 81],
although two studies found contrasting findings for women and men [72, 77]. Four studies
found no changes in PA [59, 67, 77, 79]. Changes in work conditions or jobs were associated with both decreases [71, 72] and
increases [40, 72] in PA depending on domain, intensity, age, and gender. Similarly, losing or
quitting a job or having a decreased income were associated with more [40, 72] and less [40]
PA, while two studies reported no changes [79, 83]. These results again depended on PA
domain, intensity, gender, and age. Being discharged from military was associated with a shift
from 68.2% of participants meeting PA guidelines to 50.4% [82]. Retirement was the most examined life transition with 29 studies (27.1%). In general, retire-
ment was associated with increases in leisure-time PA, (recreational) walking, and domestic
activities [40, 84, 85, 87, 88, 90, 91, 93, 96, 99, 100, 103, 104, 106–109, 132] and decreases in
occupational PA, active transportation, and total PA [83, 84, 93, 103, 104, 106, 107]. With
regard to PA intensities, an increase especially in light and moderate PA occurred [87, 96],
whereas findings on vigorous and moderate-to-vigorous PA were more ambiguous [93, 95, 96, PLOS ONE | https://doi.org/10.1371/journal.pone.0234794
June 22, 2020 10 / 24 PLOS ONE The impact of life events and transitions on physical activity 105, 132]. Qualitative data suggests that most retirees experience an increase in PA, while only
a minority reports decreases or no changes at all [97] Prevalence data similarly indicates that
there is an increased probability of meeting US government’s PA guidelines after retirement
[94]. With regard to the type of retirement, findings were heterogeneous. Transitioning to full-
time and voluntary retirement was linked to increased PA levels [89, 90], whereas transitioning
to economic inactivity due to disability led to decreases [89, 105]. For the latter, however, one
study reported declines in women only, whereas men increased their PA [96]. Retiring from a
physically demanding job was associated with a decrease in PA [86], whereas for those retiring
from a sedentary job, there was an increase [86]. Similarly, PA declined more in women and
men from manual than for those from non-manual social classes [84]. Employment-related events and transitions Some studies reported
on the long-term trajectories of PA patterns across the retirement transition indicating that
PA tends to increase right after retirement but declines later on [91, 99, 102, 104, 105]. In con-
trast, two studies found evidence that PA levels remained stable after an initial increase [101,
108]. Findings for the pre-retirement period were ambiguous with reported increases and
decreases in PA [102, 105] depending on domain and gender. Health-related events and transitions Being diagnosed with a serious illness or disease was associated with mixed outcomes. Studies
investigating changes in PA behavior after a cancer diagnosis reported both increases and
decreases [110–113], while being diagnosed with a chronic disease or condition was linked to
increases in PA [77, 117]. However, for older women, the diagnosis of an illness, having sur-
gery, developing pain, and becoming obese or “(un)healthy” were associated with a decrease in
PA [40, 77]. Additional health-related events and transitions included the menopausal transition, which
was associated with decreases in PA in two studies [118, 119] and with no changes in one
study [120], the onset of menarche, which was linked to a significant negative slope in PA
(with the gynecological age-based model but not the chronological one) [121], and the onset of
impaired sleep, which entailed an increased risk of becoming physically inactive [116]. These
findings are in line with qualitative research suggesting that the onset of health and mobility
problems is associated with constraints regarding PA [109]. Family-related events and transitions Becoming a parent was primarily associated with decreases in PA [109, 124, 136], which were
particularly pronounced in women [40, 70, 71, 139] and first-time parents [59, 76, 78, 124,
138, 142]. One study described the quality of change in PA as a smooth decline for women
before and after birth, whereas men experienced a rather abrupt drop in the year after birth
[140]. Expecting a second or subsequent child was associated with increases in light activities
[142], yet, also with decreases in leisure-time PA [124]. Data on group trajectories reflects
these tendencies as more women become inactive across the transition to parenthood than
those who became active [133, 139]. Nevertheless, one retrospective study indicated that new
parents might substitute activity domains, thus, not abandoning PA altogether and that par-
enthood might also present an opportunity to become more active [136]. Changes in PA were not only associated with parenthood but already with pregnancy as
one study reported that substantially more women decreased their PA as opposed to those
who increased theirs [137]. In general, pregnancy was linked to declines in PA [134, 141, 143–
145]. Some studies, however, indicated increases in light activities such as walking or domestic
chores [79, 141, 144, 145]. Results on long-term trajectories from pre-pregnancy to parent-
hood were ambiguous with some studies reporting rebound effects across the postpartum
period [134, 141], one study reporting no changes in PA from pregnancy to postpartum [143],
and one study indicating slight increases during maternity leave, which decreased again upon
return-to-work [135]. Despite these trends, one study indicated that individual growth trajec-
tories varied significantly from the average growth curve [134]. Relationship-related events and transitions Findings on the impact of starting a new close relationship were mixed with two studies
reporting increases in PA for young women [40] and men [72],two indicating decreases for
both genders [76, 123], and one reporting decreases only for women [122] Ending a relation-
ship was rather associated with increases in PA [72, 123] depending on domain and gender. Starting cohabitation was linked to decreases in PA [70, 76, 78], although one study found no
changes [124]. Becoming engaged and getting married were mainly associated with decreases
in PA [40, 59, 70–72, 76–78, 109, 126], depending on domain, intensity, and gender. Only one
study found a positive relationship between marriage and PA outcomes [125]. Five studies
reported no changes in PA, however, depending on domain, intensity, or gender [72, 77, 83,
124, 126]. Getting divorced or separated yielded mixed results, however, showing a trend
towards no changes [71, 83, 122, 125, 128]. In addition, two studies reported decreases in PA
in young women [40] and men [122], whereas two other studies indicated increases in PA
[126, 127]. The death of a spouse and widowhood were associated with increases in PA in three studies
[40, 129, 130] while four studies reported no changes [77, 95, 127, 128]. These relationships
were dependent on PA domain, gender, and time since bereavement. Getting re-married and PLOS ONE | https://doi.org/10.1371/journal.pone.0234794
June 22, 2020 11 / 24 PLOS ONE The impact of life events and transitions on physical activity re-coupling were associated with both decreased [122, 128] and increased [130] levels of PA,
again depending on domain and gender. Residence-related events and transitions Moving out of the parental home and gaining residential independence was linked to
decreases in PA [59, 78] or no changes [67]. Moving in general was associated with no changes
[72, 83], but with increases in transport-related walking when moving to a less urbanized area
[79]. Moving to an institution, such as retirement living, was similarly associated with signifi-
cant decreases in PA [40, 146]. Qualitative data suggested that relocations were often perceived
as disruptions in social networks, therefore leading to a drop in activities [109]. PLOS ONE | https://doi.org/10.1371/journal.pone.0234794
June 22, 2020 Limitations of current research and desiderates Limitations in the current state of research can be summarized as theoretical shortcomings on
the one hand and methodological issues on the other. Subsequently, we want to discuss these
issues and suggest some potential future directions. Theoretical limitations. On a theoretical and conceptual level, the majority of included
studies neither precisely defined the terms life event or transition nor did they provide a theo-
retical framework that conceptually relates life events and PA behavior. The lack of termino-
logical and theoretical framing became particularly apparent in vague terminology. For
instance, some studies used the term life event for processes or slow transitions (such as diffi-
culties finding a job, “becoming (un)happy”, or becoming overweight or obese), which would,
according to our definition, not account for a life event as a change in status. Yet, these studies
also assessed other events and were, therefore, included in this review. Similarly, some life
events were rather unspecific and it was not clear what they entailed (e.g. “major personal
achievement”, “receiving more education”, or “becoming (un)healthy”). Similar to the lack of
conceptual clarity regarding life events, studies rarely reflected upon the concept of PA. Most
studies acknowledged some sort of basic definition (mostly referring to Caspersen et al., [34]),
however, they focused on a variety of different PA domains and intensities often resulting in
inconclusive or mixed findings and making it rather difficult to synthesize results in a differen-
tiated, yet concise fashion. We think that the lack of conceptual clarity with regard to the terms life event and transition
and the complexity of a phenomenon like PA need to be addressed in future research. Theoret-
ical and terminological awareness are highly relevant as they help to guide research processes
and embed empirical findings within a framework for interpretation that increases scientific
rigor, reduces the risk of jumping to hasty conclusions, and allows for comparability across
studies. Theories should therefore be at “the center of research” [147, p. 2] and play a key role
in future research. Methodological limitations. Terminological and conceptual disparities resulted in quite
heterogeneous methodological approaches across studies as indicated by the variety of sam-
pling procedures and life event assessment tools that were employed. Most studies chose par-
ticipants, who found themselves in a period in which a given event or transition usually occurs
(e.g. educational transitions or retirement). Others used demographic questionnaires to assess
whether a life event (e.g. Discussion In the present scoping review, we examined the state of research on the impact of life events
and transitions on PA behavior across the life course. In this regard, we mapped the research
landscape and summarized key findings. Life events and transitions that were covered by studies spanned from early childhood to
old age and were thematically clustered into ten distinct categories. Childhood, adolescence,
and young adulthood were life phases that displayed a wide variety of life events and transi-
tions, that were oftentimes education-, relationship-, and family-related. For middle and older
aged samples, the life events and transitions that were examined the most were illness, bereave-
ment, and, above all, retirement. Our results are mostly in line with previous reviews indicating that events and transitions
during young adulthood are oftentimes accompanied by declines in PA [18–20, 23–26] and
that retirement may offer a window of opportunity [105] for positive changes in PA despite
declines in occupational PA and active commuting [19, 27]. Moreover, our results indicate a
decreasing trend in PA for the transition from primary to secondary school. This corresponds PLOS ONE | https://doi.org/10.1371/journal.pone.0234794
June 22, 2020 12 / 24 PLOS ONE The impact of life events and transitions on physical activity to one review that reports results for six studies that we have included as well [22]. Addition-
ally, despite including six more studies (including those for the transition from middle to high
school), our results similarly indicate that changes in PA across this transition are dependent
on PA domain, intensity, and time of the day or the week. Our review shows that for some life
events and transitions (e.g. changing work conditions, quitting or losing a job, starting a new
relationship, widowhood, moving, or diagnosis of illness), it is not possible to summarize clear
trends. This issue has already been emphasized in the review by Engberg et al. [19] indicating
that the causal and directional impact of life events and transitions on PA must not be over-
stated. If and how individuals adapt to the same event is a highly idiosyncratic process that
depends on a multitude of factors (including psychosocial moderators and mediators that are
oftentimes not assessed) and that might go hand in hand with increases, decreases, or no
changes in PA at all. Moreover, life events and transitions might account for substitution
effects regarding PA domains and intensities, thus, not rendering total PA. PLOS ONE | https://doi.org/10.1371/journal.pone.0234794
June 22, 2020 Limitations of current research and desiderates marriage, school enrollment, or change in employment status) had
occurred in a given period. Finally, few studies used retrospective interviews or Life-Event-
Scales that stress more on the subjective significance of an event. Independent of the PLOS ONE | https://doi.org/10.1371/journal.pone.0234794
June 22, 2020 13 / 24 PLOS ONE The impact of life events and transitions on physical activity methodological approach, the studies rarely provided theoretical justifications for the decision
on how to assess a particular life event or transition. Methodological heterogeneity also became evident with regard to the use of PA assessment
tools. Thirty-one different PA questionnaires targeting various domains and intensities and
using different question formats were applied. Additionally, 34 studies did not specify which
scales were used. Objective PA assessment tools (e.g. accelerometers or pedometers) were used
in only 21.5% of the studies–mostly with children and adolescents–and were rarely combined
with self-reports on PA habits. This fragmented and inconsistent use of assessment tools and
validated scales has already been indicated by Engberg et al. [19] and complicated the compari-
son of results across studies. For future research, we consider it as highly important to be aware of the complexity of PA
behavior and its distinct domains and intensities and to carefully choose respective assessment
tools. Although a plethora of validated PA scales exists, almost 32% of the included studies did
not specify, which questionnaires were used. Relying on validated scales that are able to differ-
entiate between various modes of PA rather than on on-the-fly creations [148] may facilitate
comparability across studies in the future. Moreover, it might be beneficial to combine objec-
tive and subjective PA assessment tools in order to measure PA levels, behavioral patterns, and
subjective perceptions to fully understand how and why behavior changes. The vast majority of studies were prospective longitudinal. These designs allow to identify
patterns within populations and reveal relationships between life events and transitions and
PA behavior. However, two issues might limit the informative value of prospective longitudi-
nal studies. First, long periods between follow-up assessments pose a challenge to the identifi-
cation of the exact timing of a life event meaning that immediate effects might remain hidden. Additionally, changes in PA levels could also be influenced by other circumstances and events
that occurred within this period but were not assessed specifically. Second, if follow-up times
are rather short, long-term developments and rebound or readjustment effects remain
concealed. PLOS ONE | https://doi.org/10.1371/journal.pone.0234794
June 22, 2020 Limitations of current research and desiderates To counteract these two potential limitations, prospective longitudinal studies should ide-
ally use repeated measures over an extended follow-up time in order to capture life events and
transitions in their temporal complexity and to account for both immediate and more persis-
tent effects. Here, a temporal division of transition periods may be helpful. Studies on retire-
ment, pregnancy, or menopause have shown that a distinction in a period before (pre), around
(peri), and after an event (post) is beneficial in order to better understand the long-term trajec-
tories of behavioral pathways. The temporal fragmentation in three phases may also help to
assess the quality of behavioral changes across a transition, meaning whether a behavior
changes in a smooth or rather abrupt fashion. A further methodological limitation of current research is that most studies did not control
for concomitant events but rather focused on singular events. Studies targeting young adult-
hood in particular have shown that this is a developmental period, which is prone to the co-
occurrence or accumulation of various life events and transitions, which might all interact in
changing PA behavior. A similar challenge that future research will have to deal with is to sepa-
rate the effects of life events on PA behavior from underlying general trends. It has been widely
acknowledged that PA declines with increasing age [149, 150] and that adolescence and young
adulthood are periods where steep declines are to be expected [9]. In this regard, PA patterns
might change due to biological maturation or other environmental factors that are not neces-
sarily related to life events and transitions. Only few studies (mostly those with a focus on preg-
nancy, parenthood, and retirement) employed either long-term follow-ups or control groups
to monitor these underlying trends. A careful choice of assessment tools, long-term follow-
ups, control group designs, and more complex statistical models might be necessary to PLOS ONE | https://doi.org/10.1371/journal.pone.0234794
June 22, 2020 14 / 24 PLOS ONE The impact of life events and transitions on physical activity disentangle potential interaction effects between different life events and transitions, underly-
ing trends, and other potential mediator or moderator effects (e.g. gender, age, socio-economic
status, etc.). In addition, we advocate that future research should further consider the use of biographi-
cal retrospective studies in order to assess life events and their impact on PA from an individu-
alized, yet socio-cultural perspective [31]. Limitations of current research and desiderates It is highly relevant to add explanatory value to the
rather descriptive results of prospective longitudinal studies. Biographical analyses may reveal
how and why life events become critical events in the sense that they are valued as personally
significant [31], how they are perceived and processed, how they account for changes in PA
behavior across the life course, and which underlying meaning is attributed to these develop-
ments. While reconstructive and self-reported PA data might be limited due to potential recall
bias or inaccuracy as well as social desirability [151, 152], the assessment of perceptions and
attitudes about behavioral pathways adds another perspective to better understand when and
why individuals take up or terminate PA. With regard to sample characteristics, a total of 20 studies included only females while only
two studies included solely men. As for the remaining studies, 17 showed an uneven gender
distribution as more than 65% of the sample consisted of women. Our findings indicate that
women and men may respond in different ways to certain events and transitions. However, as
studies showed great diversity in methodological approaches and sampling, clear gender
effects were difficult to identify. Moreover, most studies examined samples of well-educated people making it difficult to
generalize results for other populations. Particularly, research on retirement has shown that
job characteristics may have an impact on the way PA behavior evolves across this transition. Therefore, future research should also focus on socioeconomic circumstances and apply a
sampling strategy that also takes into account disadvantaged socioeconomic positions. PLOS ONE | https://doi.org/10.1371/journal.pone.0234794
June 22, 2020 S2 Table. Studies assessing the same samples.
(DOCX) S2 Table. Studies assessing the same samples. (DOCX) S1 File. Preferred reporting items for systematic reviews and meta-analyses extension for
scoping reviews (prisma-scr) checklist. (DOCX) S1 File. Preferred reporting items for systematic reviews and meta-analyses extension for
scoping reviews (prisma-scr) checklist. (DOCX) Limitations of the review With regard to our review, some limitations occur as well. First, the categorization of life
events and transitions into ten thematic categories appeared to be the most reasonable,
although we acknowledge that other categorical differentiations might be possible. Neverthe-
less, considering the heterogeneity of studies, we propose these initial distinctions in order to
facilitate the synthesis of results. In this regard, however, a second limitation occurs. While the
inclusion of 107 articles assessing 72 different life events and their relationship to various PA
domains and intensities is a notable strength for mapping the research landscape on this topic,
it also poses a considerable constraint for the depth of our analysis. Thus, the narrative synthe-
sis of results does not reflect the entire complexity of the life event-PA relationship, but rather
tries to identify emerging trends and draw out patterns across studies on a more general level. Third, only one author reviewed full-texts for eligibility. To include only studies that fit the
inclusion criteria, arguable studies were discussed with a second researcher until consensus
was reached. Fourth, we did not conduct a quality appraisal of the included studies. In this
regard, studies were given the same weight when scoping the research landscape, although
comparability might be limited. Fifth, although we extended the database search of two previ-
ous reviews [18, 19], studies that were published in another language or in journals that were
not indexed in the databases might not have been found. Through cross-referencing we tried
to lower the risk of missing any relevant studies. By focusing on peer-reviewed journal articles,
potentially relevant grey literature (e.g. dissertations that are published as monographs) might
have been missed. Eventually, by searching for terms such as life event or transition, studies
focusing on certain events and transitions (e.g. parenthood, pregnancy, retirement, etc.) but PLOS ONE | https://doi.org/10.1371/journal.pone.0234794
June 22, 2020 15 / 24 PLOS ONE The impact of life events and transitions on physical activity not using the terms life event or transition might have been missed. Again, by cross-referenc-
ing, we aimed to lower this particular risk. Conclusion This scoping review provides an overview of the current state of research on the impact of life
events and transitions on PA and a summary of key findings in this area. Considering the
health benefits of regular PA, we argue that it is crucial to understand when and why individu-
als take up or terminate PA. In this context, life events and transitions can be conceptualized
as natural interventions that inevitably occur within the life course and that are oftentimes
accompanied by changes in PA behavior for both the better and the worse. Despite some
emerging trends, the overall state of research on this topic is still characterized by heterogene-
ity and fragmentation, which is reflected in a lack of theoretical, conceptual, and terminologi-
cal clarity as well as in disparate study designs, methods, and reporting. This makes it difficult
to synthesize and compare results across studies. Eventually, we hope that our synthesis of the
research landscape will help to further develop research practices in the highly relevant fields
of PA and sport, health, and leisure studies. Acknowledgments The authors would like to thank Anna Brandauer for her assistance with the literature search. In addition, the authors want to thank the reviewers, whose profound comments helped
enhance this paper. The authors would like to thank Anna Brandauer for her assistance with the literature search. In addition, the authors want to thank the reviewers, whose profound comments helped
enhance this paper. Supporting information S1 Table. Study characteristics of the 107 included studies arranged by the assessed life
event and transition categories. (DOCX) S1 Table. Study characteristics of the 107 included studies arranged by the assessed life
event and transition categories. PLOS ONE | https://doi.org/10.1371/journal.pone.0234794
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Cardiac involvement in a patient with B-cell lymphoblastic lymphoma/acute lymphoblastic leukemia and a history of allogeneic hematopoietic stem cell transplantation and CAR T-cell therapy: A case report
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Frontiers in immunology
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cc-by
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OPEN ACCESS OPEN ACCESS
EDITED BY
Claudia Scotti,
University of Pavia, Italy
REVIEWED BY
Pouya Safarzadeh Kozani,
Guilan University of Medical
Sciences, Iran
Pooria Safarzadeh Kozani,
Tarbiat Modares University, Iran
Xi Zhang,
Xinqiao Hospital, China
*CORRESPONDENCE
Erlie Jiang
jiangerlie@ihcams.ac.cn
Xin Chen
chenxin@ihcams.ac.cn
†These authors have contributed
equally to this work
SPECIALTY SECTION
This article was submitted to
Cancer Immunity
and Immunotherapy,
a section of the journal
Frontiers in Immunology
RECEIVED 23 September 2022
ACCEPTED 28 November 2022
PUBLISHED 05 January 2023 Yigeng Cao 1†, Yadan Liu 2†, Rongli Zhang 1, Weihua Zhai 1,
Qiaoling Ma 1, Jialin Wei 1, Donglin Yang 1, Aiming Pang 1,
Yi He 1, Xin Chen 1*, Erlie Jiang 1*, Sizhou Feng 1
and Mingzhe Han 1 1State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood
Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital,
Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China,
2Hematology Department of Ningbo First Hospital, Ningbo Clinical Research Center for
Hematologic Malignancies, Ningbo, China CITATION
Cao Y, Liu Y, Zhang R, Zhai W, Ma Q,
Wei J, Yang D, Pang A, He Y, Chen X,
Jiang E, Feng S and Han M (2023)
Cardiac involvement in a patient with
B-cell lymphoblastic lymphoma/acute
lymphoblastic leukemia and a history
of allogeneic hematopoietic stem cell
transplantation and CAR T-cell
therapy: A case report. Front. Immunol. 13:1052336. doi: 10.3389/fimmu.2022.1052336 Cardiac involvement in hematological malignancies is uncommon, with only a few
cases reported to date, and it often leads to a poor prognosis. Here, we report a
case of a 42-year-old woman with a history of allogeneic hematopoietic stem cell
transplantation (allo-HSCT) and anti-CD19 chimeric antigen receptor (CAR) T-cell
therapy for B-cell lymphoblastic lymphoma/acute lymphoblastic leukemia in
whom cardiac mass and myocardial infiltration were detected. Prior to this
presentation, massive pericardial effusion had occurred 6 months after CAR T-
cell therapy, which was improved via ultrasound-guided pericardiocentesis. We
observed elevated cytokine levels and increased copy number of CAR DNA in both
pericardial effusion and serum. Upon detecting cardiac mass and myocardial
infiltration, the patient was administered tocilizumab (a humanized monoclonal
antibody against IL-6 receptor), which controlled the serum cytokine levels, and
reduced intensity chemotherapy, including vindesine, cyclophosphamide, and
prednisolone. However, the patient finally died of multiple organ failure. B cell acute lymphoblastic leukemia, B cell lymphoblastic lymphoma, allogeneic
hematopoietic stem cell transplantation, chimeric antigen receptor T cells,
cardiac involvement COPYRIGHT
© 2023 Cao, Liu, Zhang, Zhai, Ma, Wei,
Yang, Pang , He, Chen, Jiang, Feng and
Han. This is an open-access article
distributed under the terms of the
Creative Commons Attribution License
(CC BY). The use, distribution or
reproduction in other forums is
permitted, provided the original
author(s) and the copyright owner(s)
are credited and that the original
publication in this journal is cited, in
accordance with accepted academic
practice. No use, distribution or
reproduction is permitted which does
not comply with these terms. TYPE Case Report
PUBLISHED 05 January 2023
DOI 10.3389/fimmu.2022.1052336 TYPE Case Report
PUBLISHED 05 January 2023
DOI 10.3389/fimmu.2022.1052336 TYPE Case Report
PUBLISHED 05 January 2023
DOI 10.3389/fimmu.2022.1052336 TYPE Case Report
PUBLISHED 05 January 2023
DOI 10.3389/fimmu.2022.1052336 Cardiac involvement in a patient
with B-cell lymphoblastic
lymphoma/acute lymphoblastic
leukemia and a history of
allogeneic hematopoietic stem
cell transplantation and CAR T-
cell therapy: A case report Cardiac involvement in a patient
with B-cell lymphoblastic
lymphoma/acute lymphoblastic
leukemia and a history of
allogeneic hematopoietic stem
cell transplantation and CAR T-
cell therapy: A case report Introduction intense FDG uptake by these nodules. Immunohistochemical
(IHC) analysis of a left kidney biopsy specimen indicated
positivity for CD10, TdT, CD79a, Pax-5, CD43, c-myc (20%),
and Ki-67 (90%) and negativity for CD3, CD20, CD5, CD23,
Bcl-2, Bcl-6, MUM1, and CyclinD1. Neither bone marrow smear
nor flow cytometry revealed any abnormal lymphocytes, and
bone marrow biopsy revealed active proliferation of the
hematopoietic tissue. Cytogenetic analysis revealed an
abnormal karyotype: 46,XX,t(1,19)(q23;p13),-4,+mar[18]/46,
XX(2). Gene fusion analysis showed positivity for E2A/PBX1. These findings led to the diagnosis of B-LBL with E2A/PBX1. B-cell acute lymphoblastic leukemia (B-ALL) and B-cell
lymphoblastic lymphoma (B-LBL) are precursor lymphoid
neoplasms of the B lineage, which are considered to be the
same disease with different developmental stages and clinical
manifestations (1). According to version 1.2016 of the NCCN,
less than 20% bone marrow involvement can be used as a
diagnostic criterion to distinguish LBL from ALL. B-LBL is a
highly malignant non-Hodgkin’s lymphoma that commonly
occurs in children and adolescents. The most common
occurrence sites of B-LBL are the skin, soft tissue, bone, and
lymph nodes, while mediastinal or pleural involvement is rare and
cardiac involvement is even rarer (2). Some studies have shown
that cardiac involvement is common in secondary and malignant
tumors through hematogenous metastasis, lymphatic metastasis,
transvenous extension, and direct infiltration of tissues
surrounding the neoplasms and that lymphoma and leukemia
are the most common hematological malignancies involving the
heart (3–5). The heart is hidden in the mediastinum, and there are
no specific clinical manifestations of cardiac involvement, which
can easily lead to misdiagnosis or missed diagnosis relatively easy
in patients with cardiac metastasis. The patient had received two cycles of induction
chemotherapy with a combination of cyclophosphamide,
vincristine, doxorubicin, dexamethasone (hyper-CVAD A),
methotrexate, and cytarabine (hyper-CVAD B). Bone marrow
examination revealed 10% blasts, while PET-CT revealed no
intense FDG uptake. Subsequently, the patient received
vindesine, daunorubicin, cyclophosphamide, pegaspargase,
prednisone (VDCLP), cyclophosphamide, cytarabine,
6-mercaptopurine (CAM), vindesine, cyclophosphamide,
idarubicin, dexamethasone (VICD). Every bone marrow smear
after each chemotherapy showed CR, while E2A/PBX1 was
undetectable, 3.68% and 0.22% as determined via polymerase
chain reaction, respectively. Allogeneic hematopoietic stem cell transplantation (allo-
HSCT) is currently the only available methodology to treat B-
LBL, and it has been shown to greatly improve the long-term
survival of patients (6). OPEN ACCESS To the
best of our knowledge, this is the first report on the development of a cardiac mass
and occurrence of myocardial infiltration after allo-HSCT and CAR T-cell therapy. This report may provide supporting data for the early diagnosis and immediate
treatment of patients with cardiac involvement. COPYRIGHT
© 2023 Cao, Liu, Zhang, Zhai, Ma, Wei,
Yang, Pang , He, Chen, Jiang, Feng and
Han. This is an open-access article
distributed under the terms of the
Creative Commons Attribution License
(CC BY). The use, distribution or
reproduction in other forums is
permitted, provided the original
author(s) and the copyright owner(s)
are credited and that the original
publication in this journal is cited, in
accordance with accepted academic
practice. No use, distribution or
reproduction is permitted which does
not comply with these terms. B cell acute lymphoblastic leukemia, B cell lymphoblastic lymphoma, allogeneic
hematopoietic stem cell transplantation, chimeric antigen receptor T cells,
cardiac involvement frontiersin.org Frontiers in Immunology 01 Cao et al. 10.3389/fimmu.2022.1052336 Introduction However, it is difficult to obtain a complete
response (CR) or partial response (PR) in patients with B-ALL
who show relapse after transplantation, and these patients exhibit
an extremely poor prognosis (7). Chimeric antigen receptor
(CAR) T-cell therapy is an emerging cellular immunotherapy
and has revolutionized treatment modalities for relapsed or
refractory B-ALL (R/R B-ALL) with a CR rate of 70%–90% (8,
9). It has thus significantly prolonged the disease-free survival of
such patients (10, 11). Here, we present a case of a 42-year-old
woman with an initial diagnosis of B-LBL who developed cardiac
involvement after allo-HSCT and anti-CD19 CAR T-cell therapy. To the best of our knowledge, no systematic studies on the efficacy
and safety of various treatments for cardiac involvement exist. This report provides relevant data for identifying cardiac
involvement and selecting an appropriate treatment. The patient remained well for 3 months. In May 2020, bone
marrow smear revealed 47% lymphoblasts, while the rate of
E2A/PBX1 rearrangement was 45.55%. Lymphoblasts accounted
for 4.96% of the marrow cellularity, as determined using flow
cytometry. These results suggested a diagnosis of B-ALL with
E2A/PBX1 expression. Treatment with orally administered 6-
mercaptopurine and prednisone was initiated to relieve the
tumor burden. In June 2020, the patient underwent allo-HSCT from a
matched, sibling donor after a conditioning regimen with
cyclophosphamide, fludarabine, idarubicin, and antithymocyte
globulin and total body irradiation. Early post-transplantation
complications included mild diarrhea and mucositis. The bone
marrow smear indicated CR, no E2A/PBX1 amplification, and
full donor chimerism. PET-CT revealed that the treatment was
effective and that the CR of the disease was achieved, with a
Deauville score of 1 point. In September 2020, the patient complained of left breast pain
that had begun 1 week earlier. Breast ultrasound showed
multiple nodules in the left breast, while IHC of a left breast
biopsy specimen indicated extramedullary recurrence of B-ALL
after transplantation. Neither bone marrow aspirate nor flow
cytometry showed any abnormal blasts, while E2A/PBX1 was
undetectable. PET-CT revealed progressive disease, with a
maximum standard uptake value (SUVmax) of 23.0 and a
Deauville score of 5 points (Figure 1A). Then, the
administration of decitabine, fludarabine, cytarabine, and
granulocyte colony-stimulating factor was initiated. Acute
graft-versus-host-disease of the skin occurred after donor Case presentation The patient received diuretic, albumin infusion, antiviral
with ganciclovir, and supportive treatments. As part of CAR T-
cell therapy, tocilizumab was administered to control elevated
serum cytokine levels. Clinical symptoms were slightly improved
within 1 week of admission. Notably, the level of cardiac
troponin I (cTNI) was elevated prior to the administration
of tocilizumab. In May 2021, the patient complained of chest pain that
worsened when the patient breathed in or lay flat. Electrocardiogram (ECG) and myocardial enzyme levels were
normal. Echocardiography (Echo) revealed massive pericardial
effusion with rapid progression to cardiac tamponade. Ultrasound-guided pericardiocentesis, which was performed to
alleviate the clinical symptoms and drain the excess fluid around
the heart, revealed the presence of a hemorrhagic and exudative
liquid. Multiple diagnostic tests for pericardial effusion,
including bacterial gram staining, bacterial culture, fungal
culture, acid fast bacteria staining, tuberculosis (TB) antibody
examination, biopsy, and NGS, were performed. The results of
all the above mentioned tests were negative. Flow cytometry of
pericardial effusion and bone marrow aspirate did not reveal any
tumor cells or CAR T cells. However, quantitative real-time
polymerase chain reaction (qPCR) revealed that the copy
number of CAR DNA per microgram of genome was 1.92 ×
101. The results of additional laboratory tests, including those of
cytokines, are shown in Table 1. Given that some cytokines of
the pericardial fluid were notably increased, CRS was considered
as the possible cause of pericardial effusion. The pericardial
effusion was gradually reduced and removed after
immunosuppressive therapy with methylprednisolone and
rucotinib. Bone marrow aspirate and PET-CT showed CR,
with a Deauville score of 1 point. On the 12th day after presentation, PET-CT of the body and
head was performed, which showed cardiac involvement and
abnormal uptake in the liver (Figures 1B, C). Bone marrow
aspirate and cytogenetic analysis were normal, while flow
cytometry revealed lymphoblasts that accounted for 0.15% of the
marrow cellularity. E2A/PBX1 rearrangement was also positive. The results of CAR T cells are shown in Figure 2B. A cardiac mass
biopsy was needed to achieve the pathological diagnosis but was
not performed due to the patient’s poor physical condition. Owing to her poor physical condition, the patient was finally
administered a reduced intensity chemotherapy with
cyclophosphamide, vindesine, and prednisolone (VCP). Nevertheless, on the third day of chemotherapy, her blood
pressure and oxygen saturation suddenly dropped to 61/29
mmHg and 76%, respectively. Case presentation A 42-year-old woman with a history of B-LBL was admitted
to the Institute of Hematology and Blood Diseases Hospital,
Tianjin, China, because of persistent dyspnea. In May 2019, the
patient was evaluated at another hospital for recurrent pain in
her right hip, which had begun 6 months earlier and gradually
worsened with no obvious predisposing causes. PET-CT scans
revealed multiple high-density nodules in both the kidneys, left
acetabulum, left ilium, bilateral tibia, and right fibula as well as Frontiers in Immunology frontiersin.org 02 Cao et al. 10.3389/fimmu.2022.1052336 Cao et al. known drug allergies. The patient lived with her husband and
one child in rural China. The patient had previously worked as a
farmer and did not smoke or drink. lymphocyte infusion. Eventually, the breast nodules decreased in
size, and PET-CT revealed PR of the disease, with a Deauville
score of 4 points. In November 2020, the patient underwent donor-derived
anti-CD19 CAR T-cell infusion after a conditioning regimen
with fludarabine and cyclophosphamide. The sequence of CD19
CAR consists of an anti-CD19 single-chain antibody fragment
(FMC63), CD28 transmembrane domain, 4-1BB, CD3zeta, T2A
autocleavage sequences, and endodomain-deleted EGFR
(tEGFR) (12). Four days later, the patient had a recurrent high
fever with a temperature of 40°C, along with lethargy and
vomiting. Based on a previous study, a grade-2 cytokine
release syndrome (CRS) was identified (13). These clinical
issues improved after providing supportive care with
antibiotics (meropenem and caspofungin) and dexamethasone. Meanwhile, no clear etiological evidence with next-generation
sequencing (NGS) or blood culture was obtained. The patient
achieved CR and was negative for minimal residual disease on
the 14th day after CAR T-cell therapy and remained well for
5 months. On examination, the patient’s temperature was 36.6°C, her
heart rate was 104 beats per minute, blood pressure was 94/68
mmHg, respiratory rate was 25 breaths per minute, and oxygen
saturation was 93% while the patient breathed ambient air. Severe edema of the limbs was identified, while the results of
the remaining examinations were normal. The copy number of
cytomegalovirus (CMV) was 8,953 per milliliter (reference
range: <1,000). Other laboratory test results are shown in
Table 2. ECG revealed changes in the ST segment, and Echo
revealed that the left ventricular ejection fraction (LVEF) had
decreased to 49%. The level of serum cytokines was significantly
increased (Figure 2A). Chest CT revealed interstitial
inflammation of both lungs. Case presentation Along with a heart rate of 170
beats per minute, moist rales could be heard at the base of the left
lung. ECG revealed tachycardia, ST-segment depression, and
inverted T waves in the V2–6 leads. Although timely first-aid
measures were provided, the patient could not be rescued and
was eventually discharged from the hospital. The clinical course
of the patient is shown in Figure 3. On presentation to our hospital (November 2021), the
patient complained of dyspnea and fatigue that had begun 2
weeks earlier. The patient’s medical history included
hypertension. However, the patient did not need to take
medications on a regular basis to control it under the guidance
of a cardiologist. Immunosuppressive therapy with rucotinib
and dexamethasone and preventive fungal treatment with orally
administered posaconazole were continued. There were no Discussion Cardiac involvement is rare in patients with B-LBL/B-ALL,
with only a few cases having been reported in the literature. It
thus remains unclear how to identify cardiac involvement
quickly and provide effective therapies. Some summative
studies have demonstrated that cardiac metastases are detected Frontiers in Immunology frontiersin.org 03 Cao et al. Cao et al. 10.3389/fimmu.2022.1052336 FIGURE 1
PET-CT findings of the patient. (A) In September 2020, there was an irregular soft tissue mass in the upper outer quadrant of the breast (4.1 ×
2.0 cm), with an SUVmax of 23.0, as shown by the blue arrow. (B) In November 2021, there were a cardiac mass (6.0 × 4.4 cm), with an SUVmax
of 10.5, as shown by the yellow arrow and a diffusely thickened pericardium, with an SUVmax of 2.0, as shown by the green arrow. (C) In
November 2021, there was an abnormal uptake in the liver, with an SUVmax of 5.0 and a Deauville score of 5 points, as shown by the red arrow. PET-CT findings of the patient. (A) In September 2020, there was an irregular soft tissue mass in the upper outer quadrant of the breast (4.1 ×
2.0 cm), with an SUVmax of 23.0, as shown by the blue arrow. (B) In November 2021, there were a cardiac mass (6.0 × 4.4 cm), with an SUVmax
of 10.5, as shown by the yellow arrow and a diffusely thickened pericardium, with an SUVmax of 2.0, as shown by the green arrow. (C) In
November 2021, there was an abnormal uptake in the liver, with an SUVmax of 5.0 and a Deauville score of 5 points, as shown by the red arrow. techniques, such as ECG, Echo, enhanced CT, cardiac magnetic
resonance (CMR), and PET-CT, generally produced abnormal
images, and PET-CT appears to have a diagnostic value in
differentiating benign and malignant cardiac tumors and
predicting prognosis, especially for cardiac metastases and
lymphoma (17–19). Infiltration of leukemia cells into the
myocardium may lead to cardiac conduction system in many cases at the time of postmortem (4, 5). Given the rarity
of performing postmortems, only a few cases of cardiac
involvement have been reported. We reviewed recent clinical
cases with cardiac involvement and identified some common
clinical manifestations (14–16). First, patients with cardiac
involvement mostly presented non-specific symptoms, such as
chest pain, dyspnea, and edema. Second, non-invasive imaging TABLE 1 Cytokines in pericardial effusion and peripheral blood. Discussion Cytokines (pg/ml)
IL-6
IL-8
IL-10
IL-17
IL-12P70
TNF-a
IFN-g
Pericardial effusion
35,742.86
1,514.66
122.41
0.36
3.71
7.76
8.74
Peripheral blood
8.22
12.9
5.03
37.35
8.62
12.68
7.55
IL, interleukin; TNF-a, tumor necrosis factor-a; IFN-g, interferon-g. TABLE 1 Cytokines in pericardial effusion and peripheral blood. 04 Frontiers in Immunology Cao et al. 10.3389/fimmu.2022.1052336 TABLE 2 Patient characteristics on admission. TABLE 2 Patient characteristics on admission. Laboratory dataa The main reasons
why malignant cells were not detected in the pericardial
effusion sample were the small number of leukemia cells and
the powerful ability of CAR T cells to target and kill leukemia
cells. In addition, the bloody pericardial effusion also supported
our speculation of tumor invasion because inflammatory
effusions are mostly green or yellow-green. CAR DNA data
showed the persistence of CD19 CAR T cells in the patient, and
CAR T cell consistently played a role of immunologic
surveillance. Nevertheless, when leukemia cells invade the
heart again, CAR T cells cannot exert a strong antileukemia
effect as before, and the process of infiltration is accelerated to
eventually form a cardiac mass owing to the decreased ability of
CAR T-cell expansion. inflammatory diseases, metabolic diseases, and drug-related
factors, were also not considered (23). Although we found
CAR DNA copies and abnormal increases in cytokines in
pericardial effusion, no tumor cells or masses were found
using flow cytometry or CT. The reason behind the
occurrence of pericardial effusion remained unknown at the
time. However, on reviewing the disease course of the patient, we
speculated that the patient may have already had cardiac
involvement during pericardial effusion. The main reasons
why malignant cells were not detected in the pericardial
effusion sample were the small number of leukemia cells and
the powerful ability of CAR T cells to target and kill leukemia
cells. In addition, the bloody pericardial effusion also supported
our speculation of tumor invasion because inflammatory
effusions are mostly green or yellow-green. CAR DNA data
showed the persistence of CD19 CAR T cells in the patient, and
CAR T cell consistently played a role of immunologic
surveillance. Nevertheless, when leukemia cells invade the
heart again, CAR T cells cannot exert a strong antileukemia
effect as before, and the process of infiltration is accelerated to
eventually form a cardiac mass owing to the decreased ability of
CAR T-cell expansion. abnormalities and rhythmic disturbances (20, 21), which mainly
included non-specific ST–T wave changes and atrial
arrhythmias. Although CMR and PET-CT are not always
available, detection of cardiac involvement has benefited from
the development of imaging technology in recent years. Unfortunately, the Echo and CT of this patient did not
provide adequate imaging evidence of relapse. For most metastatic cardiac tumors, surgery is not suitable,
whereas surgery is a treatment option in case of primary cardiac
tumors. Laboratory dataa Laboratory dataa
Variable
On presentation
Reference rangeThis hospitalb
White cell count (per ml)
1,640
4,000–10,000
Differential count (per ml)
Neutrophils
1,430
2,000–7,000
Lymphocytes
170
800–4,000
Monocytes
40
120–1,000
Eosinophils
0
20–500
Basophils
0
0–100
Hemoglobin (g/L)
77
110–150
Platelet count (per ml)
22,000
100,000–300,000
Hematocrit (%)
23.7
37–48
Total protein (g/dl)
5.23
6.6–8.3
Albumin (g/dl)
3.14
3.5–5.2
Globulin (g/dl)
2.09
2.0–3.5
Aspartate aminotransferase (U/L)
48.70
0–35
Alanine aminotransferase (U/L)
60.30
0–35
Alkaline phosphatase (U/L)
176.30
30–120
Glutamyl transpeptidase (U/L)
291.80
8–57
Lactate dehydrogenase (U/L)
1,150.20
0–248
Total bilirubin (mg/dl)
0.9
0–1
Creatinine (mg/dl)
0.75
0.55–1.0
Cardiac troponin I (ng/ml)
0.059
<0.04
Creatine kinase isoenzyme-MB (ng/ml)
7.5
0.5–5.0
B-type natriuretic peptide (pg/ml)
352
0–100
aTo convert the values for bilirubin to micromoles per liter, multiply by 17.1. To convert the values for creatinine to micromoles per liter, multiply by 88.4. bReference values are affected by many variables, including the patient population and the laboratory methods used. They may therefore not be appropriate for all patients. Reference rangeThis hospitalb
4,000–10,000 aTo convert the values for bilirubin to micromoles per liter, multiply by 17.1. To convert the values for creatinine to micromoles per liter, multiply by 88
bReference values are affected by many variables, including the patient population and the laboratory methods used. They may therefore not be appropria B A
B
FIGURE 2
Serum cytokines and CAR19 DNA. (A) Serum cytokines of the patient during CAR T-cell therapy since admission. (B) CAR19 DNA in PB and BM
since CAR T-cell therapy. A B A FIGURE 2
Serum cytokines and CAR19 DNA. (A) Serum cytokines of the patient during CAR T-cell therapy since admission. (B) CAR19 DNA in PB and BM
since CAR T-cell therapy. frontiersin.org Frontiers in Immunology 05 Cao et al. 10.3389/fimmu.2022.1052336 Cao et al. inflammatory diseases, metabolic diseases, and drug-related
factors, were also not considered (23). Although we found
CAR DNA copies and abnormal increases in cytokines in
pericardial effusion, no tumor cells or masses were found
using flow cytometry or CT. The reason behind the
occurrence of pericardial effusion remained unknown at the
time. However, on reviewing the disease course of the patient, we
speculated that the patient may have already had cardiac
involvement during pericardial effusion. Publisher’s note All claims expressed in this article are solely those of the
authors and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed
or endorsed by the publisher. Laboratory dataa Radiation therapy and chemotherapy have been
routinely used to treat patients with cardiac metastases (3,
22). In a report by Kakefuda et al., a 61-year-old woman with
B-ALL who received hyper-CVAD chemotherapy due to a
cardiac mass finally achieved clinical remission (16). In
addition, Manabe et al. reported that a 17-year-old patient
with B-LBL with an intracardiac mass and myocardial
infiltration underwent autologous peripheral blood stem cell
transplantation after intensive chemotherapy and finally
achieved long-term remission for over 5 years (15). Kahwash
et al. reported that a 51-year-old patient with B-ALL with an
infiltrative cardiac mass treated with palliative chest radiation
ultimately died (14). The case presented in the current study
had clinical symptoms similar to those of the above cases but a
completely different course of disease progression. Thus, the
treatment strategy for this patient differed slightly from those
reported previously; this patient was administered tocilizumab
and chemotherapy. CRS and immune effector cell-associated neurotoxicity
syndrome (ICANS) are frequently reported due to the unique
and life-threatening toxicity associated with CAR T-cell therapy. The mechanisms of CRS and ICANS have been explored in
detail (24, 25). However, cardiovascular (CV) events caused by
CAR T-cell therapy have not been explored much, but CV
should also be taken seriously with limited data (26, 27). Patients treated with CAR T-cell therapy experienced
arrhythmias, decompensated heart failure, and CV-related
death. Seventeen patients had a CV event about 21 days after In the present study, we report a case of massive pericardial
effusion close to tamponade, which occurred 6 months after
CAR T treatment, and did not find common infectious causes of
pericardial effusion, such as viruses, bacteria, fungi, and
parasites, and non-infectious causes, including systemic FIGURE 3
Clinical course of the patient. TBI+CTX+Flu+IDA+ATG, total body irradiation, cyclophosphamide, fludarabine, idarubicin, and antithymocyte
globulin. BM, bone marrow aspirate; US, ultrasound; MP, methylprednisolone. course of the patient. TBI+CTX+Flu+IDA+ATG, total body irradiation, cyclophosphamide, fludarabine, idarubicin, and antithymocyte
BM, bone marrow aspirate; US, ultrasound; MP, methylprednisolone. FIGURE 3
Clinical course of the patient. TBI+CTX+Flu+IDA+ATG, total body irradiation, cyclophosphamide, fludarabine, idarubicin, and antithymocyte
globulin. BM, bone marrow aspirate; US, ultrasound; MP, methylprednisolone. frontiersin.org Frontiers in Immunology 06 Cao et al. Cao et al. 10.3389/fimmu.2022.1052336 7. Zhang M, Huang H. How to combine the two landmark treatment methods-
allogeneic hematopoietic stem cell transplantation and chimeric antigen receptor T
cell therapy together to cure high-risk b cell acute lymphoblastic leukemia? Front
Immunol (2020) 11:611710. doi: 10.3389/fimmu.2020.611710 Conclusions To the best of our knowledge, this is the first report of
cardiac involvement as an extramedullary manifestation of
relapse in a patient with B-LBL/B-ALL treated with allo-HSCT
and CAR T-cell therapy. Notably, pericardial effusion may be the
first presentation of the underlying relapse of malignancy; hence,
diagnostic testing should not be stopped after determining the
cytology of pericardial effusion in patients exhibiting CRS after
CAR T-cell therapy. The possibility of cardiac involvement
needs to be considered in time. Furthermore, simultaneous
elevations in serum cytokines and TNI should be considered
to distinguish CV associated with CRS from cardiac
involvement. There is a need to accumulate more number of
clinical cases of cardiac involvement to rapidly identify authentic
causes of atypical clinical symptoms. We hope that our
experience deepens the understanding of clinicians regarding
cardiac involvement in patients with B-BLB/B-ALL who have
undergone allo-HSCT and CAR T-cell therapy. Data availability statement CAR T-cell therapy, six patients died from CV-related causes, six
had decompensated heart failure, five had a new arrhythmia, and
some patients showed decreased LVEF after CAR T treatment. A
high grade of CRS (grade ≥2) can increase the incidence of CV
events, and an elevated troponin level has been reported as a
common clinical event associated with CRS in patients receiving
CAR T-cell therapy, which has an evident relationship with CV
events (26). Our patient showed abnormal elevations in cTNI
and myocardial enzyme levels on the first day of hospitalization,
which needed to be differentiated from myocarditis and acute
coronary syndrome. However, we surprisingly found that cTNI
began to decrease with the administration of tocilizumab, so we
believed that the initial elevation of cTNI in this patient was
mostly due to CRS associated with CV events rather than
leukemia infiltration-induced myocardial injury. Further
studies on the mechanism of TNI and CRS are needed. The original contributions presented in the study are
included in the article/supplementary material. Further
inquiries can be directed to the corresponding authors. Author contributions YL, YC, and XC were involved in the care of the patient. YL
obtained the clinical data and wrote the manuscript. YC, RZ,
WZ, QM, AP, DY, YH, and JW reviewed the data. EJ, MH, and
SF supervised the entire study. All authors contributed to the
article and approved the submitted version. Conflict of interest The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could
be construed as a potential conflict of interest. Ethics statement Written informed consent was obtained from the individual
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cardiac tumors by (18)F-fdg Pet/Ct imaging: Histological correlation and clinical
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j.jacc.2021.08.044 Frontiers in Immunology frontiersin.org 08
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Species Coexistence in Nitrifying Chemostats: A Model of Microbial Interactions
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Processes
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cc-by
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Species coexistence in nitrifying chemostats: a model of
microbial interactions Maxime Dumont, Jean-Jacques Godon, Jérôme Harmand To cite this version: Maxime Dumont,
Jean-Jacques Godon,
Jérôme Harmand. Species coexistence in nitrifying
chemostats: a model of microbial interactions. Processes, 2016, 4 (51), pp.1-17. 10.3390/pr4040051. hal-01602934 Species Coexistence in Nitrifying Chemostats:
A Model of Microbial Interactions
Maxime Dumont, Jean-Jacques Godon and Jérôme Harmand *
INRA, UR50, Laboratoire de Biotechnologie de l’Environnement, Narbonne F-11100, France;
maxime.dumont@supagro.inra.fr (M.D.); godon@inra.fr (J.-J.G.)
* Correspondence: jerome.harmand@inra.fr; Tel.: +33-468-425-159 INRA, UR50, Laboratoire de Biotechnologie de l’Environnement, Narbonne F-11100, France;
maxime.dumont@supagro.inra.fr (M.D.); godon@inra.fr (J.-J.G.)
* Correspondence: jerome.harmand@inra.fr; Tel.: +33-468-425-159 Received: 26 September 2016; Accepted: 7 December 2016; Published: 14 December 2016 Abstract: In a previous study, the two nitrifying functions (ammonia oxidizing bacteria (AOB) or
nitrite-oxidizing bacteria (NOB)) of a nitrification reactor—operated continuously over 525 days
with varying inputs—were assigned using a mathematical modeling approach together with the
monitoring of bacterial phylotypes. Based on these theoretical identifications, we develop here a
chemostat model that does not explicitly include only the resources’ dynamics (different forms of
soluble nitrogen) but also explicitly takes into account microbial inter- and intra-species interactions
for the four dominant phylotypes detected in the chemostat. A comparison of the models obtained
with and without interactions has shown that such interactions permit the coexistence of two
competing ammonium-oxidizing bacteria and two competing nitrite-oxidizing bacteria in competition
for ammonium and nitrite, respectively. These interactions are analyzed and discussed. Keywords: competitive exclusion principle; microbial interactions; nitrifying bacteria; coexistence;
chemostat models; ecosystem functions processes processes HAL Id: hal-01602934
https://hal.science/hal-01602934v1
Submitted on 27 May 2020 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
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entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers. www.mdpi.com/journal/processes Processes 2016, 4, 51; doi:10.3390/pr4040051 1. Introduction The well-known “competitive exclusion” principle states that, at equilibrium, the number of
coexisting competing species cannot exceed the number of growth-limiting resources available to
them [1,2]. However, in natural systems, the number of coexisting species often exceeds the number
of limiting resources [3]. This phenomenon is often referred to as the “paradox of the plankton” [4]. Theoretical and experimental studies of a chemostat with time-invariant operating conditions have
shown that two microbial populations in competition for a single substrate cannot coexist: the
slower-growing species in the given operating conditions will be washed out [5]. In such a case,
coexistence is predicted theoretically for discrete values of the chemostat dilution rate only when the
curves of the specific growth rate, as a function of limiting nutrient concentration, cross. The dilution
rate must have exactly the value at which the specific growth rates of the two populations in the
chemostat are equal. However, this type of coexistence is structurally unstable and cannot be realized
in practice because of random fluctuations in the chemostat dilution rate and in the physico-chemical
parameters. To bridge the gap between mathematical theory and real life, a variety of mathematical
models have been developed over recent decades. In all such models, assumptions about the idealized
chemostat have been modified (cf. for instance [6–10]). Some studies have relied on non-equilibrium
conditions to promote species diversity by preventing competitive equilibrium. One such example
is the variability in resource supply ratios: when nutrients were supplied to the chemostat in pulses,
oscillations in the abundance of species prevented the establishment of competitive equilibrium
and permitted the coexistence of a greater number of species than the number of growth-limiting
resources [11,12]. In the same way, some studies have explained coexistence in chemostats by the
heterogeneity of the medium or by variations in solid retention times [8] while others have considered Processes 2016, 4, 51; doi:10.3390/pr4040051 www.mdpi.com/journal/processes 2 of 17 Processes 2016, 4, 51 that more than one single resource is available [13]. However, only very few studies related to
the chemostat have pointed out that microbial interactions other than competition can also lead to
steady-state coexistence. When such a situation was considered, only direct interactions based on the
approach called Generalized Lotka–Volterra equations were used and no consideration was given to
resources (cf., for instance [14] for a recent study). 1. Introduction In particular, for Lotka–Volterra models, it has been
established that coexistence is possible when intra-specific competition is greater than inter-specific
competition. The ecological mechanism behind this result is that the self-limiting growth of the most
competitive species leaves some resource available for other less competitive species. While some
progress has been made towards integrating positive interactions into contemporary theory [15–17],
competition and predation still dominate ecological thinking about interspecific interaction [18–20]. The fact is that, to allow coexistence in mass-balance models, it is either necessary (i) to consider
identical kinetics parameters for all species (which is actually what we could call a “singular case” and
which would have, as a consequence, the observation of exactly the same dynamic behavior for all
species) or (ii) to add direct interaction terms between species concentrations as in the Lotka–Volterra
model, in addition to the dependence with respect to resource concentrations. Recently, interest in the study of species interactions has been renewed regarding the modern
high-throughput molecular techniques available: together with modeling, they appear as a very
promising way of investigating how ecosystems function. Available models are based on network
theory and statistical data analysis [21]. However, these proposed models usually do not consider
resources and only describe, statically, species interactions within the ecosystem of interest. Roughly speaking, all the available models could be classified as either Biodiversity-Equivalent
or Functionally-Equivalent. Biodiversity-Equivalent Models (BEM) aim to provide models able to
explain/predict/simulate biodiversity, typically as species interacting together within a microbial
ecosystem; while Functionally-Equivalent Models (FEM) are designed to predict function performances
of an ecosystem. The classic chemostat model is typically a FEM while the neutral model could be
classified as a BEM. Very few microbial-ecosystem models available in the literature have been designed
to integrate both diversity and functional considerations. In [22–24], such models were proposed for
anaerobic digestion. However, both hypothesize that the different biomasses are only in competition
for the substrate and do not include terms involving direct interactions: thus, unless the growth
functions are identical, the competitive exclusion principle applies and only one species will survive if
the system is simulated for a long enough period of time [25]. In addition, like other studies published
in the fields of ecology or mathematics, these studies remain essentially theoretical and were not
confronted to data (cf., for instance [26,27]). 1. Introduction The aim of the model proposed in the present paper is to include both diversity and functional
considerations and to validate such a model on data obtained within real experiments. More specifically,
we present here a modification of the classic model of a chemostat. It is based on Lotka–Voltera
equations and takes into account biotic interactions which can occur between species in a microbial
community (i.e., which represent inter- and intra-species interactions). Not only does this model
integrate states of more than one species per function (number of biological reactions) but it also
integrates the dynamics of available resources. This model was confronted to data obtained from a
nitrifying chemostat operated for two years under time-varying inputs. Notice, however, that for
technical reasons related to the parameter identification step, only the signs and the order of magnitude
of interaction terms between species could be obtained and not their precise values (we will come back
in detail on this important point in Section 2.5). Nitrification is an aerobic two-step microbial process (thus involving only two “functions”) in
which ammonium is oxidized to nitrate by two distinct groups of chemolithoautotrophic bacteria. Aerobic ammonium-oxidizing bacteria (AOB) first oxidize ammonium to nitrite and then aerobic
nitrite-oxidizing bacteria (NOB) oxidize the nitrite to nitrate [28,29]. During the two year experiments
used in the present study, both functional measurements (i.e., concentrations in ammonium, nitrite and
nitrate concentrations) and population measurements (i.e., total biomass concentration and relative 3 of 17 Processes 2016, 4, 51 abundances) of the bacteria present in the chemostat were made by Single Strand Conformation
Polymorphism. In a previous work, using an original mathematical approach, a functional community
assignation (i.e., in AOB or NOB community) was performed for each phylotype detected in the
chemostat [30,31]. In the present work, a microbial interaction model is optimized on the kinetics of
the two most abundant phylotypes of each functional community. In this way, we obtain a virtual
web of interactions linking four different species present in the nitrifying community. The results
obtained in this way show that the web of interactions can prevent competitive exclusion and can
explain the coexistence of the phylotypes as observed through the available measurements realized on
the nitrifying chemostat as predicted in [26,27]. 2.1. Nitrifying Chemostat Conditions The experimental set-up operated over two years consisted of two continuously-mixed 6.5 L
(working volume) all-glass chemostats inoculated beforehand with activated sludge from the municipal
sewage treatment plant of Coursan (Aude, France). The air flow-rate was maximum in order to
ensure good fluidization and provide enough oxygen which was never limiting for the nitrification
process; the pH was measured and maintained at around 7 by the automatic addition of an alkaline
solution. Both chemostats were fed with a synthetic mineral medium composed of ammonium
sulfate (its concentration varying from 0.5 to 2 g·L−1) as the nitrogen source, complemented with a
mineral solution. 2.2. Microbial Community Measurements During the experiments which lasted 525 days, two kinds of population measurements were made:
the first was the determination of the total biomass present in the chemostats; the second consisted
of the determination by Single Strand Conformation Polymorphism (SSCP) of the abundances of the
different species contained in the total biomass. Except for some well-identified periods, the experiment
was run in duplicate and both chemostats behaved similarly. For this reason, in the present study we
concentrate on the results obtained from only one device (the chemostat denoted as “chemostat B”
in [31]). The main results of this monitoring—on average over the 525 days of experiments—were
(i) 40% of the total biomass was represented by the most abundant AOB; (ii) the most abundant NOB
represented less than 10% of the total biomass; (iii) the two most abundant AOB and the two most
abundant NOB represented more than 55% of the total biomass. 2.3. Model Development The model developed in this study is based on mass balance equations for a conventional,
completely-mixed chemostat initiated from the pioneering work of Monod and of Novick and Szilard
(cf. [32,33]). It is described by the following set of differential equations: (
. X = (µ(S(t)) −D(t))X(t)
. S = (Sin(t) −S(t))D(t) −1
Yµ(S(t))X(t)
(1) (1) where the dot above a letter stands for the time-derivative, S and X represent the concentrations of
nutrient (or substrate) and biomass, respectively. Sin denotes the concentration of the nutrient in the
input flow, with dilution rate D. The function µ(S(t)) is the growth rate of the population and the yield
factor is Y. Thereafter, to simplify the notations, the time-dependence of variables is often omitted. Processes 2016, 4, 51 Processes 2016, 4, 51 4 of 17 This standard dynamic model of a chemostat can be extended easily to a 2-step nitrifying
bioprocess as follows:
. XA = (µA(S1) −D)XA
. XB = (µB(S2) −D)XB
. S1 = (Sin −S1)D −
1
YA µA(S1)XA
. S2 = −S2D +
1
YA µA(S1)XA −1
YB µB(S2)XB
. S3 = −S3D + 1
YB µB(S2)XB
,
(2) (2)
. S1 = (Sin −S1)D −
1
YA µA(S1)XA
. S2 = −S2D +
1
YA µA(S1)XA −1
YB µB(S2)XB
. S3 = −S3D + 1
YB µB(S2)XB
,
(2) where XA and XB represent the concentrations of AOB and NOB respectively, while µA(S1) and µB(S2)
are the growth rates of XA and XB, respectively. As suggested in [31], they are supposed to be Monod
functions and are thus written as µA(S1) = µmax1
S1
S1+Ks1 and µB(S2) = µmax2
S2
S2+Ks2 . where XA and XB represent the concentrations of AOB and NOB respectively, while µA(S1) and µB(S2)
are the growth rates of XA and XB, respectively. As suggested in [31], they are supposed to be Monod
functions and are thus written as µA(S1) = µmax1
S1
S1+Ks1 and µB(S2) = µmax2
S2
S2+Ks2 . S1+Ks1
S2+Ks2
From this point onwards, this model will be referred to as “model #1”. 2.3. Model Development This nitrifying chemostat
model can then be modified as follows to take into account microbial interactions:
. Xi
i=1..nA+nB
=
"
µi(.)
1 +
j=nA+nB
∑
j=1
aijXj
! −D
#
Xi
. S1 = (Sin −S1)D −
1
YA
i=nA
∑
i=1
"
µi(S1)
1 +
j=nA+nB
∑
j=1
aijXj
! Xi
#
. S2 = −S2D +
1
YA
i=nA
∑
i=1
"
µi(S1)
1 +
j=nA+nB
∑
j=1
aijXj
! Xi
#
−1
YB
i=nA+nB
∑
i=nA+1
"
µi(S2)
1 +
j=nA+nB
∑
j=1
aijXj
! Xi
#
. S3 = −S3D + 1
YB
i=nA+nB
∑
i=nA+1
"
µi(S2)
1 +
j=nA+nB
∑
j=1
aijXj
! Xi
#
,
(3) (3) where aij represents the influence of the species j on the growth rate of species i. This influence can be
positive, negative or nil according to the sign and the value of aij. From this point onwards, this last model, including intra- and interspecific interaction terms,
will be referred to as “model #2”. This interaction coefficient structure (aij) corresponds to that of
the well-known Lotka–Volterra model which is a widely used model in general ecology. Our model
differs from the classic Lotka–Volterra model in that it is coupled to a mass-balance model (the classic
chemostat model) which includes resource dynamics. Because linear systems are easier to identify,
using a linear “species by species” relationship allows a simpler way of predicting species interactions. However, in doing so, the influence of resource density is neglected. An example of the use of such an
interaction coefficient structure to describe microbial interactions within a cheese microbial community
is given in [34]. It should also be noted that, for a given species j, the interaction terms are assumed
to be constant. We are aware that this assumption may be questionable from a purely biological
point of view. However, as explained in the next section, only the sign of interactions will finally be
characterized. Said differently, we rather characterize the distributions in which these parameters live,
instead of their precise values. 2.4. Identification of the Parameters of Microbial Interaction 2.4. Identification of the Parameters of Microbial Interaction The parameters of model #1, optimized to match experimental data in [31] are recalled in Table 1 The parameters of model #1, optimized to match experimental data in [31] are recalled in Table 1 Table 1. Parameters of model #1 (adapted from [31]). Table 1. Parameters of model #1 (adapted from [31]). µmax1 (1/day)
Ks1 (mg/L)
YA
µmax2 (1/day)
Ks2 (mg/L)
YB
0.81
0.17
0.26
0.26
0.16
0.01 The identification of microbial interactions was done with the help of the model #2 described by
Equation (3). To do so, we have kept only the two major species of each group: recall the four most
abundant phylotypes (two AOBs and two NOBs) represented about 55% of total biomass. In order to
ensure a viable compromise between the complexity of the model (notably the number of parameters to 5 of 17 Processes 2016, 4, 51 be identified) and the capability of optimization algorithms to converge within a reasonable computing
time, only two phylotypes in competition for one of the two substrates S1 or S2, were studied for each
of the two nitrifying functions, thus considering only four species in total. Considering the model described by Equation (3) with nA = nB = 2, there still remains a large
number of parameters to be identified (interaction parameters between the four considered species,
that is to say 16 parameters, plus the degrees of freedom of the kinetics, representing eight additional
parameters, thus a total of 24 parameters). To minimize this number, we adopt, in the present
study, a kind of “neutral” hypothesis in considering that within a function, all the species exhibit
identical kinetics and that the only difference in growth was due to their interaction. This assumption
can be justified taking into account the following arguments. Without interactions, considering the
same growth rates for either AOBs or NOBs, all species would exhibit exactly the same dynamics:
their coexistence would be “guaranteed” (of course assuming that initial biomass concentrations are
nonzero). However, it is not what is observed in practice (cf. the data we use from [31]: we will
come back on these data later on but it will be clearly shown that the dynamics of AOB1 and AOB2,
and of NOB1 and NOB2, are significantly different). 2.4. Identification of the Parameters of Microbial Interaction Now, with different growth rates and again
no interactions, the competitive exclusion principle applies and coexistence is simply not possible. Again, it is actually not what is observed in practice. In other terms, the kinetics associated with each
species—the rate at which species i consumes its main limiting resource— should be considered to be
modulated by interactions. Referring to model #2 and assuming we assign i = 1 and 2 for AOB1 and AOB2, respectively, and
i = 3 and 4 for NOB1 and NOB2, respectively, we have: (4) µ1(S1) = µ2(S1) and µ3(S2) = µ4(S2)
(4) Thus, instead of 24, we are now left with 20 parameters needing identification. Using model #1 as
a “provider” of data (by simulation) for model #2, the identification of the interaction parameters of
model #2 can now be framed in terms of an optimization problem, as follows: find the parameters
of µi|i=1..4 under the constraints (4) and the interaction parameters aij such that the outputs of model
#2 match the simulated outputs of model #1 (S1, S2, S3 and XT = XA + XB = AOB1 + AOB2 + NOB1 +
NOB2). This optimization problem can be reformulated as follows: find the parameters of µi|i=1..4
under the constraints (4) and the interaction parameters aij in the model #2 such that: (µA(S1(t)) −D(t))XA(t)|model#1
=
i=nA
∑
i=1
"
µi(S1(t))
1 +
j=nA+nB
∑
j=1
aijXj(t)
! Xi(t)
#
−D(t)(X1(t) + X2(t))|model#2
(5) (5) and (µB(S2(t)) −D(t))XB(t)|model#1
=
i=nA+nB
∑
i=nA+1
"
µi(S2(t))
1 +
j=nA+nB
∑
j=1
aijXj(t)
! Xi(t)
#
−D(t)(X3(t) + X4(t))|model#2
(6) (6) To insist on the fact that these constraints must hold for all t, we included the time-dependance
of variables. For this problem, we used the Sum of Square Residuals (SSR) criterion consisting of
the sum of the squared difference of the terms on the left and the terms on the right in the previous
expressions—(5) and (6)—over the considered 525 days of experiments. To solve this problem, we adopted a Monte-Carlo-like approach (cf. Appendix A): 6 of 17 Processes 2016, 4, 51 •
First, we randomly ran 2000 optimizations (using more did not improve the results (i.e., did
not enable us to further decrease the final confidence intervals computed for each identified
parameter) with different initial conditions for the parameters to be estimated (all unknown
parameters in the right-hand terms of the above equations). 2.4. Identification of the Parameters of Microbial Interaction It means that, given the structure of the model
considered, a unique set of parameters cannot be found from the available data. In other words,
there exist “hidden relationships” between parameters—possibly nonlinear—which prevent the
optimization algorithm from converging towards a unique global minimum and, consequently,
from delivering a unique set of optimized parameters (cf. for instance [35] or [36] for more
information about this concept); (ii) in the results, either the parameters are centered around
0 with a large standard deviation (indicating either no interaction between the corresponding
species or an undetermined interaction) or the signs of the identified interactions are always the
same whatever the sets of optimized parameters considered and, thus, the proposed statistical
analysis of the results to obtain qualitative insights about microbial interactions in the ecosystem
is considered without considering precise parameter values. In the sequel, this procedure will be
referred to as a “Monte-Carlo-like” approach; •
Finally, following the procedure described above, 520 sets of optimized parameters over the 2000 runs
were kept and analyzed. The results are presented and discussed in the following sections. •
Finally, following the procedure described above, 520 sets of optimized parameters over the 2000 runs
were kept and analyzed. The results are presented and discussed in the following sections. 2.4. Identification of the Parameters of Microbial Interaction In other terms, we identified all the
parameters with different initial conditions 2000 times; •
In a second step, we have proceeded to a statistical analysis of the results, keeping only the
best sets of optimized parameters that are the sets for which the SSR was, at most, 5% greater
than the best solution over the 2000 optimizations. Instead of keeping only the best one, we
proceeded in this way for two reasons: (i) The problem to be solved is complex, notably because
the model is nonlinear and the number of unknowns is high. Thus, we did not necessarily obtain
the same parameter values for each of the 2000 optimizations. In other words, the problem we
consider here is said to be “non-identifiable”. It means that, given the structure of the model
considered, a unique set of parameters cannot be found from the available data. In other words,
there exist “hidden relationships” between parameters—possibly nonlinear—which prevent the
optimization algorithm from converging towards a unique global minimum and, consequently,
from delivering a unique set of optimized parameters (cf. for instance [35] or [36] for more
information about this concept); (ii) in the results, either the parameters are centered around
0 with a large standard deviation (indicating either no interaction between the corresponding
species or an undetermined interaction) or the signs of the identified interactions are always the
same whatever the sets of optimized parameters considered and, thus, the proposed statistical
analysis of the results to obtain qualitative insights about microbial interactions in the ecosystem
is considered without considering precise parameter values. In the sequel, this procedure will be
referred to as a “Monte-Carlo-like” approach; •
In a second step, we have proceeded to a statistical analysis of the results, keeping only the
best sets of optimized parameters that are the sets for which the SSR was, at most, 5% greater
than the best solution over the 2000 optimizations. Instead of keeping only the best one, we
proceeded in this way for two reasons: (i) The problem to be solved is complex, notably because
the model is nonlinear and the number of unknowns is high. Thus, we did not necessarily obtain
the same parameter values for each of the 2000 optimizations. In other words, the problem we
consider here is said to be “non-identifiable”. 2.5. Remarks •
An important question that may arise is the following: Why are outputs of the model #1 used
in the optimization problem (in the evaluation of the left-hand terms of (5) and (6)) rather than
experimental data available in [31]? The essential reason is that experimental data are corrupted
by noise. It is well known in system identification that, by definition, noise is not informative. Since the problem posed here is very sensitive to data (recall, in particular, that the problem
is non-identifiable), it is better to use noise-free data [35,36]. Here, completely noise-free data
generated with the simulation of model #1 (itself optimized with respect to real data in Dumont’s
work, cf. [31]) were used instead of using real data. •
As noted before, the sum of the four species considered within model #2 represented on average
about 55% of the total biomass in the experiments published in [31]. For practical purposes,
before proceeding to parameter identification, the relative abundances of these four species
were re-normalized in such a way that their sum equaled the total biomass as measured in the
experiments. In other words, considering that model #2 (which describes the dynamics of four
species) is equivalent to saying that the two biological reactions involved are now only attributable
to these four species and that the remaining detected species (41 −4 = 37 in the data considered
over the 525 days of experiments) are simply ignored. •
Finally, another question that may be posed is related to the parameter identification results. One may ask whether a solution to the optimization problem should be to fix all interaction
parameters to zero or not. In fact, this solution cannot give a better fit than any other because there
is no reason to have XA = X1 + X2 and XB = X3 + X4. In other terms, individual concentrations of
the four AOBs and NOB species are taken explicitly into account in the criterion used to fit model
#2 parameters while only the total biomass concentration was considered in the optimization
criterion to optimize model #1 parameters. Since the criterions are different, model parameters •
Finally, another question that may be posed is related to the parameter identification results. One may ask whether a solution to the optimization problem should be to fix all interaction
parameters to zero or not. 3. Results
paramet
Equatio 3.1. Coexistence of 2 + 2 Species on 1 + 1 Growth-Limiting Resources by Microbial Interaction
q
( )
( )
A schematic representation of the procedure followed in this study is pre The distribution of the parameter values (interactions and kinetics parameters) over the
520 optimized parameter sets are plotted in Figure 1a–e. As shown in these figures, most parameters
are randomly distributed around mean values following Gaussian-like distributions while others
present bimodal distributions. In addition, even if their precise values are not informative (for the
identifiability reasons we mentioned above), it should be noted that the intervals in which the mean
values of the kinetics parameters (cf. distributions in Figure 1e and mean values reported in Table 2)
live appear to be in accordance with those reported in the literature (cf. [37]). p
p
y
p
ppendix. . Results
.1. Coexistence of 2 + 2 Species on 1 + 1 Growth‐Limiting Resources by Microbial Interaction
The distribution of the parameter values (interactions and kinetics parameters) over the 52
ptimized parameter sets are plotted in Figure 1a–e As shown in these figures most parameters ar Table 2. Kinetics parameters of model #2. µmax1 (1/day)
Ks1 (mg/L)
µmax2 (1/day)
Ks2 (mg/L)
0.828
0.147
0.18
0.026
p
p
g
g
p
domly distributed around mean values following Gaussian‐like distributions while o
sent bimodal distributions. In addition, even if their precise values are not informative (fo
ntifiability reasons we mentioned above), it should be noted that the intervals in which the m
ues of the kinetics parameters (cf. distributions in Figure 1e and mean values reported in Tab
appear to be in accordance with those reported in the literature (cf. [37]). (a)
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0.5
0
100
200
300
400
500
600
Frequency
a11
-10
-5
0
5
10
0
50
100
150
200
250
300
Frequency
a12
-2.5
-2
-1.5
-1
-0.5
0
0.5
1
0
50
100
150
200
250
300
350
400
450
Frequency
a13
2
3
4
5
6
7
0
50
100
150
200
250
300
350
400
450
Frequency
a14
Figure 1. Cont. Table 2. Kinetics parameters of model #2. 2.5. Remarks In fact, this solution cannot give a better fit than any other because there
is no reason to have XA = X1 + X2 and XB = X3 + X4. In other terms, individual concentrations of
the four AOBs and NOB species are taken explicitly into account in the criterion used to fit model
#2 parameters while only the total biomass concentration was considered in the optimization
criterion to optimize model #1 parameters. Since the criterions are different, model parameters 7 of 17
n result
teractio 7 of 17
n result
teractio Processes 2016, 4, 51
Finally, anot
One may ask (i.e., interaction terms) are tuned so that the differences between the two sides of Equations (5)
and (6) are minimized. p
g
y
there is no reason to have XA = X1 + X2 and XB = X3 + X4. In other terms, individual concentration
of the four AOBs and NOB species are taken explicitly into account in the criterion used to f
d l #2
hil
l
h
l bi
i
id
d i
h (i.e., interaction terms) are tuned so that the differences between the two sides of Equations (5)
and (6) are minimized. there is no reason to have XA = X1 + X2 and XB = X3 + X4. In other terms, individual concentration
of the four AOBs and NOB species are taken explicitly into account in the criterion used to f
d l
h l
l
h
l b
d
d
h A schematic representation of the procedure followed in this study is presented in the appendix. Results
model #2 parameters while only the total biomass concentration was considered in th
optimization criterion to optimize model #1 parameters. Since the criterions are different, mode
parameters (i.e., interaction terms) are tuned so that the differences between the two sides o 3. Results
paramet
Equatio 9 of 17
9 of 17 Processes 2016, 4, 51
Processes 2016, 4, 51 Processes 2016, 4, 51
9 of 1
(d)
(e)
-0.685
-0.68
-0.675
-0.67
-0.665
-0.66
-0.655
0
50
100
150
200
250
300
Frequency
a41
-0.36
-0.355
-0.35
-0.345
-0.34
-0.335
-0.33
0
50
100
150
200
250
300
350
Frequency
a42
-10
-5
0
5
10
0
50
100
150
200
250
300
350
400
Frequency
a43
3.22
3.24
3.26
3.28
3.3
3.32
0
50
100
150
200
250
300
Frequency
a44
0.75
0.8
0.85
0.9
0.95
1
1.05
1.1
0
100
200
300
400
500
600
Frequency
max1
0.16
0.18
0.2
0.22
0.24
0.26
0.28
0.3
0
100
200
300
400
500
600
Frequency
max2
0.146
0.1465
0.147
0.1475
0.148
0.1485
0.149
0
50
100
150
200
250
300
350
Frequency
Ks1
0.02
0.022
0.024
0.026
0.028
0.03
0.032
0
50
100
150
200
250
300
350
Frequency
Ks2
Figure 1. Distributions of the optimized interaction parameters for model #2 after Monte-Carlo-like
optimization: (a) a11 to a14; (b) a21 to a24; (c) a31 to a34; (d) a41 to a44 and for the two ammonia oxidizing
bacteria (AOB) and nitrite-oxidizing bacteria (NOB) of model #2 (e) µmax1, µmax2, Ks1 and Ks2. 3. Results
paramet
Equatio mean values following Gaussian‐l
I
dditi
if th i
i
l -0.2
-0.1
0
0.1
0.2
0.3
0.4
0.5
0
100
200
300
400
500
600
Frequency
a11 -10
-5
0
5
10
0
50
100
150
200
250
300
Frequency
a12 ( )
-2.5
-2
-1.5
-1
-0.5
0
0.5
1
0
50
100
150
200
250
300
350
400
450
Frequency
a13
2
3
4
5
6
7
0
50
100
150
200
250
300
350
400
450
Frequency
a14 (a) Figure 1. Cont. 8 of 17
8 of 17 8 of 17
8 of 17 Processes 2016, 4, 51
Processes 2016, 4, 51 (b)
(c)
-10
-5
0
5
10
0
50
100
150
200
250
300
Frequency
a21
-0.2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0
50
100
150
200
250
300
350
400
450
Frequency
a22
-3
-2
-1
0
1
2
3
4
5
0
50
100
150
200
250
300
350
400
450
Frequency
a23
-10
-8
-6
-4
-2
0
2
0
50
100
150
200
250
300
350
400
450
Frequency
a24
0.43
0.435
0.44
0.445
0.45
0.455
0.46
0
50
100
150
200
250
Frequency
a31
0.08
0.09
0.1
0.11
0.12
0.13
0.14
0.15
0
50
100
150
200
250
300
Frequency
a32
-0.3
-0.25
-0.2
-0.15
-0.1
0
50
100
150
200
250
300
350
Frequency
a33
-10
-5
0
5
10
0
50
100
150
200
250
300
350
400
Frequency
a34
Figure 1. Cont. -0.2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0
50
100
150
200
250
300
350
400
450
Frequency
a22 -10
-5
0
5
10
0
50
100
150
200
250
300
Frequency
a21 -3
-2
-1
0
1
2
3
4
5
0
50
100
150
200
250
300
350
400
450
Frequency
a23 -10
-8
-6
-4
-2
0
2
0
50
100
150
200
250
300
350
400
450
Frequency
a24 (
0.43
0.435
0.44
0.445
0.45
0.455
0.46
0
50
100
150
200
250
Frequency
a31 b)
0.08
0.09
0.1
0.11
0.12
0.13
0.14
0.15
0
50
100
150
200
250
300
Frequency
a32 (b) -0.3
-0.25
-0.2
-0.15
-0.1
0
50
100
150
200
250
300
350
Frequency
a33
-10
-5
0
5
10
0
50
100
150
200
250
300
350
400
Frequency
a34 (c) Figure 1. Cont. Figure 1. Cont. 3. Results
paramet
Equatio -0.685
-0.68
-0.675
-0.67
-0.665
-0.66
-0.655
0
50
100
150
200
250
300
Frequency
a41
-0.36
-0.355
-0.35
-0.345
-0.34
-0.335
-0.33
0
50
100
150
200
250
300
350
Frequency
a42
-10
-5
0
5
10
0
50
100
150
200
250
300
350
400
Frequency
a43
3.22
3.24
3.26
3.28
3.3
3.32
0
50
100
150
200
250
300
Frequency
a44 -0.685
-0.68
-0.675
-0.67
-0.665
-0.66
-0.655
0
50
100
150
200
250
300
Frequency
a41 -0.36
-0.355
-0.35
-0.345
-0.34
-0.335
-0.33
0
50
100
150
200
250
300
350
Frequency
a42 -10
-5
0
5
10
0
50
100
150
200
250
300
350
400
Frequency
a43
3.22
3.24
3.26
3.28
3.3
3.32
0
50
100
150
200
250
300
Frequency
a44 -10
-5
0
5
10
0
50
100
150
200
250
300
350
400
Frequency
a43 3.22
3.24
3.26
3.28
3.3
3.32
0
50
100
150
200
250
300
Frequency
a44 d)
0.16
0.18
0.2
0.22
0.24
0.26
0.28
0.3
0
100
200
300
400
500
600
Frequency
max2 (d) (
0.75
0.8
0.85
0.9
0.95
1
1.05
1.1
0
100
200
300
400
500
600
Frequency
max1 (e)
0.146
0.1465
0.147
0.1475
0.148
0.1485
0.149
0
50
100
150
200
250
300
350
Frequency
Ks1
0.02
0.022
0.024
0.026
0.028
0.03
0.032
0
50
100
150
200
250
300
350
Frequency
Ks2 (e) Figure 1. Distributions of the optimized interaction parameters for model #2 after Monte-Carlo-like
optimization: (a) a11 to a14; (b) a21 to a24; (c) a31 to a34; (d) a41 to a44 and for the two ammonia oxidizing
bacteria (AOB) and nitrite-oxidizing bacteria (NOB) of model #2 (e) µmax1, µmax2, Ks1 and Ks2. Processes 2016, 4, 51
Tables 2 an
the kinetics para Processes 2016, 4, 51
Tables 2 an
the kinetics para Processes 2016, 4, 51
Tables 2 an
the kinetics para 10 of 17
her with Tables 2 and 3 present the mean values for the microbial interaction parameters together with the
kinetics parameters identified. Table 2. Kinetics parameters of model #2. μmax1 (1/day)
Ks1 (mg/L)
μmax2 (1/day)
Ks2 (mg/L) Table 3. Mean values of interaction parameters of model #2 (“B” stands for “bimodal distribution”,
the sign of the interaction being specified by B/+ or B/−). 0.828
0.147
0.18
0.026
Table 3. 3. Results
paramet
Equatio Mean values of interaction parameters of model #2 (“B” stands for “bimodal distribution”, a11
a12
a13
a14
a21
a22
a23
a24
−0.087
~0
−0.697
4.815
~0
0.476
0.995
−6.362
a31
a32
a33
a34
a41
a42
a43
a44
B/+
B/+
B/−
−1.225
B/−
−0.351
−0.837
B/+
the sign of the interaction being specified by B/+ or B/−). a11
a12
a13
a14
a21
a22
a23
a24
0.087
~0
−0.697
4.815
~0
0.476
0.995
−6.362
a31
a32
a33
a34
a41
a42
a43
a44
B/+
B/+
B/−
−1.225
B/−
−0.351
−0.837
B/+ Concerning its main outputs, the interaction model presents a very good fit with the simulated
data obtained from model #1 (cf. Figure 2). Concerning its main outputs, the interaction model presents a very good fit with the simulated
data obtained from model #1 (cf. Figure 2). Figure 2. Simulations of main outputs of models #1 and #2 in appropriate concentration units
(obtained with the mean values of the optimized parameters given in Table 1 for model#1 and Table
4 for model #2, the mean values over the 520 values were used as well for parameters with bimodal
distributions). XA, XB, S1 (a negative part for the y‐axis was considered for clarity), S2 and S3
concentrations from the top to the bottom, predictions of model #1 in red crosses, predictions of
model #2 in green where XA = X1 + X2 and XB = X3 + X4. Figure 2. Simulations of main outputs of models #1 and #2 in appropriate concentration units (obtained
with the mean values of the optimized parameters given in Table 1 for model#1 and Table 4 for model
#2, the mean values over the 520 values were used as well for parameters with bimodal distributions). XA, XB, S1 (a negative part for the y-axis was considered for clarity), S2 and S3 concentrations from
the top to the bottom, predictions of model #1 in red crosses, predictions of model #2 in green where
XA = X1 + X2 and XB = X3 + X4. Figure 2. Simulations of main outputs of models #1 and #2 in appropriate concentration units
(obtained with the mean values of the optimized parameters given in Table 1 for model#1 and Table
4 for model #2, the mean values over the 520 values were used as well for parameters with bimodal
distributions). 3. Results
paramet
Equatio 11 of 17
ameters
in Table Processes 2016, 4, 51
unique and one
among the 520 s Table 4. A particular set of optimized parameters taken randomly from the final optimized
parameter sets. Table 4. A particular set of optimized parameters taken randomly from the final optimized µmax1 (1/day)
Ks1 (mg/L)
µmax2 (1/day)
Ks2 (mg/L)
0.81
0.17
0.26
0.016
μmax1 (1/day)
Ks1 (mg/L)
μmax2 (1/day)
Ks2 (mg/L)
0.81
0.17
0.26
0.016 The predictions of model #2 using this specific set of parameters together with the experimental
data presented in [31], are plotted in Figure 3. Because of the uncertainty of measurements, it is clear
that only the qualitative behavior of these simulation results should be considered within the context
of the actual work. The predictions of model #2 using this specific set of parameters together with the experimental
data presented in [31], are plotted in Figure 3. Because of the uncertainty of measurements, it is clear
that only the qualitative behavior of these simulation results should be considered within the context
of the actual work. Figure 3. Simulations of model #2 (with the optimized set parameters given in Table 4) and
measurements of species abundances for AOB1, AOB2, NOB1 and NOB2 (which correspond to
peaks 38, 35, 5 and 9 from [31], respectively). Figure 3. Simulations of model #2 (with the optimized set parameters given in Table 4) and
measurements of species abundances for AOB1, AOB2, NOB1 and NOB2 (which correspond to peaks
38, 35, 5 and 9 from [31], respectively). Figure 3. Simulations of model #2 (with the optimized set parameters given in Table 4) and
measurements of species abundances for AOB1, AOB2, NOB1 and NOB2 (which correspond to
peaks 38, 35, 5 and 9 from [31], respectively). Figure 3. Simulations of model #2 (with the optimized set parameters given in Table 4) and
measurements of species abundances for AOB1, AOB2, NOB1 and NOB2 (which correspond to peaks
38, 35, 5 and 9 from [31], respectively). Whatever the species considered, it should be noted that the models with interactions always
better fitted data than the predictions of the same models in which interactions were fixed at 0. This
result can easily be understood given that a model with more tuning parameters than another
should be able to deliver better predictions. 3. Results
paramet
Equatio In addition, from an ecological point of view, when
microbial interactions are not considered (fixed to 0), the model does not allow the coexistence of 2 +
2 species on 1 + 1 growth‐limiting resources as predicted by the theory of the chemostat. Yet this
coexistence was observed in practice (cf. the measurements of species abundances reported in [31] or
in Figure 3 here above). The divergence between experimental data and estimates of the models
between t = 375 days and t = 430 days for the AOB2 can be explained by a washout of the chemostat
in reaction to the increase in the flow rate made on t = 337 days which was already mentioned in [31]. Whatever the species considered, it should be noted that the models with interactions always
better fitted data than the predictions of the same models in which interactions were fixed at 0. This
result can easily be understood given that a model with more tuning parameters than another should
be able to deliver better predictions. In addition, from an ecological point of view, when microbial
interactions are not considered (fixed to 0), the model does not allow the coexistence of 2 + 2 species on
1 + 1 growth-limiting resources as predicted by the theory of the chemostat. Yet this coexistence was
observed in practice (cf. the measurements of species abundances reported in [31] or in Figure 3 here
above). The divergence between experimental data and estimates of the models between t = 375 days
and t = 430 days for the AOB2 can be explained by a washout of the chemostat in reaction to the
increase in the flow rate made on t = 337 days which was already mentioned in [31]. 3. Results
paramet
Equatio XA, XB, S1 (a negative part for the y‐axis was considered for clarity), S2 and S3
concentrations from the top to the bottom, predictions of model #1 in red crosses, predictions of
model #2 in green where XA = X1 + X2 and XB = X3 + X4. Figure 2. Simulations of main outputs of models #1 and #2 in appropriate concentration units (obtained
with the mean values of the optimized parameters given in Table 1 for model#1 and Table 4 for model
#2, the mean values over the 520 values were used as well for parameters with bimodal distributions). XA, XB, S1 (a negative part for the y-axis was considered for clarity), S2 and S3 concentrations from
the top to the bottom, predictions of model #1 in red crosses, predictions of model #2 in green where
XA = X1 + X2 and XB = X3 + X4. As explained in the Materials and Methods section, we can compare the experimental data and
the predictions for the dynamics of AOB1, AOB2, NOB1 and NOB2 that can be simulated using
model #2. However, notice that the behavior over time of these species, e.g., the concentrations of
these species, may vary depending on the sets of interaction and kinetics parameters identified. Indeed, recall that the best 520 parameter sets have been kept for the statistical analysis of results. All
As explained in the Materials and Methods section, we can compare the experimental data and
the predictions for the dynamics of AOB1, AOB2, NOB1 and NOB2 that can be simulated using model
#2. However, notice that the behavior over time of these species, e.g., the concentrations of these
species, may vary depending on the sets of interaction and kinetics parameters identified. Indeed,
recall that the best 520 parameter sets have been kept for the statistical analysis of results. All permit a
good fit with the outputs of both models #1 and #2 (otherwise, they would not have been kept after the
parameter identification process) while predicting different individual behavior for the two AOB and
the two NOB species. In other words, the set of optimized parameters is not unique and one may only
compare simulations of model#2 using a particular set of parameters among the 520 sets that have
been kept for the statistical analysis. Such a specific set is given in Table 4. 3.2. Web of Microbial Interactions
3.2. Web of Microbial Interactions On the basis of the identified microbial interactions model, we can build a web of microbial
interactions linking the different phylotypes with their mean values as reported in Table 3. Over the network of 16 interactions contained in the model #2, six are positive and eight are
negative (cf. Figure 4). Only two almost-null interactions emerge. Concerning interactions between 12 of 17
ght are Processes 2016, 4, 51
interactions linki
O
h both AOB, neither competition nor facilitation was highlighted. However, strong intra-specific
facilitation appears for the second AOB with a22 = +0.47. These two ammonium-oxidizing phylotypes
exercise interactions of both facilitation and competition with both of the nitrite-oxidizing phylotypes. However, a stronger, mostly negative interaction (a24 = −6.36, a34 = −1.23 and a14 = +4.81) of all
bacteria with NOB2 is to be noted which seems to be compensated for by a strong intra-specific
relationship of NOB2 with itself (a44 = +3.26/Bimodal distribution). Concerning interactions
between the two nitrite-oxidizing phylotypes, NOB1 imposes a high negative interaction on NOB2
(a34 = −1.23) while NOB2 forces a moderately negative relationship on NOB1 (a43 = −0.84). A moderate
intra-specific competition appears for NOB1 (a33 = −0.18/Bimodal distribution) whereas a high
intra-specific facilitation appears for NOB2 (a44 = +3.26). Concerning the type of interactions which
maintain NOB in relation to AOB, a supplementary “balanced structure” appears. Indeed, if we
take into account only the signs of interaction, there are as many positive as negative interactions. Whereas NOB1 presents fairly positive interactions with AOBs (a31 = +0.44 and a32 = +0.11/Bimodal
distributions), NOB2 seems to interact rather negatively with AOBs (a41 = −0.67/Bimodal distribution
and a42 = −0.35). negative (cf. Figure 4). Only two almost‐null interactions emerge. Concerning interactions between
both AOB, neither competition nor facilitation was highlighted. However, strong intra‐specific
facilitation appears for the second AOB with a22 = +0.47. These two ammonium‐oxidizing phylotypes
exercise interactions of both facilitation and competition with both of the nitrite‐oxidizing
phylotypes. However, a stronger, mostly negative interaction (a24 = −6.36, a34 = −1.23 and a14 = +4.81)
of all bacteria with NOB2 is to be noted which seems to be compensated for by a strong intra‐specific
relationship of NOB2 with itself (a44 = +3.26/Bimodal distribution). Concerning interactions between
the two nitrite‐oxidizing phylotypes, NOB1 imposes a high negative interaction on NOB2 (a34 = −1.23)
while NOB2 forces a moderately negative relationship on NOB1 (a43 = −0.84). 3.2. Web of Microbial Interactions
3.2. Web of Microbial Interactions A moderate
intra‐specific competition appears for NOB1 (a33 = −0.18/Bimodal distribution) whereas a high
intra‐specific facilitation appears for NOB2 (a44 = +3.26). Concerning the type of interactions which
maintain NOB in relation to AOB, a supplementary “balanced structure” appears. Indeed, if we take
into account only the signs of interaction, there are as many positive as negative interactions. Whereas NOB1 presents fairly positive interactions with AOBs (a31 = +0.44 and a32 = +0.11/Bimodal
distributions), NOB2 seems to interact rather negatively with AOBs (a41 = −0.67/Bimodal distribution
and a42 = −0.35). The actual results underline positive interactions only from AOB1 to NOB2 and AOB2 to NOB1
(cf. Figure 4). However, in the same time, negative interactions were observed between AOB1/NOB1
and AOB2/NOB2. In addition, competition was not observed between AOBs, whereas there was
strong competition between NOBs. and a42 0.35). The actual results underline positive interactions only from AOB1 to NOB2 and AOB2 to NOB1
(cf. Figure 4). However, in the same time, negative interactions were observed between AOB1/NOB1
and AOB2/NOB2. In addition, competition was not observed between AOBs, whereas there was
strong competition between NOBs. Figure 4. Interaction web (based on the statistical analysis of the signs of interactions). +, − and 0
correspond to positive, negative and neutral interactions, respectively. The size of bacteria “oval
greys” and the thickness of lines indicate the trends of the bacterial abundances and the levels of
interaction (based on the mean values reported in Table 3), respectively. Figure 4. Interaction web (based on the statistical analysis of the signs of interactions). +, −and 0
correspond to positive, negative and neutral interactions, respectively. The size of bacteria “oval greys”
and the thickness of lines indicate the trends of the bacterial abundances and the levels of interaction
(based on the mean values reported in Table 3), respectively. Figure 4. Interaction web (based on the statistical analysis of the signs of interactions). +, − and 0
correspond to positive, negative and neutral interactions, respectively. The size of bacteria “oval
greys” and the thickness of lines indicate the trends of the bacterial abundances and the levels of
interaction (based on the mean values reported in Table 3), respectively. Figure 4. Interaction web (based on the statistical analysis of the signs of interactions). +, −and 0
correspond to positive, negative and neutral interactions, respectively. 3.2. Web of Microbial Interactions
3.2. Web of Microbial Interactions The size of bacteria “oval greys”
and the thickness of lines indicate the trends of the bacterial abundances and the levels of interaction
(based on the mean values reported in Table 3), respectively. 4. Discussion
4. Discussion Monitoring over two years the population and functioning of a nitrifying chemostat under
time‐varying environmental conditions highlighted the coexistence of numerous species in a culture
fed with ammonium as the nitrogen source. The data acquired indicate that, throughout the period
Monitoring over two years the population and functioning of a nitrifying chemostat under
time-varying environmental conditions highlighted the coexistence of numerous species in a culture
fed with ammonium as the nitrogen source. The data acquired indicate that, throughout the period of
the study, there occurred rapid, significant shifts in the structure of bacterial population. A comparison
with the experimental data of the two proposed models—with and without microbial interactions—has 13 of 17 Processes 2016, 4, 51 shown that microbial interactions lead to the coexistence of a greater number of species than predicted
by the classical chemostat model in which the competitive exclusion principle applies. In macrosystem
ecology, several models have been presented that represent intra- and inter-species interactions in
food webs (cf. [15]). The most frequently-used model is the multispecies Lotka–Volterra. For a given
species, it is based on a linear relationship between its growth rate parameters and the population of
each member of the community. For instance, with respect to real data, this model has been used to
describe microbial interactions within a cheese microbial community [34]. In the present study, we
combined this interaction coefficient structure (aij) with a classic model of a nitrifying chemostat. To the
best of our knowledge, it is one of the first models of microbial ecosystems which tries (i) to integrate
the dynamics of several interacting bacterial in the chemostat and (ii) tunes its parameters to match
data together with the modeling of functions (here, nitritation and nitratation). To date, the prime
focus of predator–prey models has been applied to models describing food webs in which non-trophic
interactions, such as competition, facilitation and biotic disturbance, have been largely ignored [15]. Because food web models focus—by their very nature—exclusively on trophic interactions, they
assume implicitly that predation is the most important process regulating community structure and
dynamics. Moreover, while models of complex food webs incorporate only competition among species,
they generally ignore any form of resource competition among the species (cf. [38,39]). 4. Discussion
4. Discussion This can be
explained by the fact that, in food webs, nutrients flow via trophic links and, for this reason, trophic
interactions have a fundamental character due to the principle of mass conservation. p
p
Concerning the “competitive exclusion principle” (i.e., the “paradox of the plankton”), a variety
of mathematical models have been proposed over recent decades, as underlined in the introduction. However, once again, intra- as well as inter-specific interactions have not been taken into consideration
and in all such models, the assumptions about an idealized chemostat have been modified to permit
coexistence [10]. Some studies have relied on non-equilibrium conditions to promote species diversity
by preventing competitive exclusion. Examples include variability in resource supply ratios. When
nutrients were supplied to the chemostat in pulses, oscillations in the abundance of species prevented
competitive equilibrium from occurring and led to the coexistence of a greater number of species
than the number of growth-limiting resources (cf. [11,12]). The only recent theoretical study that
does not contradict the competitive exclusion principle and, at the same time, permits what has
been called “practical coexistence” with neither variations in the environment nor any interaction
through species or foodweb is [25]. In this study, it is the diversity itself—which is considered to
be high, as observed in most microbial natural ecosystems—which creates the conditions under
which the system can allow species coexistence over an arbitrarily long period of time as long as
growth rate functions for each species are close enough. Their conclusions were that possibly we
observe ecosystems that are never close to an equilibrium. Some other studies explain coexistence in a
chemostat by variation in solid retention times [8]. Only a very small number of studies have taken
an interest in microbial interactions. In [10], Hebeler et al. explained coexistence in the chemostat
as a result of metabolic by-products. In their study, they found experimental evidence for a specific
metabolic property of Staphylococcus aureus which produced acetate (a by-product). In a mixed
culture, this can have an activating effect (as a secondary substrate) as well as an inhibiting effect
(by reducing pH) on the other species. In [12], it is shown, using a modeling study, that the number
of coexisting species may exceed the number of limiting resources when internal system feedbacks
induce oscillations and chaos. This occurs if the species displace each other in a cyclic fashion. 4. Discussion
4. Discussion However, this can be considered as a very rare phenomenon—for not saying singular—since it requires
a very precise parameterization of the parameters of community members [7]. Nevertheless, few
authors have considered interactions empirically and directly in microbial ecology, even though a long
history of experimental and theoretical ecology has elucidated how predation interacts with other
non-trophic processes—including interference competition, facilitation, disturbance, environmental
stress, productivity and recruitment—to regulate species distribution and abundance (cf. [40,41]). Finally, the microbial interaction model proposed here makes it possible to obtain a virtual web of Processes 2016, 4, 51 14 of 17 interactions linking the main species present in the ecosystem of interest. It appears that we have
underlined a “balanced interacting-structure model” in the sense that there are as many positive as
negative interactions in the virtual web. The experiments reported in [31] in the chemostat clearly show the coexistence of bacteria
performing the same function (41 phylotypes observed) for a long period (525 days). These results can
be compared to those presented in [42]. These authors considered a two-competitor/one prey model
and explained the coexistence of the two competitors by density-dependent mortality for one of the
two species. The interaction web also shows preferential “couples” or “pairs” of micro-organisms
(AOB1/NOB2 and AOB2/NOB1) that are not “interchangeable” (AOB1/NOB1 and AOB2/NOB2)
and which exhibit negative interactions. With a model built at a population level, it is obviously very
difficult to interpret biologically direct interaction terms aij. However, an example of a configuration in
which such interactions could develop is as follows. Consider—for some unknown reason—that AOB1
and NOB1, on the one hand, and AO2 and NOB2, on the other hand, develop specific interactions
(for instance in forming ”bi- specific” flocs). Because of their proximity, AOB1 and NOB1 (resp. AOB2
and NOB2) could develop specific biotic interactions while each pair would now be in competition
with the other. If such an explanation remains highly speculative, it is not inconceivable that such
interactions are developing in a complex natural ecosystem. There is no competition for NH4+ between AOBs. Unfortunately, some interactions, such as
NOB2/NOB2, as well as the low abundance of NOBs compared to AOBs, often underlined in the
literature, cannot be explained. In conclusion, we developed a new model of nitrifying chemostat to study the interactions
within two functional groups of four species: two AOBs and two NOBs. 4. Discussion
4. Discussion The network of virtual
interactions obtained can explain the coexistence of these 2 + 2 species on 1 + 1 growth-limiting
resources. Of the perspectives deriving from this work, one could design other more complete models
including multiple functional groups of organisms, such as nitrifying and denitrifying heterotrophs,
phosphorus-accumulating organisms, etc. In addition, this study focused on the effects of interactions
within a single trophic level (bacteria). Effects of the presence of higher trophic levels, for instance
predation by virus or protozoa, could also be considered. However, in all these cases, a number of
issues, including the identification of a large number of model parameters, need to be dealt with from
a theoretical viewpoint before such more complicated models can be developed. Acknowledgments: All experiments and Maxime Dumont’s PhD grant were financed by INRA. Author Contributions: All authors conceived and designed the experiments; Maxime Dumont performed
the experiments; all authors analyzed the data; Jérôme Harmand and Maxime Dumont designed models. Jérôme Harmand wrote the paper. Conflicts of Interest: The authors declare no conflict of interest. Procedure followed for the identification of the virtual interaction web. Procedure followed for the identification of the virtual interaction web. 15 of 17
15
f 17 Processes 2016, 4, 51 Figure A1. Identification procedure. Figure A1. Identification procedure. Figure A1. Identification procedure. Figure A1. Identification procedure. References
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(CC-BY) license (http://creativecommons.org/licenses/by/4.0/). © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
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(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
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Factors associated with medication adherence of hypertensive patients in the Philippines: a systematic review
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Clinical hypertension
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REVIEW Open Access Factors associated with medication
adherence of hypertensive patients in the
Philippines: a systematic review Margarita M. Gutierrez1,2*
and Rungpetch Sakulbumrungsil2 Abstract Background: Diseases of the heart and vascular system are the leading cause of mortality in the Philippines. Hypertension, the most important modifiable risk factor, has a prevalence rate of 28% and a control rate of 20%. Despite the proven efficacy of pharmacologic treatment, medication adherence is reported to be as low as 66%. While there are publications that reported factors that affect adherence in Filipinos, there are no existing research
that evaluated them systematically. This review is conducted to present and synthesize findings of published
literatures. Methods: Databases—PubMed, Scopus, Wiley Online library, Science Direct, JSTOR, Web of Science, SAGE journals,
and Cochrane—were used to search for articles published from 2000 to 2020 that studied medication adherence in
adult Filipino hypertensive population. Out of the initial 1514 articles, 15 articles met the criteria and were included
in the analysis. The evidence from the included studies was summarized and discussed in a narrative review using
the World Health Organization framework for adherence to long-term therapies as the framework. Result: The factors that were positively associated with adherence were health care system-related factors: good
patient-health provider relationship, accessibility of health services, use of specialty clinics and programs for
hypertension, and health insurance. The factors found to be negatively associated with adherence are (1) social
economic factors: younger age, single civil status, low educational attainment, and unemployment; (2) patient-
related factors: low in health literacy and awareness, knowledge on hypertension, attitude towards hypertension,
self-efficacy, and social support; (3) therapy-related factors: inconsistent drug regimen schedule, use of Thiazide and
complementary and alternative medicines; (4) condition-related factors: low illness perception, and absence of
comorbidities. Conclusions: Findings should be interpreted with caution because of methodological limitations. Despite this,
given that health systems related factors are modifiable, they can be the focus of interventions and future
researches to increase medication adherence. Clinicians may also want to screen their Filipino hypertensive patients
for factors that are associated to low adherence in order to provide a tailored advice. Longitudinal research studies
with heterogeneous samples of hypertensive Filipinos are imperative so that targeted interventions can be
developed for the population. Keywords: Hypertension, Medication adherence, Patient compliance, Pharmacy, Philippines * Correspondence: mmgutierrez2@up.edu.ph
1University of the Philippines Manila College of Pharmacy, Manila, Philippines
2Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok,
Thailand * Correspondence: mmgutierrez2@up.edu.ph
1University of the Philippines Manila College of Pharmacy, Manila, Philippines
2Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok,
Thailand Gutierrez and Sakulbumrungsil Clinical Hypertension (2021) 27:19
https://doi.org/10.1186/s40885-021-00176-0 Gutierrez and Sakulbumrungsil Clinical Hypertension (2021) 27:19
https://doi.org/10.1186/s40885-021-00176-0 (2021) 27:19 Gutierrez and Sakulbumrungsil Clinical Hypertension
https://doi.org/10.1186/s40885-021-00176-0 * Correspondence: mmgutierrez2@up.edu.ph
1University of the Philippines Manila College of Pharmacy, Manila, Philippines
2Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok,
Thailand © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,
which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give
appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if
changes were made. The images or other third party material in this article are included in the article's Creative Commons
licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons
licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain
permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the
data made available in this article, unless otherwise stated in a credit line to the data. Page 2 of 15 Gutierrez and Sakulbumrungsil Clinical Hypertension (2021) 27:19 Gutierrez and Sakulbumrungsil Clinical Hypertension (2021) 27:19 Data sources and search strategy The
search
strategy
was
run
on
eight
databases:
PubMed, Scopus, Wiley Online library, Science Direct,
JSTOR, Web of Science, SAGE journals, Cochrane li-
brary between the year 2000 to 2020. A facet analysis
broke the review question into its component parts
population (Filipino), intervention (for hypertension and
anti-hypertensive) and outcome (medication adherence). Index terms such as ‘hypertension’ and ‘medication ad-
herence’ were explored and all sub-headings were in-
cluded. The
resulting
search
term
is
((((((“patient
compliance”) OR “patient adherence”) OR “medication
compliance”) OR “medication adherence”)) AND ((Anti-
hypertensive) OR Hypertension)) AND ((Philippines)
OR Filipino). Subsequently, the reference list of included
studies were scrutinized to identify additional relevant
studies. Corresponding authors were searched and con-
tacted to include these literatures in the review as well. Adherence, as defined in chronic disorders context by
the World Health Organization (WHO) [8] is “the extent
to which a person’s behavior with respect to taking
medication, following a diet, and/or executing lifestyle
changes,
corresponds
with
agreed
recommendations
from a healthcare provider”. High adherence is defined
objectively as medication possession ratio of 80 to 100%. Historically, high adherence to hypertensive medications
is associated with higher blood pressure control [9]. In
2007, Philippine Heart Association-Council on Hyper-
tension Report on Survey of Hypertension showed treat-
ment rates for hypertension is at 65%, however, of those
receiving treatment the adherence rate is at 66% result-
ing to a hypertension control rates of only 20% [10]. This is consistent to the findings of de Guzman et al. [11] where they found out that the elderly Filipino pa-
tients have low adherence at only 41.54%. Background weaknesses in the implementation of these program it is
still worth exploring if there are other factor beyond
medicine access that contribute to medication adherence
[19]. g
Six of the ten leading cause of death in the Philippines
are non-communicable in etiology, the leading cause be-
ing cardiovascular diseases [1]. A systematic review by
Kearney et al. PM Kearney, M Whelton, K Reynolds, P
Muntner, PK Whelton and J He [2] found that hyperten-
sion is a the most important modifiable risk factor for
stroke and myocardial infarction that applies to both de-
veloped and developing countries. A prospective, multi-
staged, stratified nationwide survey on hypertension was
conducted in the Philippines found that the prevalence
of hypertension is 28% or approximately 29 million citi-
zens, and it is projected to increase over time [3–5]. Ac-
cording to Musini et al. VM Musini, AM Tejani, K
Bassett and JM Wright [6], treating healthy persons (60
years or older) with moderate to severe systolic and/or
diastolic hypertension with antihypertensive drug, re-
duces all-cause mortality and cardiovascular morbidity
and mortality. Despite the proven efficacy of antihyper-
tensive drugs in controlling blood pressure, patient ad-
herence in clinical practice is reported to be as low as 20
to 50% and therefore it can be presumed that many pa-
tients, experience difficulty in following long-term treat-
ment recommendations [7]. The purpose of this article was to critically review the
literature on factors associated with medication adher-
ence in hypertensive Filipinos. No such systematic re-
view appears to have been previously conducted. The
review may be used to recommend focus of future re-
searches and propose interventions to improve it in
hypertensive Filipino patients. Study selection
d
l Study samples included in the review were individuals
aged 18 years and above of Filipino ethnicity, with hyper-
tension and receiving antihypertensive treatment from
primary care, outpatient or community setting. The out-
comes considered in the review was medication adher-
ence that is measured using objective measures or
validated subjective (self-reported) instruments, summa-
rized in Table 1 [14, 16, 20–28]. Review articles, confer-
ence presentations and discussion papers were excluded. The process of selecting these studies are illustrated in
Fig. 1 flow diagram of the search strategy based on
PRISMA [29]. While it is easy to assume that perhaps medicine ac-
cess is the main barrier to adherence, there are findings
that suggest that even Filipino immigrants in developed
countries are also non adherent to hypertensive medica-
tion therapy, when compared to other Asian American
counterparts despite the availability of medications [12–
16]. Thus raising the question if there culturally or
uniquely Filipino characteristics or factors that contrib-
ute to phenomenon of non-adherence beyond barriers of
access. Moreover, the Philippine government in the last
10 years have medicine access programs to respond to
the persistently high out of pocket spending and barriers
to access to essential drugs [17–19]. While there are Data extraction and screening All retrieved articles were organized and screened using
a Microsoft Excel spread sheet and EndNote manage-
ment software. After removing duplicates, articles’ titles
and abstracts were assessed for eligibility. Then, the full-
text articles were screened and data of the eligible arti-
cles were extracted into a standardized data extraction
table. Extraction of the data was based on the following Gutierrez and Sakulbumrungsil Clinical Hypertension (2021) 27:19 Page 3 of 15 Table 1 Outcome measures for medication adherence
Code
Description
Subjective measures (self-reported adherence)
Hill-Bone high blood pressure
compliance scale [20]
HB-HCT [20]
This scale uses a 14-item Likert scale to measure three behavioral domains a) reduced-sodium
intake b) appointment keeping c) medication taking [20]. Cronbach’s alpha = 0.74 and 0.84. Self-structured questionnaires (ten
items) [21]
SSQ-10 [21]
Researcher created tool which was validated with 23 respondents who served as the pilot
study of this research [21]. Cronbach’s alpha value = 0.7. Morisky Medication Adherence
Scale [22]
MMAS-8 [22]
MMAS-8 is a series of eight binary questions, a “No” is one point. A score of 8 indicates high
adherence, 6–7 is medium, and < 6 is poor adherence [22]. Cronbach’s alpha = 0.83. 11 Items adapted MMAS-8 [23]
Adapted
MMAS-8 [23]
10- to 11-item questionnaire with scores ranging from 0 to 44. Adherent have scores, 0 to 21
and non-adherent scores, 22 to 44 [23]. Cronbach’s alpha = 0.932. Medical Outcomes Study Specific
Adherence Scale [24]
MAOSS [24]
Assesses of participant’s tendency to adhere to eight behaviors associated with hypertension
self-care that include patient’s ability to follow a salt and low fat or weight loss diet, take pre-
scribed medications, cut down or stop smoking, curtail or avoid alcohol, exercise regularly,
avoid stress, and use relaxation techniques for the past 4 weeks measured using a 6-point
Likert scale. The higher the mean score, the greater the adherence [24]. Cronbach’s alpha =
0.811. Binary adherence question
Binary [25]
Is the respondents taking the right medications at the right dosages at the right time,
answerable by yes or no [25]. Medication Adherence
Questionnaire [26]
MAQ [26]
A total score of 0–1 was defined as adherent while 2 or above was considered as non-
adherent [26]. Adherence Self-Report Question-
naire [27]
ASRQ [27]
A brief self-administered questionnaire measuring “timing adherence,” defined as taking medi-
cations at the correct dose and intervals. Has six items describing adherence to the timing of
medication intake. Data extraction and screening Adherence was defined as an ASRQ score of less than or equal to 2 [27]. Specificity 90.3% and sensitivity 14.6%. Objective measures
Proportion of days covered
PDC [14]
Days the patient was covered by at least one drug for each type of medication, based on the
prescription fill date and days of supply divided by the number of days of drug coverage and
multiplied by 100. Medication adherence was defined using the standard threshold of PCD
greater than 80% [14]. Data on filled medications including medication names, fills and days of
supply were obtained from pharmacy claims databases [14]. Medication Possession ratio
MPR [28]
Calculated by dividing the no of day supply dispensed by the no of days evaluated multiplied
by 100%. For the research they used the formula Possession ratio = days supplied for 1stRX/
(filldateof2ndRX-filldateof1stRX). Possession ratio of 0.8 or greater was considered adherent [16,
28]
HB-HCT: Hill-Bone High Blood Pressure Compliance Scale; SSQ-10: Self-structured questionnaires (ten items); MMAS-8: Morisky Medication Adherence Scale;
MAOSS: Medical Outcomes Study Specific Adherence Scale; MAQ: Medication Adherence Questionnaire; ASRQ: Adherence Self-Report Questionnaire; PDC:
Proportion of days covered; MPR: Medication Possession Ratio Table 1 Outcome measures for medication adherence
Code
Description Table 1 Outcome measures for medication adherence A total score of 0–1 was defined as adherent while 2 or above was considered as non-
adherent [26]. Days the patient was covered by at least one drug for each type of medication, based on the
prescription fill date and days of supply divided by the number of days of drug coverage and
multiplied by 100. Medication adherence was defined using the standard threshold of PCD
greater than 80% [14]. Data on filled medications including medication names, fills and days of
supply were obtained from pharmacy claims databases [14]. HB-HCT: Hill-Bone High Blood Pressure Compliance Scale; SSQ-10: Self-structured questionnaires (ten items); MMAS-8: Morisky Medication Adherence Scale;
MAOSS: Medical Outcomes Study Specific Adherence Scale; MAQ: Medication Adherence Questionnaire; ASRQ: Adherence Self-Report Questionnaire; PDC:
Proportion of days covered; MPR: Medication Possession Ratio Descriptive analysis of studies examining similar vari-
ables and any association observed were considered to
offer a simple indication of the level of evidence. Data extraction and screening Sum-
mary ranges of quantitative proportions and measures
relating to prevalence and factors associated with medi-
cation adherence were compiled and presented on Table
1 [14, 16, 20–28] and Table 2 [11, 14–16, 21, 23, 25–27,
31, 34–38] The evidence from the studies was synthe-
sized and presented in a narrative review following the
WHO’s report on adherence as the framework of the re-
view. In this framework, there are five interacting di-
mensions for medication adherence: (1) social/economic
factors, (2) patient-related factors, (3) health team and
system-related factors, (4) therapy-related factors, and
(5) condition-related factors [8]. categories: research design, objective, study setting, sam-
pling technique, sample size, eligibility criteria, measure
of medication adherence, independent variables tested,
medication adherence result, key findings and any statis-
tical information (odds ratios, 95% confidence interval,
P-value, correlation coefficients). Quality of the studies
was evaluated and reported using the Strengthening the
Reporting of Observational Studies in Epidemiology
(STROBE) for observational studies, Consolidated Stan-
dards of Reporting Trials (CONSORT) 2010 for parallel
group randomized trials and the Joanna Briggs Institute
Checklist for Quasi-Experimental Studies. categories: research design, objective, study setting, sam-
pling technique, sample size, eligibility criteria, measure
of medication adherence, independent variables tested,
medication adherence result, key findings and any statis-
tical information (odds ratios, 95% confidence interval,
P-value, correlation coefficients). Quality of the studies
was evaluated and reported using the Strengthening the
Reporting of Observational Studies in Epidemiology
(STROBE) for observational studies, Consolidated Stan-
dards of Reporting Trials (CONSORT) 2010 for parallel
group randomized trials and the Joanna Briggs Institute
Checklist for Quasi-Experimental Studies. Data analysis A meta-analysis of the findings was not possible due to
the heterogeneity of important aspects of the method-
ology of the selected studies including sampling proced-
ure, population ages, study settings, study design and
measurement
procedure
of
medication
adherence. Social/economic factors screening, 93 articles were full-text reviewed. The refer-
ence list of the selected literatures was then scrutinized
that resulted in the inclusion of 42 articles for further
screening. The final number of articles included in this
systematic review is 15: one randomized controlled trial
[38], three quasi-experimental [21, 26, 36], three obser-
vational retrospective analysis [14–16], and eight obser-
vational cross-sectional study [11, 23, 25, 27, 31, 34, 35,
37]. The description of the included studies is summa-
rized in Table 2. The cumulative number of study partic-
ipants is 21,668 with age of ranging between ages 18 to
75 years old. The systematic review identified eight studies in which
social/economic
factors
from
the
WHO
adherence
model was examined. Eight studies examined the associ-
ation between age and medication adherence; Five of the
studies determined that there is a significant negative as-
sociation between younger age and adherence rates [14–
16, 25, 26]. Three of the five use retrospective analysis
and objective measures of medication adherence [14–
16]. In two of the studies the age group below 40 years
old is twice less likely to be adherent when compared to
40 to 64 years old [16, 26]. It should be noted however
that three studies that use cross-sectional study design
did not find an association between age and medication
adherence [21, 23, 27]. Results A total of 1472 citations from the databases were identi-
fied. After elimination of duplicate records and abstract Gutierrez and Sakulbumrungsil Clinical Hypertension (2021) 27:19 Gutierrez and Sakulbumrungsil Clinical Hypertension Page 4 of 15 (2021) 27:19 Fig. 1 Flow diagram of the search strategy based on PRISMA Fig. 1 Flow diagram of the search strategy based on PRISMA Social/economic factors Medication adherence Adult patients
with chronic
illness (including
hypertension)
Cluster
sampling
method
504
Inclusion: households in sixMIUs from
Northern Philippines, Metro Manila,
Central Visayas and Mindanao,
Philippines
Ea 2018 [35] Science
Direct
Q2 (nursing)
STROBE
[33] (18/
22)
Observational
(cross-
sectional)
To explore self-care among Filipino
immigrants in the United States who
have hypertension. Adult with
hypertension
(Filipino
immigrants in the
United States)
Convenience
sampling
163
Inclusion: at least 18 years old, first
generation Filipino immigrant, able to
speak and write in English, and who
had a current diagnosis of hypertension
or were currently taking an
tih
t
i
di
ti
t th ti First author
(year)
Source
Quality of research
Study design
Journal rank
(SJR 2018) [30]
Score
based on
checklist
Research
design
Objective
Population
Sampling
technique
Filipino
sample
size
Eligibility criteria
Calano
(2019) [31]
PubMed
Q1 (nursing)
JBI [32] (8/
9)
Quasi-
experimental
(pre-test–
post-test)
To determine the effectiveness of a
community-based health program
grounded on the knowledge, adher-
ence and blood pressure control of
adults with hypertension. Adults with
hypertension
Purposive
sampling
50
Inclusion: 40–59 years old patients
diagnosed with hypertension for at least
6 months from Bulacan, Philippines
Exclusion: physical and/or mental
disabilities
Coyoca
(2013) [21]
Google
Scholar
Not available
STROBE
[33] (17/
22)
Observational
(cross-
sectional)
To explore the barriers and factors
that have an effect of the adherence
to therapeutic regimen. Adults with
hypertension and
type II diabetes
Convenience
sampling
56
Inclusion: 18 years old and above
patients diagnosed with hypertension
and type II diabetes recruited from
private and public clinics of Iligan City
Philippines
de Guzman
(2013) [11]
Research
Gate
Q3 (education)
STROBE
[33] (14/
22)
Observational
(cross-
sectional)
To develop and test a model of
medication adherence among
Filipino elderly. Geriatric patients
with chronic
illness (including
hypertension)
Random
sampling
325
Inclusion: > 60 years old, diagnosed
with chronic illness, and with at least
one long-term medication
Exclusion: mental or cognitive
disabilities
Dror (2005)
[34]
Science
Direct
Q1 (health
policy)
JBI [32] (7/
9)
Observational
(cross-
sectional)
To examine evidence of association
between affiliation with Micro
Insurance Units (MIU) and healthcare
utilization. Adult patients
with chronic
illness (including
hypertension)
Cluster
sampling
method
504
Inclusion: households in sixMIUs from
Northern Philippines, Metro Manila,
Central Visayas and Mindanao,
Philippines
Ea 2018 [35] Science
Direct
Q2 (nursing)
STROBE
[33] (18/
22)
Observational
(cross-
sectional)
To explore self-care among Filipino
immigrants in the United States who
have hypertension. Medication adherence Of the 15 studies on Table 3 [11, 14–16, 21, 23, 25–27,
31, 34–38] only two studies [23, 27] reported a baseline
medication adherence to be high, however in both re-
searches, a subjective or self-reported adherence method
is used for measurement. The rest of the studies re-
ported low adherence, four [14–16, 34] of them utilize
objective measure for medication adherence. Four studies examined the association between gender
and medication adherence; two of them determined that
there is a significant association while the other two
stated that they found none. The conclusions of both
studies are inconsistent, in terms of which sex (male or
female) has lower adherence [14, 21]. Overall, the result
is in inconclusive. Page 5 of 15 Gutierrez and Sakulbumrungsil Clinical Hypertension (2021) 27:19 (2021) 27:19 Gutierrez and Sakulbumrungsil Clinical Hypertension p
First author
(year)
Source
Quality of research
Study design
Journal rank
(SJR 2018) [30]
Score
based on
checklist
Research
design
Objective
Population
Sampling
technique
Filipino
sample
size
Eligibility criteria
Calano
(2019) [31]
PubMed
Q1 (nursing)
JBI [32] (8/
9)
Quasi-
experimental
(pre-test–
post-test)
To determine the effectiveness of a
community-based health program
grounded on the knowledge, adher-
ence and blood pressure control of
adults with hypertension. Adults with
hypertension
Purposive
sampling
50
Inclusion: 40–59 years old patients
diagnosed with hypertension for at least
6 months from Bulacan, Philippines
Exclusion: physical and/or mental
disabilities
Coyoca
(2013) [21]
Google
Scholar
Not available
STROBE
[33] (17/
22)
Observational
(cross-
sectional)
To explore the barriers and factors
that have an effect of the adherence
to therapeutic regimen. Adults with
hypertension and
type II diabetes
Convenience
sampling
56
Inclusion: 18 years old and above
patients diagnosed with hypertension
and type II diabetes recruited from
private and public clinics of Iligan City
Philippines
de Guzman
(2013) [11]
Research
Gate
Q3 (education)
STROBE
[33] (14/
22)
Observational
(cross-
sectional)
To develop and test a model of
medication adherence among
Filipino elderly. Geriatric patients
with chronic
illness (including
hypertension)
Random
sampling
325
Inclusion: > 60 years old, diagnosed
with chronic illness, and with at least
one long-term medication
Exclusion: mental or cognitive
disabilities
Dror (2005)
[34]
Science
Direct
Q1 (health
policy)
JBI [32] (7/
9)
Observational
(cross-
sectional)
To examine evidence of association
between affiliation with Micro
Insurance Units (MIU) and healthcare
utilization. Medication adherence Adult with
hypertension
(Filipino
immigrants in the
United States)
Convenience
sampling
163
Inclusion: at least 18 years old, first
generation Filipino immigrant, able to
speak and write in English, and who
had a current diagnosis of hypertension
or were currently taking an
antihypertensive medication at the time
of recruitment
Exclusion: pregnant or taking a
contraceptive medication
Encabo
(2017) [23]
CU
Library
Q4
(pharmaceutical
science)
STROBE
[33] (2.5/9)
Observational
(cross-
sectional)
To know the percentage of the
people who were adherent to their
medication and know the factors that
contributed to the medication
adherence. Adult with
hypertension
(member of
hypertensive
club)
Purposive
sampling
94
Inclusion: at least 19 years old,
diagnosed with hypertension for at least
a month, and are either taking or
prescribed with maintenance
medications for hypertension from
Caloocan, Philippines recruited from
hypertensive club and barangay and the
local municipality recommendation
Juarez
(2013) [14]
Wiley
Online
Library
Q2 (economics,
econometrics)
STROBE
[33] (22/
22)
Observational
(retrospective
analysis)
To identify factors associated with
years of medication adherence and
to examine the relationship between
years of adherence and healthcare
utilization. Adult patients
with diabetes and
hypertension
(Filipino
immigrants in the
United States)
Purposive
sampling
1748
Inclusion: adult patients with diabetes
who were enrolled in a large health
plan in Hawaii for 4 years between 2007
and 2010 and at least one prescription
medication Page 6 of 15 Gutierrez and Sakulbumrungsil Clinical Hypertension (2021) 27:19 (2021) 27:19 Gutierrez and Sakulbumrungsil Clinical Hypertension p
(
)
First author
(year)
Source
Quality of research
Study design
Journal rank
(SJR 2018) [30]
Score
based on
checklist
Research
design
Objective
Population
Sampling
technique
Filipino
sample
size
Eligibility criteria
Ku (2015)
[25]
SAGE
Library
Q3 (health
policy)
STROBE
[33] (19/
22)
Observational
(cross-
sectional)
To measure factors that could be
associated with self-management
practices of people with type 2 dia-
betes and hypertension. Adults with
hypertension and
diabetes
Purposive
sampling
549
Inclusion: at least 20 years old,
diagnosed with type 2 diabetes and
hypertension for at least six months,
recruited from local government unit in
Luzon, Philippines
Mamangon
(2018) [26]
Grey
literature
Not available
STROBE
[33] (20/
22)
Observational
(cross-
sectional)
To identify the proportion of the
patients who are non-adherent and
identify factors associated with medi-
cation adherence behavior. Adults with
hypertension
Convenience
sampling
47
Inclusion: 25–59 years old diagnosed
hypertensive in Barangay 898, Manila and
recipients of free antihypertensive
medication from J. Medication adherence Vicencio Health Center
Exclusion: not able to speak in English
or Filipino, advised to discontinue
medications by physicians
Pablo
(2018) [36]
Research
Gate
Not available
JBI [32] (8/
9)
Quasi-
experimental
(pre-test–
post-test)
To determine the most prevalent
complementary and alternative
medicine (CAM) and determine if
associated with medication
adherence. To assess whether medication
adherence seminar significantly
increased the medication adherence. Adults with
hypertension
and/or diabetes
Purposive
sampling
66
Inclusion: at least 18 years old clinically
diagnosed with hypertension and/or
diabetes, currently prescribed with their
maintenance medicine while practicing
or using CAM, may or may not have not
more than 2 comorbidities recruited
from National Government Center,
Quezon City, Philippines
Palileo-
Villanueva
(2011) [27]
Grey
literature
Not available
STROBE
[33] (18/
22)
Observational
(cross-
sectional)
To determine the prevalence of
adherence to blood pressure
lowering medications and identify
factors affecting it. Adult with
hypertension
(outpatient
specialty clinic)
Convenience
sampling
276
Inclusion: patients with hypertension
consulting at the General Medicine
Outpatient Continuity Clinic of the
Philippine General Hospital
Exclusion: newly diagnosed to have
hypertension or not yet maintained on
antihypertensive medications at the
time of the interview
Taira (2006)
[15]
PubMed
Q1 (health
policy)
STROBE
[33] (22/
22)
Observational
(retrospective
analysis)
To measure the impact of medication
copayment level and other predictors
on compliance with antihypertensive
medications. Adult with
hypertension
(members of a
managed care
organization)
Purposive
sampling
13,708
Inclusion: diagnosed hypertensive with
at least 1 antihypertensive medication
prescription with at least a 15-day supply
between January 1999 and June 2004
Taira (2007)
[16]
PubMed
Q1 (arts and
humanities)
STROBE
[33] (22/
22)
Observational
(retrospective
analysis)
To identify predictors of adherence
for specific groups, particularly Asian
Americans and Pacific Islanders. Adult with
hypertension
(members of a
managed care
organization)
Purposive
sampling
3812
Inclusion: 18 years old diagnosed
hypertensive with drug coverage in a
large health plan. Must have filled at
least one prescription for one of five
classes of identified antihypertensive
medications. Low adherence 41.54% (n = 135)
Medium adherence 39.69% (n = 129)
High adherence 18.15% (n = 59) Medication adherence Blood sugar level
11. Awareness of the
disease
12. Relationship
towards their
doctors
13. Availability and
accessibility to
healthcare services
14. Consultation with
consultation in
public clinics
Values cannot be determined. 1. Gender (P-value =
adherence [21]. 2. Civil status (P-valu
higher adherence [2
3. Work status (P-va
patients has higher
4. Social support sys
value = 0.028) – dire
5. Accessibility to he
(Spearman rho P-va
tionship [21]
6. Consultation with
clinics (P-value = 0.0
[21]
7. Health care provid
value = 0.016) – dire
8. Health awareness
0.000)- direct relatio
de Guzman
(2013) [11]
MMAS-8 [22]
1. Social support
2. Medication belief
3. Follow-up visits
4. Consultation
satisfaction,
5. Memory task
6. Trust with
physician
7. Perceived stress
8. Memory strategies
9. Memory load
10. Depression
11. Length of time
taking the
medications
12. Number of
conditions
13. Self-efficacy
Low adherence 41.54% (n = 135)
Medium adherence 39.69% (n = 129)
High adherence 18.15% (n = 59)
1. Trust with physici
2. Consultation satis
inverse [11]
3. No. of conditions
4. No. of medication
[11]
5. Event-based mem
[11]
6. External memory
inverse [11]
7. Depression (β = 0
Dror (2005)
[34]
Compliance
(binary question
taking drugs
prescribed)
Uninsured chronically ill not taking drugs is
32.6% while insureds is lower at 20.2%. Insured persons rep
compliance among
0.0015) [34]. Ea (2018)
[35]
MAOSS [24]
1. Acculturation
2. Acculturative
stress
3. Hypertension self-
efficacy
4. Patient activation
1. Self-efficacy (by se
0.003) (β = 270) – d
2. Patient activation
– direct
Encabo
(2017) [23]
Adapted MMA8
[23]
1. Age
2. Gender
3. Marital status
4. Employment
5. History of
hypertension
14.5 (SD = 13.6), with scores ranging from 0
to 44 (scores of 22 to 44 is non adherent)
77.7% (n = 73) patients were adherent and
22.3% (n = 21) are non-adherent. Use of maintenance
on odds ratio (OR) 1. Trust with physician (β = 1.168) – direct [11]
2. Consultation satisfaction (β = −0.215) –
inverse [11]
3. No. of conditions (β = 0.693) – direct [11]
4. No. of medication (β = −0.151) – inverse
[11]
5. Event-based memory(β = −0.329) – inverse
[11]
6. External memory strategy (β = −0.186) –
inverse [11]
7. Depression (β = 0.215) – direct [11] 1. Trust with physician (β = 1.168) – direct [11] 2. Uninsured chronically ill not taking drugs is
32.6% while insureds is lower at 20.2%. Medication adherence Exclusion: Patients using other therapeutic
classes, including adrenergic inhibitors,
alpha-adrenergic blocking agents, other di-
uretics or combination drugs and/or newly
shifted to a different medication Page 7 of 15 Gutierrez and Sakulbumrungsil Clinical Hypertension (2021) 27:19 Table 2 Description of the studies (Continued)
First author
(year)
Source
Quality of research
Study design
Journal rank
(SJR 2018) [30]
Score
based on
checklist
Research
design
Objective
Population
Sampling
technique
Filipino
sample
size
Eligibility criteria
Ursua
(2014) [37]
PubMed
Q2 (medicine)
JBI [32] (8/
9)
Quasi-
experimental
(pre-test–
post-test with
control)
To assess the feasibility and efficacy
of a 4-month community health
worker intervention to improve
hypertension management among
Filipino immigrants in New York and
New Jersey. Adult with
hypertension
(Filipino
immigrants in
Unites States)
Purposive
sampling
88
Inclusion: 25–75 years old, self-
identified as Filipino in New York City or
Jersey City, US (had one systolic blood
pressure (SBP) reading of 132 mmHg or
one diastolic blood pressure (DBP) read-
ing of 82 mmHg
Exclusion: on renal dialysis, had
participated in a previous cardiovascular
disease (CVD) study, or had experienced
a heart attack or stroke
Ursua
(2018) [38]
Science
Direct
Q1 (health
informatics)
CONSORT
[39] (19/
25)
Randomized
clinical trial
To assess the efficacy of the
intervention on blood pressure
control, SBP and DBP, and
compliance to appointment keeping. Adult with
hypertension
(Filipino
immigrants in
United States)
Random
Sampling
182
Inclusion: 25–75 years old, self-
identified as Filipino in New York city,
and diagnosed hypertensive or on anti-
hypertensive medication use
Exclusion: on renal dialysis, had an
acute or terminal illness or serious
mental illness, had participated in a
previous CVD study, or had a history of
heart attack, stroke, or congestive heart
failure
SJR, SCImago Journal Rank; JBI, Joanna Briggs Institute; STROBE, Strengthening the Reporting of Observational Studies in Epidemiology; CONSORT, Consolidated Standards of Reporting Trials Gutierrez and Sakulbumrungsil Clinical Hypertension (2021) 27:19 Gutierrez and Sakulbumrungsil Clinical Hypertension (2021) 27:19 Page 8 of 15 Table 3 Summary of findings
First
author
(year of
study)
Measure of
medication
adherence
Independent
variables tested
Medication adherence of Filipino sample
Statistically signific
Calano
(2019) [31]
HB-HCT [20]
Not applicable
Pre-test 1.40 (8.56)
Post-test 17.98 (5.74)
Community-based h
0.03) F-value = 5.00)
Coyoca
(2013) [21]
SSQ-10 [21]
1. Age
2. Gender
3. Religion
4. Civil status
5. Educational
attainment
6. Work status
7. Family monthly
income
8. Social support
system
9. Length of
diagnosis
10. Insured persons reported better drug
compliance among chronically ill (P-value =
0.0015) [34]. 14.5 (SD = 13.6), with scores ranging from 0
to 44 (scores of 22 to 44 is non adherent)
77.7% (n = 73) patients were adherent and
22.3% (n = 21) are non-adherent. 1. Trust with physician (β = 1.168) – direct [11]
2. Consultation satisfaction (β = −0.215) –
inverse [11]
3. No. of conditions (β = 0.693) – direct [11]
4. No. of medication (β = −0.151) – inverse
[11]
5. Event-based memory(β = −0.329) – inverse
[11]
6. External memory strategy (β = −0.186) –
inverse [11]
7. Depression (β = 0.215) – direct [11] Mean score pre-intervention period is 2
(mean = 1.8939; SD = 0.86164) equivalent to
“Sometimes” non-adherent to their
medication. 51.06% were non adherent to
antihypertensive medication, 61.70% have
forgotten to take antihypertensive medicine
during the last two weeks. Medication adherence Age (P = 0.002) older more adherent [25]
2. Specialty clinic had better adherence [25]
(Fisher’s exact test P < 0.001) [25]. 3. Knowledge (P = 0.007) higher more
adherent [25]
4. Positive attitude (P < 0.001) higher more
adherent [25]
5. Perception of support (P < 0.001) higher
more adherent [25]
6. Perception of self-efficacy (P = 0.004) higher
more adherent Ku (2015)
[25]
Binary [25]
1. Age
2. Knowledge
3. Attitudes
4. Perceptions of
support
5. Perception of self-
efficacy
6. Obesity/adiposity
7. Specialty clinic
8. Body mass index
(BMI)
9. Waist
circumference, and
waist–hip ratio
Mamangon
(2018) [26]
MAQ [26] and
MMAS-8 [22]
1. Presence of
comorbidities
2. Illness perception
3. Patient-doctor
relationship
4. Health literacy
51.06% were non adherent to
antihypertensive medication, 61.70% have
forgotten to take antihypertensive medicine
during the last two weeks. Ku (2015)
[25]
Binary [25]
1. Age
2. Knowledge
3. Attitudes
4. Perceptions of
support
5. Perception of self-
efficacy
6. Obesity/adiposity
7. Specialty clinic
8. Body mass index
(BMI)
9. Waist
circumference, and
waist–hip ratio
Mamangon
(2018) [26]
MAQ [26] and
MMAS-8 [22]
1. Presence of
comorbidities
2. Illness perception
3. Patient-doctor
relationship
4. Health literacy
51.0
ant
forg
dur 5. Perception of support (P < 0.001) higher
more adherent [25] 6. Perception of self-efficacy (P = 0.004) higher
more adherent 1. Age aged 25–59 years old were non-
adherent to antihypertensive medication [26]. 2. Patient-doctor relationship (PR, 1.6; 95%
confidence interval [CI], 0.96–2.75) associations
but not statistically significant [26]. 3. Comorbidity (PR, 1.15; 95% CI, 0.66–2.01)
associations but not statistically significant
[26]. 4. Illness perception associations but not
statistically significant (PR, 1.61; 95% CI, 0.50–
5.17) [26]. 5. Health literacy (PR, 1.96; 95% CI. 0.90–4.27)
associations but not statistically significant
[26]. 1. Age aged 25–59 years old were non-
adherent to antihypertensive medication [26]. 2. Patient-doctor relationship (PR, 1.6; 95%
confidence interval [CI], 0.96–2.75) associations
but not statistically significant [26]. 3. Comorbidity (PR, 1.15; 95% CI, 0.66–2.01)
associations but not statistically significant
[26]. 4. Illness perception associations but not
statistically significant (PR, 1.61; 95% CI, 0.50–
5.17) [26]. 5. Health literacy (PR, 1.96; 95% CI. 0.90–4.27)
associations but not statistically significant
[26]. Pablo
(2018) [36]
Adapted MMAS-
8 [23]
1. Attitude towards
complementary and
alternative medicine
(CAM)
2. Medication
adherence seminar
Mean score pre-intervention period is 2
(mean = 1.8939; SD = 0.86164) equivalent
“Sometimes” non-adherent to their
medication. 2. A significant negative correlation between
medication adherence and CAM Attitude
Pearson correlation r value = −0.730, P-
value = 0.049), increase in medication
adherence if low CAM attitude. Inverse
relationship [36] Medication adherence Consultation satisfaction (β = −0.215) –
inverse [11] Insured persons reported better drug
compliance among chronically ill (P-value =
0.0015) [34]. 1. Self-efficacy (by self-efficacy scale) (P-value =
0.003) (β = 270) – direct
2. Patient activation P-value = 0.024) (β = 205)
– direct Use of maintenance drugs (P = 0.016) based
on odds ratio (OR) Gutierrez and Sakulbumrungsil Clinical Hypertension (2021) 27:19 Page 9 of 15 Table 3 Summary of findings (Continued)
First
author
(year of
study)
Measure of
medication
adherence
Independent
variables tested
Medication adherence of Filipino sample
Statistically significant findings
diseases
7. Medication
Juarez
(2013) [14]
PDC [14]
1. Years of
adherence
2. Healthcare
utilization
Mean years of adherence for
antihypertensive medication is 2.17 years. 1. Age older was significantly associated w
greater adherence 1.33 (1.26, 1.42) [14]. 2. Female sex was significantly associated
fewer years of adherence 0.9 (0.89, 0.99) [1
3 Ethnicity Filipino negatively associated w Medication adherence of Filipino sample
Statistically significant findings 1. Age older was significantly associated with
greater adherence 1.33 (1.26, 1.42) [14]. 2. Female sex was significantly associated with
fewer years of adherence 0.9 (0.89, 0.99) [14]. 3. Ethnicity Filipino negatively associated with
adherence. OR 0.90 (080–1.00) [14]
4. Comorbidities history of either coronary
artery disease 1.19 (1.11, 1.28) or congestive
heart failure 1.20 (1.09, 1.32) was significantly
associated with more years of adherence to
antihypertensive medications [14]. 5. Poly pharmacy being on lipid-lowering 1.38
(1.28, 1.49) and antidiabetic medications 1.34
(1.26, 1.43) increased adherence on
antihypertensive. 1. Age (P = 0.002) older more adherent [25]
2. Specialty clinic had better adherence [25]
(Fisher’s exact test P < 0.001) [25]. 3. Knowledge (P = 0.007) higher more
adherent [25]
4. Positive attitude (P < 0.001) higher more
adherent [25]
5. Perception of support (P < 0.001) higher
more adherent [25]
6. Perception of self-efficacy (P = 0.004) higher
more adherent 1. Age older was significantly associated with
greater adherence 1.33 (1.26, 1.42) [14]. 2. Female sex was significantly associated with
fewer years of adherence 0.9 (0.89, 0.99) [14]. 3. Ethnicity Filipino negatively associated with
adherence. OR 0.90 (080–1.00) [14]
4. Comorbidities history of either coronary
artery disease 1.19 (1.11, 1.28) or congestive
heart failure 1.20 (1.09, 1.32) was significantly
associated with more years of adherence to
antihypertensive medications [14]. 5. Poly pharmacy being on lipid-lowering 1.38
(1.28, 1.49) and antidiabetic medications 1.34
(1.26, 1.43) increased adherence on
antihypertensive. 1. 1. Post-seminar intervention (P = 0.000), in-
crease in the medication adherence of pa-
tients [36] 1. Age aged 25–59 years old were non-
adherent to antihypertensive medication [26].
2. Patient-doctor relationship (PR, 1.6; 95%
confidence interval [CI], 0.96–2.75) associations
but not statistically significant [26].
3. Comorbidity (PR, 1.15; 95% CI, 0.66–2.01)
associations but not statistically significant
[26].
4. Illness perception associations but not
statistically significant (PR, 1.61; 95% CI, 0.50–
5.17) [26].
5. Health literacy (PR, 1.96; 95% CI. 0.90–4.27)
associations but not statistically significant
[26]. 1. Post-seminar intervention (P = 0.000), in-
crease in the medication adherence of pa-
tients [36]
2. A significant negative correlation between
medication adherence and CAM Attitude
Pearson correlation r value = −0.730, P-
value = 0.049), increase in medication
adherence if low CAM attitude. Inverse
relationship [36] Adherence rates were less than 65% among
all racial/ethnic groups. Medication adherence Chronicity of
hypertension
b. Blood pressure
(BP)
c. BP control
d. Number of
comorbidities
e. Comorbidities
3. Therapy related
a. Number of
medications
b. Number of anti
c. Class of
antihypertensive
medications
d. Cost – weekly cost
of antihypertensive
medications
1. Patient factors
a. Educational
attainment
b. Knowledge scores
Taira (2006)
[15]
MPR [28] and
binary
1. Age
2. Gender
3. Ethnicity
4. Morbidity level,
health plan type
5. Isle of residence
6. Comorbidities
7. Year of treatment
8. Physician ethnicity
Adherence rates were less than 65% among
all racial/ethnic groups. Taira (2007)
[16]
MPR [28]
1. Copayment level
2. Age
3. Ethnicity
4. Morbidity level
5. Therapeutic class
Age low compliance, age 40 years (42.5%
compliance), 40 to 64 years were nearly
twice as likely to be compliant with
medications [16]. Ethnicity low compliance, Filipino ethnicity
(58.7% compliance) [16] 0.002) [27]. 1. Ethnicity Filipino patients were least
adherent, compared to whites (P < 0.001) [15]. 2. Age lower adherence in younger age (P <
0.001) [15]
3. Educational attainment. Adherence
improved with increase (P < 0.001) [15]. 4. Patients seeing cardiologists or other
specialists were less adherent than patients
seeing primary care doctors (P < 0.001) [15]. 5. Patients seeing female physicians were less
adherent than those seeing male physicians
(P < 0.001) [15]. 6. Patients seeing Filipino physicians tended to
be less adherent than patients seeing white
physicians (P < 0.001) [15]. 7. Patients with a history of diabetes tended
to be more adherent (P < 0.001) [15]. 8. History of heart disease has lower
adherence (P < 0.001) [15]. 9. Adherence to be highest for beta blockers
and calcium channel blockers, followed by
Angiotensin receptor blocker and Angiotensin
converting enzyme inhibitors. Adherence to all
these therapeutic classes was significantly
higher than adherence to thiazide diuretics
(P < 0.001) [15]. 1. Ethnicity Filipino patients were least
adherent, compared to whites (P < 0.001) [15]. 2. Age lower adherence in younger age (P <
0.001) [15] 3. Educational attainment. Adherence
improved with increase (P < 0.001) [15]. 4. Patients seeing cardiologists or other
specialists were less adherent than patients
seeing primary care doctors (P < 0.001) [15]. 5. Patients seeing female physicians were less
adherent than those seeing male physicians
(P < 0.001) [15]. 6. Patients seeing Filipino physicians tended to
be less adherent than patients seeing white
physicians (P < 0.001) [15]. Taira (2007)
[16]
MPR [28]
1. Taira (2007)
[16]
MPR [28]
1. Copayment level
2. Age
3. Ethnicity
4. Morbidity level
5. Therapeutic class
Age low compliance, age 40 years (42.5%
compliance), 40 to 64 years were nearly
twice as likely to be compliant with
medications [16].
Ethnicity low compliance, Filipino ethnicity
(58.7% compliance) [16] 1. Low compliance members wit Health
maintenance organization coverage (59.7%
compliance). Members of HMOs had lower
compliance than members of preferred
provider organization [16].
2. Copayment level, independent of other
determinants, was found to be a strong
predictor of compliance with antihypertensive
medications (P < 0.05) [16].
3. Greater compliance seen among patients
filing pharmacy claims for drugs that required
lower copayments [16].
4. Compliance was lower for drugs in less
preferred tiers [16].
5. Lower medication compliance was seen in
those patients with high morbidity (i.e., 1. Ethnicity Filipino patients were least
adherent, compared to whites (P < 0.001) [15].
2. Age lower adherence in younger age (P <
0.001) [15]
3. Educational attainment. Adherence
improved with increase (P < 0.001) [15].
4. Patients seeing cardiologists or other
specialists were less adherent than patients
seeing primary care doctors (P < 0.001) [15].
5. Patients seeing female physicians were less
adherent than those seeing male physicians
(P < 0.001) [15].
6. Patients seeing Filipino physicians tended to
be less adherent than patients seeing white
physicians (P < 0.001) [15].
7. Patients with a history of diabetes tended
to be more adherent (P < 0.001) [15].
8. History of heart disease has lower
adherence (P < 0.001) [15].
9. Adherence to be highest for beta blockers
and calcium channel blockers, followed by
Angiotensin receptor blocker and Angiotensin
converting enzyme inhibitors. Adherence to all
these therapeutic classes was significantly
higher than adherence to thiazide diuretics
(P < 0.001) [15]. Medication adherence Palileo-
Villanueva
(2011) [27]
ASRQ [27]
1. Socioeconomic
factors
a. Sex
b. Age
c. Employment
status
d. Economic status
2. Condition related
Adherence is 72% in a specialty clinic. 1. Post-seminar intervention (P = 0.000), in-
crease in the medication adherence of pa-
tients [36]
2. A significant negative correlation between
medication adherence and CAM Attitude
Pearson correlation r value = −0.730, P-
value = 0.049), increase in medication
adherence if low CAM attitude. Inverse
relationship [36] 2. A significant negative correlation between
medication adherence and CAM Attitude
Pearson correlation r value = −0.730, P-
value = 0.049), increase in medication
adherence if low CAM attitude. Inverse
relationship [36] 1. Number of maintenance medications
increasing has more adherence increase in the
number of drugs a patient has, the odds of
being more adherent increases by 1.15 times
(P = 0.05) [27]. 2. Financial support from children, patients
that were being supported by their children
were twice more likely to be adherent (P = Gutierrez and Sakulbumrungsil Clinical Hypertension (2021) 27:19 Page 10 of 15 Page 10 of 15 Page 10 of 15 Table 3 Summary of findings (Continued)
First
author
(year of
study)
Measure of
medication
adherence
Independent
variables tested
Medication adherence of Filipino sample
Statistically significant findings
a. Chronicity of
hypertension
b. Blood pressure
(BP)
c. BP control
d. Number of
comorbidities
e. Comorbidities
3. Therapy related
a. Number of
medications
b. Number of anti
c. Class of
antihypertensive
medications
d. Cost – weekly cost
of antihypertensive
medications
1. Patient factors
a. Educational
attainment
b. Knowledge scores
0.002) [27]. Taira (2006)
[15]
MPR [28] and
binary
1. Age
2. Gender
3. Ethnicity
4. Morbidity level,
health plan type
5. Isle of residence
6. Comorbidities
7. Year of treatment
8. Physician ethnicity
Adherence rates were less than 65% among
all racial/ethnic groups. 1. Ethnicity Filipino patients were least
adherent, compared to whites (P < 0.001) [15]. 2. Age lower adherence in younger age (P <
0.001) [15]
3. Educational attainment. Adherence
improved with increase (P < 0.001) [15]. 4. Patients seeing cardiologists or other
specialists were less adherent than patients
seeing primary care doctors (P < 0.001) [15]. 5. Patients seeing female physicians were less
Gutierrez and Sakulbumrungsil Clinical Hypertension (2021) 27:19
Page 10 of 15 Table 3 Summary of findings (Continued)
First
author
(year of
study)
Measure of
medication
adherence
Independent
variables tested
Medication adherence of Filipino sample
a. Taira (2007)
[16]
MPR [28] Health team and system-related factors y
Healthcare team and system-related factors include pa-
tient provider relationship and logistical barriers to
healthcare [8]. Accessibility to health services and insur-
ance whether locally or abroad showed positive relation-
ship with adherence. Locally, Coyoca et al. [21] and Dror
et al. [34], statistically proven the link where insured
persons reported better drug compliance among chron-
ically ill. Consistent findings were observed in Filipinos
residing abroad, they determined that copayment level,
independent of other determinants, was found to be a
strong predictor of compliance with antihypertensive
medications specifically when offered by the patient’s
preferred organization. In addition, greater compliance Medication adherence Weight, and BMI improvement (P < 0.01)
[38] HB-HCT, Hill-Bone high blood pressure compliance scale; SSQ-10, Self-structured questionnaires (ten items); PR, Prevalence ratio; MMAS-8, Morisky Medication
Adherence Scale; MAOSS, Medical Outcomes Study Specific Adherence Scale; SD, standard deviation; PDC, proportion of days covered; MAQ, Medication
Adherence Questionnaire; ASRQ, Adherence Self-Report Questionnaire; MPR, Medication Possession ratio; CHW, community health worker pressure compliance scale; SSQ-10, Self-structured questionnaires (ten items); PR, Prevalence ratio; MMAS-8, Morisky Medication
ical Outcomes Study Specific Adherence Scale; SD, standard deviation; PDC, proportion of days covered; MAQ, Medication
Q, Adherence Self-Report Questionnaire; MPR, Medication Possession ratio; CHW, community health worker HB-HCT, Hill-Bone high blood pressure compliance scale; SSQ-10, Self-structured questionnaires (ten items); PR, Prevalence ratio; MMAS-8, Morisky Medication
Adherence Scale; MAOSS, Medical Outcomes Study Specific Adherence Scale; SD, standard deviation; PDC, proportion of days covered; MAQ, Medication
Adherence Questionnaire; ASRQ, Adherence Self-Report Questionnaire; MPR, Medication Possession ratio; CHW, community health worker Only one study examined the association of civil sta-
tus, they determined that in sample of 56 participants
married female participants has higher adherence [21]. Two studies examined if the educational attainment im-
pacts the medication adherence; Taira et al. [15] deter-
mined
that
adherence
improved
with
increased
educational attainment, however in both Coyoca et al. [21] and Palileo-Villanueva et al. [27] no association was
found. Coyoca et al. [21] found an association between
work and adherence where working patients has higher
adherence. Result of studies on self-efficacy is mixed, one of the
studies did not find an association, but in two of them
concluded that higher patient’s perceived self-efficacy is
associated with higher adherence [11, 25, 35]. In the case
of social support, three studies found a direct relation-
ship between social support system and higher adher-
ence [21, 25, 27]. Specifically, in the study of Palileo-
Villanueva et al. [27], they specifically found that pa-
tients that were being supported by their children finan-
cially were twice more likely to be adherent. Medication adherence Copayment level
2. Age
3. Ethnicity
4. Morbidity level
5. Therapeutic class 1. Low compliance members wit Health
maintenance organization coverage (59.7%
compliance). Members of HMOs had lower
compliance than members of preferred
provider organization [16]. 1. Low compliance members wit Health
maintenance organization coverage (59.7%
compliance). Members of HMOs had lower
compliance than members of preferred
provider organization [16]. 2. Copayment level, independent of other
determinants, was found to be a strong
predictor of compliance with antihypertensive
medications (P < 0.05) [16]. 3. Greater compliance seen among patients
filing pharmacy claims for drugs that required
lower copayments [16]. 4. Compliance was lower for drugs in less
preferred tiers [16]. 5. Lower medication compliance was seen in
those patients with high morbidity (i e 2. Copayment level, independent of other
determinants, was found to be a strong
predictor of compliance with antihypertensive
medications (P < 0.05) [16]. 3. Greater compliance seen among patients
filing pharmacy claims for drugs that required
lower copayments [16]. 4. Compliance was lower for drugs in less
preferred tiers [16]. 4. Compliance was lower for drugs in less
preferred tiers [16]. 5. Lower medication compliance was seen in
those patients with high morbidity (i.e., Table 3 Summary of findings (Continued)
First
author
(year of
study)
Measure of
medication
adherence
Independent
variables tested
Medication adherence of Filipino sample
Statistically significant findings
i di
i
h
f
h
bid
Gutierrez and Sakulbumrungsil Clinical Hypertension (2021) 27:19
Page 11 of 15 Gutierrez and Sakulbumrungsil Clinical Hypertension (2021) 27:19 Page 11 of 15 Table 3 Summary of findings (Continued)
First
author
(year of
study)
Measure of
medication
adherence
Independent
variables tested
Medication adherence of Filipino sample
Statistically significant findings Table 3 Summary of findings (Continued)
First
author
(year of
study)
Measure of
medication
adherence
Independent
variables tested
Medication adherence of Filipino sample
Statistically significant findings
indicating the presence of other comorbid
conditions) compared with patients with low
comorbidity [16]. 6. Best compliance observed for angiotensin
receptor blockers, followed by calcium
channel blockers, [beta] adrenergic receptor
antagonists ([beta]-blockers), angiotensin-
converting enzyme inhibitors, and last, thia-
zide diuretics [16]. 7. Filipino patients were more likely than other
ethnic groups to have received tier 3 (for
medications with a $20 to $165 copayment
most expensive) medications (13.4% vs. 12%)
[16]. Medication adherence Ursua
(2014)
HB-HCT [20]
Pre-test 11.15 (2.73)
Post-test 11.54 (2.15)
CHW intervention significant changes were
exhibited for systolic and diastolic BP, weight,
and BMI (P < 0.01) but not significant for
medication adherence. Ursua
(2018)
HB-HCT [20]
Pre-test intervention 3.6 (0.5) vs. control 3.6
(0.5) P-value = 0.867
Post-test intervention 3.8 (0.5) vs. control 3.7
(0.3)
1. Community-based intervention delivered by
CHWs improve BP and related factors [38]. 2. Adjusted odds of controlled BP for the
treatment group was 3.2 times the odds of
the control group (P < 0.001) and individuals
in the treatment group showed significant
changes in appointment keeping [38]. 3. Weight, and BMI improvement (P < 0.01)
[38]
HB-HCT, Hill-Bone high blood pressure compliance scale; SSQ-10, Self-structured questionnaires (ten items); PR, Prevalence ratio; MMAS-8, Morisky Medication
Adherence Scale; MAOSS, Medical Outcomes Study Specific Adherence Scale; SD, standard deviation; PDC, proportion of days covered; MAQ, Medication
Adherence Questionnaire; ASRQ, Adherence Self-Report Questionnaire; MPR, Medication Possession ratio; CHW, community health worker Medication adherence of Filipino sample
Statistically significant findings indicating the presence of other comorbid
conditions) compared with patients with low
comorbidity [16]. indicating the presence of other comorbid
conditions) compared with patients with low
comorbidity [16]. 6. Best compliance observed for angiotensin
receptor blockers, followed by calcium
channel blockers, [beta] adrenergic receptor
antagonists ([beta]-blockers), angiotensin-
converting enzyme inhibitors, and last, thia-
zide diuretics [16]. 7. Filipino patients were more likely than other
ethnic groups to have received tier 3 (for
medications with a $20 to $165 copayment
most expensive) medications (13.4% vs. 12%)
[16]. Ursua
(2014)
HB-HCT [20]
Pre-test 11.15 (2.73)
Post-test 11.54 (2.15)
Ursua
(2018)
HB-HCT [20]
Pre-test intervention 3.6 (0.5) v
(0.5) P-value = 0.867
Post-test intervention 3.8 (0.5) v
(0.3) CHW intervention significant changes were
exhibited for systolic and diastolic BP, weight,
and BMI (P < 0.01) but not significant for
medication adherence. Pre-test intervention 3.6 (0.5) vs. control 3.6
(0.5) P-value = 0.867
Post-test intervention 3.8 (0.5) vs. control 3.7
(0.3) 1. Community-based intervention delivered by
CHWs improve BP and related factors [38]. 2. Adjusted odds of controlled BP for the
treatment group was 3.2 times the odds of
the control group (P < 0.001) and individuals
in the treatment group showed significant
changes in appointment keeping [38]. 3. Patient-related factors Patient-related factors are the knowledge, attitudes, be-
liefs, and perceptions of hypertensive patients [8]. There
are six literatures that examined patient-related factors. Patient’s health literacy has positive relationship but not
statistically
significant
in
the
study
conducted
by
Mamangon et al. [26]. Disease awareness on the other
hand is statistically significant in the study of Coyoca
et al. [21] where they related this to the coping mechan-
ism to stress of the participants. In two literatures,
knowledge is directly associated with medication adher-
ence [25, 35]. For attitude, three studies concluded that
positive attitude lead to higher adherence [11, 25, 35]. Page 12 of 15 Page 12 of 15 Gutierrez and Sakulbumrungsil Clinical Hypertension (2021) 27:19 Gutierrez and Sakulbumrungsil Clinical Hypertension was seen among patients filing pharmacy claims for
drugs that required lower copayments. They also found
an inverse relationship with compliance to medications
in the most expensive tiers [16]. order to validate the result and to avoid hasty conclu-
sions, it is recommended for future researchers that
when studying the association of adherence and poly-
pharmacy, instead of running a univariate analysis it
would be better to use multivariate regression models in
order to account for potential sources of bias from con-
founders. This recommendation is consistent with stud-
ies conducted in other countries where it has shown
repeatedly that fewer medications improved adherence. Four literatures examined the effect of patient and
health care provider relationship, Coyoca et al. [21] de-
termined that there is a positive relationship between
good relationship and adherence, this is also consistent
with the findings of Mamangon et al. [21, 26]. In the de
Guzman et al. [11]‘s study, an interesting finding is that
higher trust with physician resulted to higher adherence,
but curiously if the patient has high satisfaction with the
doctor consultation the adherence tend to be low. In
Taira et al. [15]‘s study, a study conducted abroad, found
out that adherence was higher if patients are consulting
with primary care doctors over specialist. Filipino pa-
tients also tend to have lower adherence with a female
and Filipino physicians. In Encabo et al. [23], the use of maintenance drugs
that means consistent scheduling of medication had a
positive relationship with adherence. An interesting find-
ings of de Guzman et al. [11] appeared that Filipino eld-
erly preferred no assistance in remembering medication
intake whether using event-based memory or external
memory based strategy. Condition-related factors Therapy-related factors include the complexity of the
medical
regimen,
duration
of
treatment,
frequent
changes in treatment, and adverse effects [8]. The find-
ings with regards to number of medications or polyphar-
macy is mixed in three of the studies; de Guzman et al. [11], found that the number of medication is inversely
proportional with medication adherence. But in two of
the studies, it appears that the increasing number of
medication
led
to
higher
adherence. In
Palileo-
Villanueva et al. [27], they specifically stated that the
odds of being more adherent increases by 1.15 times if
the patient has polypharmacy. In Juarez et al. [14], they
emphasized that poly pharmacy on lipid-lowering and
antidiabetic medications are statistically significantly in-
creased adherence on antihypertensive. Condition-related factors are those illness-related de-
mands faced by the patient, such as severity of blood
pressure, absence or presence of symptoms, and level of
disability and/or comorbidities [8]. For the number of
comorbidities, it appears that the finding for Filipinos
abroad have different pattern compared to patients lo-
cally. In the finding of de Guzman et al. [11] and
Mamangon et al. [26] as the number of other conditions
increased, adherence also increased. But in Taira et al. [16], a study conducted abroad, lower medication com-
pliance was seen in those patients with high morbidity. As for the type of comorbidity, Juarez et al. [14] deter-
mined that history of either coronary artery disease or
congestive heart failure was significantly associated with
more years of adherence to antihypertensive medica-
tions. The result conducted abroad however, showed an
opposite relationship. For Taira et al. [15], the history of
heart disease has lower adherence but history of diabetes
tended to be more adherent. Despite the two latter findings that associated poly-
pharmacy and adherence positively it should be noted
that, certainly confounders, such as multiple comorbidi-
ties, advance stage of illness, and patient’s illness repre-
sentation among others may have contributed to this. In
other words, the statement “odds of being more adher-
ent increases by 1.15 times if the patient has polyphar-
macy” should have be more accurately reported as
“patients in the advance stages of their illness with mul-
tiple medications are more adherent by 1.15 times”. In Patient-related factors In Taira et al. [15], they sug-
gested that adherence is highest for beta blockers and
calcium channel blockers, followed by angiotensin recep-
tor blockers and angiotensin-converting enzyme inhibi-
tors and that adherence is lower in thiazide diuretics. This finding is somewhat consistent with Taira et al. [16], thiazide diuretics also has the lowest adherence,
but in this paper best compliance observed for angioten-
sin receptor blockers, followed by calcium channel
blockers, beta blockers, angiotensin-converting enzyme
inhibitors in that order. With regards to CAM, Pablo
et al. [36] found that medication adherence is higher if
the patients has low attitude towards CAM. In three studies, consultation in specialty continuity
clinics for hypertension showed positive relationship
with adherence whether it is public or privately managed
institution [21, 25, 27]. The same positive relationship is
observed in the four community-based health programs
researched, regardless whether they are a short term (2-
h lecture) or long-term (6 months program). There is an
observed increase in adherence post-intervention in
these researches [31, 36–38]. Discussion The result of medication adherence is consistent with
the national survey reported in PRESYON2 that the ad-
herence among those treated for hypertension is 66% Page 13 of 15 Page 13 of 15 Page 13 of 15 Gutierrez and Sakulbumrungsil Clinical Hypertension (2021) 27:19 Gutierrez and Sakulbumrungsil Clinical Hypertension most were cross-sectional as well. Healthcare providers
may still consider screening for these issues when deal-
ing with their hypertensive patients. [10]. The factors found that are consistently associated
with adherence across the included studies were all
health systems related. The factors that increase adher-
ence are (1) accessibility of health services, (2) positive
relationship with providers of health services, (3) spe-
cialty clinics and programs for hypertension, and (4)
health insurance. This is comparable to the finding in
the United States, where patients reporting better access
to health care, satisfaction with their care and a good
patient-doctor relationship were significantly more likely
to be more adherent [40]. This review revealed that more researches are needed
to determine which factors influence antihypertensive
medication adherence in Filipinos. Several concerns
were identified after this systematic review, first concern
regarding these studies is that most of the studies were
based on homogeneous samples of hypertensive Filipi-
nos, often they are even collected within the same locale
or they are recruited from the same organization or
health insurance providers. This will therefore limit
generalizability. Another concern in interpreting the re-
sults of this review is that most of the studies measured
medication adherence using subjective methods or by
self-report, and there are inherent biases when using this
tool. Consistent objective measurements of adherence
are lacking and need to be incorporated as an outcome
for future studies when exploring factors associated with
medication adherence. The third concern is related to
research design, since adherence is a multidimensional
and dynamic process, it cannot be assumed that medica-
tion adherence and factors associated with medication
adherence are stable over time. Most of the studies ex-
ploring medication adherence were observational and
cross-sectional. There is a need for more longitudinal
analyses of factors associated with medication adherence
so that we can determine if an important factor remains
associated with adherence at different time periods
across a patient’s hypertensive diagnosis. Consent for publication
Not applicable. Consent for publication
Not applicable. Consent for publication
Not applicable. Funding 14. Juarez DT, Tan C, Davis J, Mau M. Factors affecting sustained medication
adherence and its impact on health care utilization in patients with
diabetes. J Pharm Health Serv Res. 2013;4(2):89–94. https://doi.org/10.1111/
jphs.12016. Funding for the conduct of the research is shouldered by the authors. Research grant from Chulalongkorn University and/or University of the
Philippines will be requested after publication. Funding agencies plays no
role in the design of the study and collection, analysis, and interpretation of
data and in writing the manuscript. 15. Taira DA, Wong KS, Frech-Tamas F, Chung RS. Copayment level and
compliance with antihypertensive medication: analysis and policy
implications for managed care. Am J Manag Care. 2006;12(11):678–83. 15. Taira DA, Wong KS, Frech-Tamas F, Chung RS. Copayment level and
compliance with antihypertensive medication: analysis and policy
implications for managed care. Am J Manag Care. 2006;12(11):678–83. Acknowledgements
h
h 11. de Guzman AB, Guevara KI, Guiang FJ, Gutierrez AL, Habaluyas AS, Hizon
MA, et al. Developing a model of medication adherence among Filipino
elderly. Educ Gerontol. 2013;39(5):298–313. https://doi.org/10.1080/036012
77.2012.661336. g
The research was supported by Chulalongkorn University and University of
the Philippines. We are grateful to the department of social and
administrative pharmacy and Department of pharmacy for their
contributions to the study: The research was supported by Chulalongkorn University and University of
the Philippines. We are grateful to the department of social and
administrative pharmacy and Department of pharmacy for their
contributions to the study: 12. Zhao B, Jose PO, Pu J, Chung S, Ancheta IB, Fortmann SP, et al. Racial/ethnic
differences in hypertension prevalence, treatment, and control for
outpatients in northern California 2010-2012. Am J Hypertens. 2015;28(5):
631–9. https://doi.org/10.1093/ajh/hpu189. Discussion Finally, there
are other factors and interventions for enhancing medi-
cation adherence in hypertension like unit dose dispens-
ing, combination pills, use of technology for dosing The number of studies conducted exploring reasons
for non-adherence in hypertensive Filipinos is limited
and majority are also cross-sectional which makes it dif-
ficult to assess causality. Further, we don’t know if the
relationships are sustained over time. Despite these limi-
tations, given that health team and system-related fac-
tors
are
modifiable,
these
can
be
the
focus
of
interventions and further research to increase medica-
tion adherence. The other four dimensions of medication adherence,
have patterns of association as well, but are not consist-
ent in all included studies. The factors that are associ-
ated to poor adherence are younger age, single in civil
status, low educational attainment, unemployment, low
health literacy and awareness, low knowledge on hyper-
tension, negative attitude towards hypertension, low self-
efficacy, low social support, inconsistent drug regimen
schedule, taking Thiazide diuretics, taking CAMs, low
illness perception, and no comorbidities as seen in Fig. 2. Factors that are associated with Adherence in Filipinos
These findings, must be interpreted with caution be-
cause of the small number of studies and the fact that Fig. 2 Factors that are associated with Adherence in Filipinos Fig. 2 Factors that are associated with Adherence in Filipinos Page 14 of 15 Gutierrez and Sakulbumrungsil Clinical Hypertension (2021) 27:19 reminders, and blood pressure monitoring that have not
been examined for their associations to medication ad-
herence in Filipinos. Abbreviations
ASRQ Adh 9. Bramley TJ, Gerbino PP, Nightengale BS, Frech-Tamas F. Relationship of
blood pressure control to adherence with antihypertensive monotherapy in
13 managed care organizations. J Manag Care Pharm. 2006;12(3):239–45. https://doi.org/10.18553/jmcp.2006.12.3.239. 9. Bramley TJ, Gerbino PP, Nightengale BS, Frech-Tamas F. Relationship of
blood pressure control to adherence with antihypertensive monotherapy in
13 managed care organizations. J Manag Care Pharm. 2006;12(3):239–45. https://doi.org/10.18553/jmcp.2006.12.3.239. ASRQ: Adherence Self-Report Questionnaire; CAM: Complementary and
alternative medicines; HB-HCT: Hill-Bone High Blood Pressure Compliance
Scale; MAOSS: Medical Outcomes Study Specific Adherence Scale;
MAQ: Medication Adherence Questionnaire; MMAS-8: Morisky Medication
Adherence Scale; MPR: Medication Possession Ratio; PDC: Proportion of days
covered; SSQ-10: Self-structured questionnaires (ten items); WHO: World
Health Organization 10. Sison J, Arceo LP, Trinidad E, Bautista AJ, Buan E, Chua P, et al. Philippine
heart association-council on hypertension report on survey of hypertension
and target organ damage (PRESYON 2-TOD*) a report on prevalence of
hypertension, awareness, treatment profile and control rate. Philipp J
Cardiol. 2007;35:1–9. Competing interests
The authors declare no competing interests. Competing interests
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community in Caloocan City, Philippines. Thai J Pharm Sci. 2017;41:173–6. Availability of data and materials 16. Taira DA, Gelber RP, Davis J, Gronley K, Chung RS, Seto TB. Antihypertensive
adherence and drug class among Asian Pacific Americans. Ethn Health. 2007;12(3):265–81. https://doi.org/10.1080/13557850701234955. 16. Taira DA, Gelber RP, Davis J, Gronley K, Chung RS, Seto TB. Antihypertensive
adherence and drug class among Asian Pacific Americans. Ethn Health. 2007;12(3):265–81. https://doi.org/10.1080/13557850701234955. Data sharing is not applicable to this article as no datasets were generated
or analyzed during the current study. Page 15 of 15 Page 15 of 15 Gutierrez and Sakulbumrungsil Clinical Hypertension (2021) 27:19 (2021) 27:19 17. Paz-Pacheco E, Jimeno C. Diabetes care in the Philippines. J ASEAN Fed
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among Filipino Americans with hypertension: a randomized controlled trial. Prev Med Rep. 2018;11:42–8. https://doi.org/10.1016/j.pmedr.2018.05.002. 21. Coyoca GS, Chan CC, Jamero HJ, Teves GF, Tabil VG. Barriers to therapeutic
regimen adherence of type II diabetes mellitus patients in Iligan City,
Philippines. Proc Annu Int Conf Syiah Kuala Univ - Life Sci Eng Chapter. 2013;3:204–9. 39. Altman DG, Schulz KF, Moher D, Egger M, Davidoff F, Elbourne D, et al. The
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6/0003-4819-134-8-200104170-00012. 22. Morisky DE, Ang A, Krousel-Wood M, Ward HJ. Predictive validity of a
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(Greenwich). 2008;10(5):348–54. https://doi.org/10.1111/j.1751-7176.2008. 07572.x. 40. Publisher’s Note Ea EE, Colbert A, Turk M, Dickson VV. Self-care among Filipinos in the United
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Of Creatures Great and Small: The Advantages of Farm Animal Models in Immunology Research
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Edited by: Effective vaccines have
been developed for many acute viral and
bacterial infections, whereas for others,
like tuberculosis (TB), there is still a need
for reliable vaccines. Pathology seen in the
murine models of TB currently used is very
different to the pathology seen in humans
(Gupta and Katoch, 2009), whereas the
bovine and human diseases share many
similarities (Waters et al., 2011). For such
diseases it is crucial that we use natural dis-
ease-models, with the appropriate pathogen In recent decades the inappropriate use
of antibiotics in farm animals, either as
growth enhancers or for treatment of infec-
tious diseases, has resulted both in higher
concentrations of antibiotic residues in
meat destined for the human food chain
and in an increasing prevalence of antibi-
otic-resistant bacteria (Carlet et al., 2012). Increased public concerns regarding their
potential impact on human health may
result in a reduction or cessation in the use
of antibiotics in farm animals (Veterinary
Record, 2011, 2012). Thus, alternative treat-
ments for the control of infectious diseases
in farm animals need to be identified as a
research priority. In achieving this goal, the
physiological relevance of an animal model
to the intended target population becomes
a key factor. The majority of models cur-
rently used for pharmaceutical research are
murine and it is possible that potentially
valuable therapeutics are discounted on
the basis of lack of responses generated in
this system, as shown very recently for sep-
sis (Seok et al., 2013). Several non-rodent
species, such as cattle, pigs, and chicken,
are both valuable models for infectious
diseases in humans (Hein and Griebel,
2003; Visscher and Goddard, 2011; Waters
et al., 2011; Costa et al., 2012; Meurens
et al., 2012) and important clinical targets
in their own right. Effective vaccines have
been developed for many acute viral and
bacterial infections, whereas for others,
like tuberculosis (TB), there is still a need
for reliable vaccines. Pathology seen in the
murine models of TB currently used is very
different to the pathology seen in humans
(Gupta and Katoch, 2009), whereas the
bovine and human diseases share many
similarities (Waters et al., 2011). Opinion Article Acknowledgment We thank D. McKeever (RVC) for help-
ful discussions and revisions. Amanda J. Gibson, Tracey J. Coffey and Dirk Werling
are funded through the BBSRC, espe-
cially the “Combating Endemic Diseases
of Farmed Animals for Sustainability
(CEDFAS)” initiative, DEFRA, Wellcome
Trust and industrial grants. This manuscript
represents number PID_00272 of the RVC. We thank D. McKeever (RVC) for help-
ful discussions and revisions. Amanda J. Gibson, Tracey J. Coffey and Dirk Werling
are funded through the BBSRC, espe-
cially the “Combating Endemic Diseases
of Farmed Animals for Sustainability
(CEDFAS)” initiative, DEFRA, Wellcome
Trust and industrial grants. This manuscript
represents number PID_00272 of the RVC. References Edited by: Virgil Schijns, Wageningen University, Netherlands in the most appropriate host, to examine the
host-pathogen interactions that occur in
outbred populations. A fundamental point
in the development of new and improved
intervention strategies is the understanding
of host differences in the innate immune
response, which primes the subsequent
adaptive immune response. Although the
innate immune system has been largely
conserved during evolution, marked
variations and diversity exist between dif-
ferent mammalian species within Pattern-
Recognition-Receptor (PRR) structure
(Jungi et al., 2011). These differences are
based on evolutionary pressure within the
innate immune system, potentially reflect-
ing the specific threats encountered by each
species (Zhang et al., 2010). This selective
pressure appears to be absent in the avail-
able murine sequences (Werling et al.,
2009). The similarity between human and
farm animal PRR (Jungi et al., 2011) is fur-
ther supported by their similar response to
ligands (Kapetanovic et al., 2012), in con-
trast to murine PRR (Hajjar et al., 2002;
Grabiec et al., 2004; Farhat et al., 2010). Since recognition by PRR is associated with
adaptive immunity by providing optimal
immunostimulation, learning more about
these key molecules in farm animals might
inform us about their adjuvant effect in
vaccines for use in these animals as well
as humans. Given the size and blood vol-
umes of farm animals, there are also greater
opportunities to repeatedly access to differ-
ent cell types – an asset which would facili-
tate the assessment of cell specific effects of
such immunomodulatory agents on autol-
ogous cells (Hein and Griebel, 2003). Farm
animal models do have their disadvantages
such as dedicated housing, biosecurity,
and the confinement of infected animals. However, it is misleading to rely on murine in the most appropriate host, to examine the
host-pathogen interactions that occur in
outbred populations. A fundamental point
in the development of new and improved
intervention strategies is the understanding
of host differences in the innate immune
response, which primes the subsequent
adaptive immune response. Although the
innate immune system has been largely
conserved during evolution, marked
variations and diversity exist between dif-
ferent mammalian species within Pattern-
Recognition-Receptor (PRR) structure
(Jungi et al., 2011). These differences are
based on evolutionary pressure within the
innate immune system, potentially reflect-
ing the specific threats encountered by each
species (Zhang et al., 2010). This selective
pressure appears to be absent in the avail-
able murine sequences (Werling et al.,
2009). Edited by: The similarity between human and
farm animal PRR (Jungi et al., 2011) is fur-
ther supported by their similar response to
ligands (Kapetanovic et al., 2012), in con-
trast to murine PRR (Hajjar et al., 2002;
Grabiec et al., 2004; Farhat et al., 2010). Since recognition by PRR is associated with
adaptive immunity by providing optimal
immunostimulation, learning more about
these key molecules in farm animals might
inform us about their adjuvant effect in
vaccines for use in these animals as well
as humans. Given the size and blood vol-
umes of farm animals, there are also greater
opportunities to repeatedly access to differ-
ent cell types – an asset which would facili-
tate the assessment of cell specific effects of
such immunomodulatory agents on autol-
ogous cells (Hein and Griebel, 2003). Farm
animal models do have their disadvantages
such as dedicated housing, biosecurity,
and the confinement of infected animals. However, it is misleading to rely on murine models to fill the gaps in our knowledge
of disease-pathogenesis, and choosing a
research model should be more than just
a matter of convenience and convention
(Bolker, 2012). In recent decades the inappropriate use
of antibiotics in farm animals, either as
growth enhancers or for treatment of infec-
tious diseases, has resulted both in higher
concentrations of antibiotic residues in
meat destined for the human food chain
and in an increasing prevalence of antibi-
otic-resistant bacteria (Carlet et al., 2012). Increased public concerns regarding their
potential impact on human health may
result in a reduction or cessation in the use
of antibiotics in farm animals (Veterinary
Record, 2011, 2012). Thus, alternative treat-
ments for the control of infectious diseases
in farm animals need to be identified as a
research priority. In achieving this goal, the
physiological relevance of an animal model
to the intended target population becomes
a key factor. The majority of models cur-
rently used for pharmaceutical research are
murine and it is possible that potentially
valuable therapeutics are discounted on
the basis of lack of responses generated in
this system, as shown very recently for sep-
sis (Seok et al., 2013). Several non-rodent
species, such as cattle, pigs, and chicken,
are both valuable models for infectious
diseases in humans (Hein and Griebel,
2003; Visscher and Goddard, 2011; Waters
et al., 2011; Costa et al., 2012; Meurens
et al., 2012) and important clinical targets
in their own right. www.frontiersin.org Edited by: For such
diseases it is crucial that we use natural dis-
ease-models, with the appropriate pathogen However, in addition to their potential
use as models for human diseases, research
in farm-animals models have a “right-on-
their-own,” taken the predicted increase in
food supply necessary by 70% to support
an ever-increasing global population as well
as the occurrence of new emerging diseases,
such as Schmallenberg virus into account. These challenges can be faced and overcome
by an increased readiness of farm animal
research centers to apply their models in
novel ways, a willingness of the medical
profession to accept more suitable disease-
models, and a willingness of funding agen-
cies and companies to invest in this type of
research partnership. Amanda J. Gibson1, Tracey J. Coffey 2 and Dirk Werling1* Amanda J. Gibson1, Tracey J. Coffey 2 and Dirk Werling1* Amanda J. Gibson1, Tracey J. Coffey 2 and D
1 Department of Pathology and Infectious Diseases, Royal Veterin
2 School of Veterinary Medicine and Sciences, University of Notti
*Correspondence: dwerling@rvc.ac.uk
Edited by:
Virgil Schijns, Wageningen University, Netherlands
Reviewed by:
Virgil Schijns, Wageningen University, Netherlands 1 Department of Pathology and Infectious Diseases, Royal Veterinary College, London, UK
2 School of Veterinary Medicine and Sciences, University of Nottingham, Sutton Bonington, UK
*Correspondence: dwerling@rvc.ac.uk 1 Department of Pathology and Infectious Diseases,
2 School of Veterinary Medicine and Sciences, Univer
*Correspondence: dwerling@rvc.ac.uk
Edited by:
Virgil Schijns, Wageningen University, Netherlands
Reviewed by:
Virgil Schijns, Wageningen University, Netherlands Opinion Article
published: 27 May 2013
doi: 10.3389/fimmu.2013.00124 Opinion Article
published: 27 May 2013
doi: 10.3389/fimmu.2013.00124 Of creatures great and small: the advantages of farm animal
models in immunology research Amanda J. Gibson1, Tracey J. Coffey 2 and Dirk Werling1* References Bolker, J. (2012). Model organisms: there’s more to
life than rats and flies. Nature 491, 31–33. doi:
10.1038/491031a Carlet, J., Jarlier, V., Harbarth, S., Voss, A., Goossens, H.,
and Pittet, D. (2012). Ready for a world without anti-
biotics? The Pensieres Antibiotic Resistance Call to
Action. Antimicrob. Resist. Infect. Control 1, 11. doi:
10.1186/2047-2994-1-11 Costa, L. F., Paixao, T. A., Tsolis, R. M., Baumler, A. J.,
and Santos, R. L. (2012). Salmonellosis in cattle: May 2013 | Volume 4 | Article 124 | 1 www.frontiersin.org Lessons learned from farm animals Gibson et al. advantages of being an experimental model. Res. Vet. Sci. 93, 1–6. doi: 10.1016/j.rvsc.2012.03.002 with cattle. Clin. Dev. Immunol. 2011, 768542. doi:
10.1155/2011/768542 advantages of being an experimental model. Res. Vet. Sci. 93, 1–6. doi: 10.1016/j.rvsc.2012.03.002 Kapetanovic, R., Fairbairn, L., Beraldi, D., Sester, D. P.,
Archibald, A. L., Tuggle, C. K., et al. (2012). Pig bone
marrow-derived macrophages resemble human mac-
rophages in their response to bacterial lipopolysac-
charide. J. Immunol. 188, 3382–3394. doi: 10.4049/
jimmunol.1102649 Werling, D., Jann, O. C., Offord, V., Glass, E. J., and Coffey,
T. J. (2009). Variation matters: TLR structure and spe-
cies-specific pathogen recognition. Trends Immunol. 30, 124–130. doi: 10.1016/j.it.2008.12.001 Farhat, K., Riekenberg, S., Jung, G., Wiesmuller, K. H.,
Jungi, T. W., and Ulmer, A. J. (2010). Identification of
full length bovine TLR1 and functional characteriza-
tion of lipopeptide recognition by bovine TLR2/1 het-
erodimer. Vet. Res. 41, 34. doi: 10.1051/vetres/2010006 Meurens, F., Summerfield, A., Nauwynck, H., Saif, L.,
and Gerdts, V. (2012). The pig: a model for human
infectious diseases. Trends Microbiol. 20, 50–57. doi:
10.1016/j.tim.2011.11.002 Zhang, Q., Zmasek, C. M., and Godzik, A. (2010). Domain
architecture evolution of pattern-recognition recep-
tors. Immunogenetics 62, 263–272. doi: 10.1007/
s00251-010-0428-1 Grabiec, A., Meng, G., Fichte, S., Bessler, W., Wagner, H.,
and Kirschning, C. J. (2004). Human but not murine
toll-like receptor 2 discriminates between tri-palmi-
toylated and tri-lauroylated peptides. J. Biol. Chem. 279, 48004–48012. doi: 10.1074/jbc.M405311200 Seok, J., Warren, H. S., Cuenca, A. G., Mindrinos, M. N.,
Baker, H. V., Xu, W., et al. (2013). Genomic responses
in mouse models poorly mimic human inflammatory
diseases. Proc. Natl. Acad. Sci. U.S.A. 110, 3507–3512. doi: 10.1073/pnas.1222878110 Received: 09 May 2013; accepted: 11 May 2013; published
online: 27 May 2013. Gupta, U. D., and Katoch, V. M. (2009). Animal models of
tuberculosis for vaccine development. Indian J. Med. Res. 129, 11–18. Frontiers in Immunology | Immunotherapies and Vaccines References Citation: Gibson AJ, Coffey TJ and Werling D (2013) Of
creatures great and small: the advantages of farm animal Veterinary Record. (2011). Call to phase out prophylac-
tic use of antimicrobials in livestock. Vet. Rec. 169,
479–480. doi: 10.1136/vr.d7090 models in immunology research. Front. Immunol. 4:124. doi: 10.3389/fimmu.2013.00124 Hajjar, A. M., Ernst, R. K., Tsai, J. H., Wilson, C. B.,
and Miller, S. I. (2002). Human Toll-like receptor
4 recognizes host-specific LPS modifications. Nat. Immunol. 3, 354–359. doi: 10.1038/ni777 This article was submitted to Frontiers in Immunotherapies
and Vaccines, a specialty of Frontiers in Immunology. Veterinary Record. (2012). US FDA told to stop cer-
tain antibiotics being used as growth promoters. Vet. Rec. 170, 348. doi: 10.1136/vr.e2472 and Vaccines, a specialty of Frontiers in Immunology. Copyright © 2013 Gibson, Coffey and Werling. This is Copyright © 2013 Gibson, Coffey and Werling. This is
an open-access article distributed under the terms of the Copyright © 2013 Gibson, Coffey and Werling. This is
an open-access article distributed under the terms of the
Creative Commons Attribution License, which permits
use, distribution and reproduction in other forums, pro-
vided the original authors and source are credited and
subject to any copyright notices concerning any third-
party graphics etc. Hein, W. R., and Griebel, P. J. (2003). A road less travelled:
large animal models in immunological research. Nat. Rev. Immunol. 3, 79–84. doi: 10.1038/nri977 Visscher, P. M., and Goddard, M. E. (2011). Cattle gain
stature. Nat. Genet. 43, 397–398. doi: 10.1038/ng.819 Jungi, T. W., Farhat, K., Burgener, I. A., and Werling,
D. (2011). Toll-like receptors in domestic ani-
mals. Cell Tissue Res. 343, 107–120. doi: 10.1007/
s00441-010-1047-8 Waters, W. R., Palmer, M. V., Thacker, T. C., Davis,
W. C., Sreevatsan, S., Coussens, P., et al. (2011). Tuberculosis immunity: opportunities from studies May 2013 | Volume 4 | Article 124 | 2 Frontiers in Immunology | Immunotherapies and Vaccines
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Heritability, combining ability and inheritance of storage root dry matter in yam beans
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African crop science journal
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cc-by
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ISSN 1021-9730/2017 $4.00
© 2017, African Crop Science Society ISSN 1021-9730/2017 $4.00
© 2017, African Crop Science Society African Crop Science Journal, Vol. 25, No. 1, pp. 83 - 95
Printed in Uganda. All rights reserved ISSN 1021-9730/2017 $4.00
© 2017, African Crop Science Society African Crop Science Journal by African Crop Science Society is licensed under
a Creative Commons Attribution 3.0 Uganda License. Based on a work
at www.ajol.info/ and www.bioline.org.br/cs
DOI: http://dx.doi.org/10.4314/acsj.v25i1.6 ABSTRACT Storage root dry matter content (RDM) is central to the improvement of consumer and industrial attributes of
root crops. Yam bean (Pachyrhizus species) is a legume root crop newly introduced in Uganda, but its adoption
may be constrained by low RDM. The objective of this study was to investigate the magnitude of variance for
general combining ability (GCA) and specific combining ability (SCA) effects, heritability estimates and inheritance
of RDM in yam beans. Progenies of sixteen crosses, generated using the North Carolina (NCII) mating scheme,
were evaluated in F1 and F2 field experiments, with three replications at the National Crops Resources Research
Institute (NaCRRI), Namulonge in Central Uganda, during the first cropping seasons of April, 2013 and 2014,
respectively. Significant (p<0.001) mean squares for general combining ability (GCA), and specific combining
ability (SCA) were observed in F1 and F2 generations, demonstrating additive and non-additive genetic inheritance
of RDM. High Baker’s ratio in F1 (0.76) and F2 (0.78), and large σ2
GCA/σ2
SCA (3.13) indicate predominance of
additive gene effects. Narrow sense heritability was high in both F1 (0.74) and F2 (0.75), implying that RDM can
be improved in yam beans through selection. The results provide a basis for selection of suitable parents for
hybridisation and breeding programmes to develop high dry matter yam bean varieties in Uganda. Key Words: Baker’s ratio, gene action, Pachyrhizus species R. AGABA, P. TUKAMUHABWA, P. RUBAIHAYO, R.O.M. MWANGA1, A. SSENYONJO1,
J. NDIRIGWE2, S. TUMWEGAMIRE1,3 and W. GRÜNEBERG4
Department of Agricultural Production, Makerere University, P. O. Box 7062, Kampala, Uganda
1International Potato Center (CIP), P. O. Box 22274, Kampala,Uganda
2Rwanda Agriculture Board (RAB), P. O. Box 5016, Kigali, Rwanda
3Current address: International Institute of Tropical Agriculture (IITA), P. O. Box 34441,
Dar es Salaam, Tanzania
4International Potato Center, Apartado 1558, Lima 12, Peru
Corresponding author: rondessblessed@gmail.com (Received 12 November, 2016; accepted 13 February, 2017 Key Words: Baker’s ratio, gene action, Pachyrhizus species HERITABILITY, COMBINING ABILITY AND INHERITANCE OF STORAGE
ROOT DRY MATTER IN YAM BEANS R. AGABA, P. TUKAMUHABWA, P. RUBAIHAYO, R.O.M. MWANGA1, A. SSENYONJO1,
J. NDIRIGWE2, S. TUMWEGAMIRE1,3 and W. GRÜNEBERG4
Department of Agricultural Production, Makerere University, P. O. Box 7062, Kampala, Uganda
1International Potato Center (CIP), P. O. Box 22274, Kampala,Uganda
2Rwanda Agriculture Board (RAB), P. O. Box 5016, Kigali, Rwanda
3Current address: International Institute of Tropical Agriculture (IITA), P. O. Box 34441,
Dar es Salaam, Tanzania
4International Potato Center, Apartado 1558, Lima 12, Peru
Corresponding author: rondessblessed@gmail.com INTRODUCTION Rodríguez-Navarro et al., 2009) because of
its capability to improve soil fertility through
Rhizobium-Bradyrhizobium-facilitated
nitrogen-fixation (Castellanos et al., 1997),
minimal pest incidences due to insecticidal
rotenone in seeds and shoot (Grüneberg et al.,
1999; Lautié et al., 2013), relatively short
growth cycles, and wide geographic adaptation
even in semi-arid conditions (Diouf et al.,
1998; Belford et al., 2001). Yam bean gives
high and stable yields of up to 80 t ha-1 of edible
storage roots (Sørensen, 1996; Karuniawan,
2004); making its yield potential comparable
to other root and tuber crops, such as cassava
(Manihot esculenta Crantz) and sweetpotato
(Ipomoea batatas) (Zanklan et al., 2007). Rodríguez-Navarro et al., 2009) because of
its capability to improve soil fertility through
Rhizobium-Bradyrhizobium-facilitated
nitrogen-fixation (Castellanos et al., 1997),
minimal pest incidences due to insecticidal
rotenone in seeds and shoot (Grüneberg et al.,
1999; Lautié et al., 2013), relatively short
growth cycles, and wide geographic adaptation
even in semi-arid conditions (Diouf et al.,
1998; Belford et al., 2001). Yam bean gives
high and stable yields of up to 80 t ha-1 of edible
storage roots (Sørensen, 1996; Karuniawan,
2004); making its yield potential comparable
to other root and tuber crops, such as cassava
(Manihot esculenta Crantz) and sweetpotato
(Ipomoea batatas) (Zanklan et al., 2007). Yam bean (Pachyrhizus sp.) is a storage root-
forming legume of neo-tropical origin
(Sørensen, 1996), but was recently introduced
in East and Central Africa for food production
(Heider et al., 2011). The crop belongs to the
genus Pachyrhizus Rich. ex DC; subtribe
Diocleinae, tribe Phaseoleae and the Fabaceae
legume family (Sørensen, 1988). The genus
consists of five species of which two, namely
P. ferrugineous (Piper) Sørensen and P. panamensis Clausen are still wild; and P. erosus
(L.) Urban, P. ahipa (Wedd.) Parodi and P. tuberosus (Lam.) Spreng are in cultivation
(Lackey,1977; Ingham, 1990). All Pachyrhizus
species are propagated by true seed, diploid
(2n=2x=22), self-compatible and permit
successful interspecific hybridisation
(Sorensen, 1990; Grüneberg et al.,2003). Storage root dry matter content in root
crops is important for consumption and
industrial processing (Tumwegamire et al.,
2011), yet most yam bean varieties have low
RDM which limits adoption (Zanklan et al.,
2007; Agaba et al., 2016). Potential for
improvement of RDM lies in hybridisation with
the high RDM cultivar of Pachyrhizus tuberosus
called the chuin (Ørting et al., 1996;
Grüneberg et al., 2003; Zanklan et al., 2007). RÉSUMÉ La teneur en matières èche du stock racinaire (MSR) est essentiel en terme d’amélioration des qualités des cultures
racinaires pour les consommateurs et d’industriels. Le haricot-igname (espèce Pachyrhizus) est une culture
racinaire légumineuse nouvellement introduite en Ouganda, mais son adoption pourrait être handicapée par
sateneur basse en matières èche racinaire. Cette étude avait pour objectif d’étudier le niveau de la variance pour
les effets de la capacité de combinaison générale et la capacité de combinaison spécifique, estimations de l’héritabilité
et l’héritage de MSR dans les haricots ignames. Les progénies de seize croisements générés à l’aide du système
de croisement de la Caroline du Nord (NCII), ont été évalués dans des essais en champs de F1 et F2 avec trois
répétitions à l’Institut National de Recherche sur les Ressources Végétales (NaCRRI), Namulonge en Ouganda,
pendant les premières saisons culturalesd’ Avril 2013 et 2014, respectivement. Les carrés moyens significatifs
(P<0,001) de capacité généralede combinaison et capacité spécifique de combinaison étaient observés dans les 84 R. AGABA et al. générations F1 et F2 démontrant ainsi un heritage génétique additif et non additive de MSR. Le rapport élevé de
Baker en F1 (0,76) et F2 (0,78), et σ2
GCA/σ2
SCA (3.13) large indiquent la prédominance des effets de gènes additifs. Une étroite héritabilité d’ordre de F1 (0.74) et F2 (0.75) était observée, ce qui implique que MSR peut être
amélioré dans le haricot-igname par voie de sélection. Les résultats constituent une base pour la sélection des
parents appropriés pour les programmes d’hybridation et d’amélioration génétique pour développer des variétés
de haricot-ignames à haute teneur en matièresè che en Ouganda. Mots Clés: Ratpport de Baker, action génique, espèce Pachyrhizus Mots Clés: Ratpport de Baker, action génique, espèce Pachyrhizus INTRODUCTION The chuin is known for high RDM (26 to 36%)
and can be cooked or processed like cassava
to “gari”, a food staple for millions of people
in West Africa (Zanklan et al., 2007; Padonou
et al., 2013). g
Yam bean is traditionally a minor and
underutilised crop (Sørensen, 1996; Jacobsen
et al., 2015), mainly because of low storage
root dry matter (RDM) content that limits its
food and industrial use (Rizky et al., 2013;
Agaba et al., 2016). Nonetheless, the crop has
attracted scientific and nutritional interest
because its storage roots contain vitamins and
micronutrients, such as iron and zinc (Noman
et al., 2007; Dini et al., 2013), 56 to 58%
starch (Forsyth et al., 2002) and 8 to 18%
proteins (Velasco and Grüneberg, 1999;
Zanklan et al., 2007) on dry weight basis. Yam
bean may offer nutritional benefits to local
populations, where micro-nutrient deficiencies
are rampant such as communities in the new
introduction areas of East and Central Africa. The crop is suitable for low-input agriculture
on small farm holdings (Zanklan et al., 2007; Through hybridisation and selection, the
chuin may improve dry matter content of P. erosus accessions with high yields, wide
adaptation, and drought tolerance (Belford et
al ., 2001) and the P. ahipa accessions, with
early maturity and erect or semi-erect growth
habit (Zanklan et al., 2007). While studying
the possibility of incorporating high dry matter Storage root dry matter in yam beans 85 and three P. erosus accessions. The crosses
were done following North Carolina II (NC
II) mating design, with no reciprocals in
accordance with Comstock and Robinson
(1952) to generate a mixture of 16 full- and
half-sib F1 families. from the chuin into the broader yam bean gene
pool, Grüneberg et al. (2003) observed fertile
and vigorous interspecific hybrids between P. tuberosus-chuin and P. erosus and P. ahipa
accessions. The improvement of storage RDM has been
identified as a major breeding goal for yam
beans (Grüneberg et al., 2003; Zanklan et al.,
2007), but the crop remains genetically under-
exploited; with a general lack of genetic studies
on dry matter content. A recent study by Jha
and Singh (2014) reported positive and
significant combining abilities (GCA and SCA
effects) for quantitative traits in Mexican yam
bean (Pachyrhizus erosus L. Urban) such as
root yield, root length and root girth. INTRODUCTION However,
these results have not been corroborated
across the cultivated yam bean species and no
progress has been made to study inheritance
of RDM in yam beans. This study, therefore,
was conducted to estimate variance
components, combining ability and heritability
so as to infer the inheritance of RDM in yam
bean as a basis for improvement through
selection. Field evaluations. The seeds of each of the
16 F1 progenies and their 9 parents were
planted under field conditions at Namulonge,
during the first rains of April, 2013. Plots
consisted of two ridges, each measuring 3
metres long and 1 metre apart. Seeds were
sown on ridges by hand at a spacing of 0.3
metre between plants. Plots were arranged in
a randomised complete block design (RCBD)
and replicated twice due to limited seed. The experiments were kept weed free and
no reproductive pruning of flowers was done
or fertilisers or agrochemicals applied during
the crop growth cycle. From the F1
experiment, seeds were allowed to mature and
were harvested on single plant basis, dried and
planted in an F2 experiment during the first
rains of 2014. However, two crosses, namely
209022 x 209015 and 209031 x 209015, did
not produce F1 seed and were, therefore,
excluded from the evaluations (Table 1). MATERIALS AND METHODS Experimental sites. The field study was
carried out at the National Crops Resources
Research Institute (NaCRRI) at Namulonge in
Central Uganda. It is located at 00 32’N and
320 35’E, 1150 metres above sea level, and
experiences a mean annual temperature of 22.2
oC and a bimodal rainfall pattern with a mean
of 1270 mm. Namulonge is characterised by
red ferralitic soils with low pH (4.9-5.0)
(Tumwegamire et al., 2011) Data collection. The F1 and F2 experiments
were harvested six months after planting in
2013 and 2014, respectively. Three storage
roots were randomly selected from each
genotype and taken to the NaCRRI nutrition
quality laboratory for determination of RDM. The sampled roots were washed thoroughly
using flowing tap-water to minimise soil
contamination, then peeled and sectioned into
multiple longitudinal slices. The slices were
mixed together and a sample of 100 g fresh
weight was packed in polythene bags and
vacuum-freeze dried at -31oC for 72 hours,
using a vacuum freeze drier, YK-118-50, True-
Ten Industries, Korea, 1995. The freeze dried
samples were weighed immediately after
leaving the freeze drier and their dry weight Development of breeding populations. Nine yam bean accessions, with varying RDM,
were crossed to generate F1 progenies (Table
1). Three high dry matter cultivars belonging
to P. tuberosus-chuin were used as male
parents and crossed with six low dry matter
female parents; three of which were P. ahipa 86 R. AGABA et al. TABLE 1. CIP code names and characteristics of parents and crosses evaluated in F1 and F2 under field
conditions at NaCRRI, Namulonge in Central Uganda
CIP code names
Species
Plant type
bRDM Classification
Crosses
Parents
209076
209004
P. ahipa
Erect
Low RDM
209013
P. tuberosus - chuin cultivar
Spreading
High RDM
209077
209004
P. ahipa
Erect
Low RDM
209014
P. tuberosus - chuin cultivar
Spreading
High RDM
209079
209022
P. ahipa
Erect
Low RDM
209013
P. tuberosus - chuin cultivar
Spreading
High RDM
209078
209004
P. ahipa
Erect
Low RDM
209015
P. tuberosus - chuin cultivar
Spreading
High RDM
209080
209022
P. ahipa
Erect
Low RDM
209014
P. tuberosus - chuin cultivar
Spreading
High RDM
209082
209031
P. ahipa
Erect
Low RDM
209013
P. tuberosus - chuin cultivar
Spreading
High RDM
209083
209031
P. ahipa
Erect
Low RDM
209014
P. tuberosus - chuin cultivar
Spreading
High RDM
209085
209016
P. erosus
Spreading
Low RDM
209013
P. MATERIALS AND METHODS tuberosus - chuin cultivar
Spreading
High RDM
209086
209016
P. erosus
Spreading
Low RDM
209014
P. tuberosus - chuin cultivar
Spreading
High RDM
209087
209016
P. erosus
Spreading
Low RDM
209015
P. tuberosus - chuin cultivar
Spreading
High RDM
209088
209018
P. erosus
Spreading
Low RDM
209013
P. tuberosus - chuin cultivar
Spreading
High RDM
209089
209018
P. erosus
Spreading
Low RDM
209014
P. tuberosus - chuin cultivar
Spreading
High RDM
209090
209018
P. erosus
Spreading
Low RDM
209015
P. tuberosus - chuin cultivar
Spreading
High RDM
209091
209019
P. erosus
Spreading
Low RDM
209013
P. tuberosus - chuin cultivar
Spreading
High RDM
209092
209019
P. erosus
Spreading
Low RDM
209014
P. tuberosus - chuin cultivar
Spreading
High RDM
209093
209019
P. erosus
Spreading
Low RDM
209015
P. tuberosus - chuin cultivar
Spreading
High RDM
a CIP = International Potato Center; b RDM = storage root dry matter content; Low RDM< 20%; High RDM >
25% TABLE 1. CIP code names and characteristics of parents and crosses evaluated in F1 and F2 under field
conditions at NaCRRI, Namulonge in Central Uganda a CIP = International Potato Center; b RDM = storage root dry matter content; Low RDM< 20%; High RDM >
25% recorded. RDM was calculated as the average
difference between flesh and dry weight
estimates (Wilken et al., 2008). were further processed using the general
combining ability model in which male and
female parents were fited as fixed factors and
the residual taken as an estimate of SCA effects
(female x male interaction). GCA effects were
estimated from the parameter estimates for
parents from the ANOVA of North Carolina II
design (NCII); while the SCA effects were
calculated as the difference between the
observed and predicted means of RDM in Statistical and genetic analyses. Mean
performance of the crosses and parents were
obtained based on the general analysis of
variance model in GenStat 14th edition (VSN
International Ltd, Hemel Hempstead, UK)
(Payne et al., 2011). The means of crosses Storage root dry matter in yam beans 87 The gene action controlling inheritance of
RDM in yam bean was inferred from the
relative magnitudes of GCA and SCA. Baker’s
ratio was used to estimate the relative
significance of additive and non-additive gene
effects (Baker, 1978). MATERIALS AND METHODS In the estimation of
genetic parameters using NCII ANOVA, we
assumed no maternal effects and epistasis
(Kearsey and Pooni, 1998) in the inheritance
of RDM in yam beans. crosses. The significance of GCA and SCA
effects were tested using a standard t-test. The
estimates of narrow sense heritability (NSH),
broad sense heritability (BSH) and Baker’s ratio
were calculated from the GCA and SCA
variance components analysis following
Falconer et al. (1996); NSH = [ σ2
GCA( Female) + σ2
GCA(Male)] / [σ2
GCA(Female) NSH = [ σ2
GCA( Female) + σ2
GCA(Male)] / [σ2
GCA(Female) + σ2
GCA( Male)+ σ2
SCA+ σ2
e], + σ2
GCA( Male)+ σ2
SCA+ σ2
e], σ2 = Sample variance, σ2 = Sample variance, [σ2
GCA (Female) + σ2
GCA (Male)] = Additive gene
effects, [σ2
GCA(Female) + σ2
GCA(Male)+ σ2
SCA+ σ2
e] =
Phenotypic effects, and [σ2
GCA(Female) + σ2
GCA(Male)+ σ2
SCA+ σ2
e] =
Phenotypic effects, and σ2
e = Error variance While BSH, which estimates the proportion of
phenotypic variation due to total genetic
effects: BSH = [σ2
GCA (Female) + σ2
GCA (Male) + σ2
SCA] /
[σ2
GCA (Female) + σ2
GCA (Male) + σ2
SCA+ σ2
e], RESULTS Where: Where: Analysis of variance. The mean squares of
crosses were highly significant (P< 0.001) in
both F1 and F2 generations (Table 2). Also,
the mean squares for male general combining
ability (GCAmale) were significant (P<0.01) for
RDM in both F1 and F2 generations. However,
the GCA estimates for female parents were
not consistent across F1 and F2 generations,
giving significant (P<0.001) mean squares only
in F2. Moreover, the mean squares for female
GCA affects were four times higher than for
the male parents in the F2 generation (Table
2). The interaction mean squares between male
and female parents (SCA effects) were highly
significant (P<0.001) in both F1 and F2
generations. The variance components for
GCA effects were higher than SCA effects
giving a predictability ratio (σ2
GCA/σ2
SCA) higher
than unity for males in F1, as well as females
in F2 (Table 2). The predictability ratio for
GCAmale were three times as large as the SCA
effects (σ2
GCA/σ2
SCA = 3.13) in F1 generation. However, the combining ability of both parents
was not consistent because the predictability
ratio for female parents and male parents in F1
and F2, respectively, were lower than unity. Where: [σ2
GCA (Female) + σ2
GCA (Male) + σ2
SCA] = [σ2
GCA (Female) + σ2
GCA (Male) + σ2
SCA] = Total genetic effects, and [σ2
GCA (Female) + σ2
GCA
(Male) + σ2
SCA+ σ2
e] = Phenotypic effects [σ2
GCA (Female) + σ2
GCA Heritability and Bakars’ ratio. The RDM
showed high narrow sense heritability
estimates namely, 0.74 in F1 and 0.75 in F2
generation. Similarly, the broad sense
heritability estimates were high (0.97) across
F1 and F2 generations. Baker’s ratio estimates
for RDM were also consistently high at 0.76
and 0.78 in F1 and F2, respectively (Table 2). (Male) + σ2
SCA+ σ2
e] = Phenotypic effects (Male) + σ2
SCA+ σ2
e] = Phenotypic effects Then Baker’s ratio (BR) was calculated as: BR = [σ2
GCA (Female) + σ2
GCA (Male)]/ [σ2
GCA (Female) + σ2
GCA (Male) + σ2
SCA] 88 R. AGABA et al. TABLE 2. Mean squares, combining abilities, heritabilities and Baker’s ratios for RDM in F1 and F2 yam bean
progenies evaluated at Namulonge in Central Uganda
df
F1 Population
F2 Population
Source of variation Mean squares F test Mean squares F test
Crosses
15
22.23***
9.18E-08
22.79***
2.85E-13
GCAfemale
5
0.91
0.757449
39.37
6.56E-13
GCAmale
2
72.25***
2.39E-08
10.54**
0.001394
SCA
8
23.05***
5.99E-07
14.44***
1.02E-07
Residual
23
1.745
1.38
CV%
6.16
6.63
Variance components
σ2GCAF
0.52
28.53
σ2GCAM
41.40
7.64
σ2SCA
13.21
10.46
σ2GCAF/σ2SCA
0.04
2.73
σ2GCAM/σ2SCA
3.13
0.73
NSH
0.74
0.75
BSH
0.97
0.97
Bakers’ ratio
0.76
0.78
df = degrees of freedom; CV = coefficient of variation; GCA = General combining ability; SCA = specific
combining ability; NSH = Narrow sense heritability; BSH = Broad sense heritability; σ2 = variance; F = Female;
M= Male; ** and *** significant at P<0.01 and<0.001, respectively TABLE 2. Mean squares, combining abilities, heritabilities and Baker’s ratios for RDM in F1 and F2 yam bean
progenies evaluated at Namulonge in Central Uganda df = degrees of freedom; CV = coefficient of variation; GCA = General combining ability; SCA = specific
combining ability; NSH = Narrow sense heritability; BSH = Broad sense heritability; σ2 = variance; F = Female;
M= Male; ** and *** significant at P<0.01 and<0.001, respectively the cross 209019 x 209014 had negative
SCA effects in both F1and F2 generations. Combining ability effects. Where: The evaluated parents could be classified into
two groups, namely high dry matter genotypes
(209013, 209014 and 209015) with a range
of 29.32 to 33.21% and the low dry matter
group (209004, 209016, 209018, 209019,
209022 and 209031) with RDM of 7.83 to
15.72% (Fig. 1). RDM values at 23.5 and 22.4% respectively,
in comparison to the mean of 17.73% in the
F2 generation. Crosses, 209004 x 209015,
209018 x 209014, 209018 x 209015 and
209031 x 209014, had higher dry matter values
than their respective generation means across
F1 and F2 populations. The cross 209022 x
209013 had the lowest RDM at 10.32 and
5.89% in F1 and F2 generations, respectively. The evaluated parents could be classified into
two groups, namely high dry matter genotypes
(209013, 209014 and 209015) with a range
of 29.32 to 33.21% and the low dry matter
group (209004, 209016, 209018, 209019,
209022 and 209031) with RDM of 7.83 to
15.72% (Fig. 1). Parent
GCA effects
F1 Population
F2 Population
209013
-3.701***
-1.137***
209014
3.169***
1.683***
209015
0.799
-0.397
209004
0.376
0.373
209016
-0.924
3.323***
209018
0.116
3.163***
209019
-0.124
-0.887
209022
1.056
-10.307***
209031
-0.224
-0.817
SE
1.159
2.985
SE = Standard error; *** Significant at P<0.001 S.E = standard error; *** significant at P<0.001 Where: On the basis of
the estimated GCA effects (Table 3), male
parent 209014 contributed positive and
significant GCA effects (P<0.001) in F1 and
F2 generation while female parents; 209016 and
209018 showed significant and positive GCA
effects only in F2 generation. Negative and
significant GCA effects were recorded for male
parent 209013 in both F1 and F2, as well as
female parent, 209022 in F2 generation. Performance of parents and progenies. The RDM values for crosses in F1
generation varied from 10.32% in 209022
x 209013 to 34.11% in 209022 x 209014
(Fig. 1). However, the best performing
cross (209022 x 209014) in F1 generation
did not produce F2 seed and was eliminated
from further analysis. SCA effects were highly significant
(P<0.001) for crosses 209022 x 209013 and
209022 x 209014 in F1 generation, but these
crosses did not produce F2 seed and could not
be evaluated in the F2 experiment (Table 4). Crosses that gave positive SCA effects across
F1 and F2 included 209004 x 209014, 209016
x 209013, 209018 x 209013, 209018 x 209015
and 209019 x 209013 (Table 4). Conversely, y
The cross 209004 x 209014 with RDM
of 25.43% was the next best, and
consistently performed better than the
respective means in F1 and F2 populations. Whereas crosses, 209016 x 209013 and
209016 x 209014 had less RDM than the
population mean of 21.43% in F1 generation,
the same crosses rebounded with higher Storage root dry matter in yam beans 89 TABLE 3. General combining ability (GCA effects)
for RDM in the F1 and F2 yam bean progenies
evaluated at Namulonge in Central Uganda TABLE 3. General combining ability (GCA effects)
for RDM in the F1 and F2 yam bean progenies
evaluated at Namulonge in Central Uganda RDM values at 23.5 and 22.4% respectively,
in comparison to the mean of 17.73% in the
F2 generation. Crosses, 209004 x 209015,
209018 x 209014, 209018 x 209015 and
209031 x 209014, had higher dry matter values
than their respective generation means across
F1 and F2 populations. The cross 209022 x
209013 had the lowest RDM at 10.32 and
5.89% in F1 and F2 generations, respectively. Genetic variability and inheritance of RDM. Genetic variability and inheritance of RDM. The combining ability analysis revealed
significant variances for both GCA and SCA
effects demonstrating that both additive and
non-additive gene effects play vital roles in the
expression of RDM in early generations of yam
bean breeding. The results revealed the
presence of significant genetic variation
between crosses and among both male and
female parents indicating potential for selection
of high dry matter lines to improve RDM in
yam beans. GCA mean squares were three and
two times higher than SCA mean squares for
males and females in F1 and F2, respectively
(Table 2) which suggested preponderance of
additive gene action in the inheritance of RDM. Nonetheless, significant SCA mean squares for
RDM in F1 and F2,indicated non-allelic
interaction among the parents and
demonstrated inconsistence of males over
different female parents across the two
generations. TABLE 4. Specific combining ability (SCA) effects
for RDM in F1 and F2 yam bean progenies evaluated at
Namulonge in Central Uganda
SCA estimates
Crosses
F1 Population F2 Population
209004 x 209013
0.086
-2.197
209004 x 209014
0.46
0.499
209004 x 209015
-0.546
1.698
209016 x 209013
1.423
3.59
209016 x 209014
-2.134
0.716
209016 x 209015
0.711
-4.306
209018 x 209013
2.22
1.383
209018 x 209014
-2.627
-2.711
209018 x 209015
0.407
1.328
209019 x 209013
3.09
0.327
209019 x 209014
-2.517
-1.607
209019 x 209015
-0.573
1.281
209022 x 209013
-8.462***
-
209022 x 209014
8.462***
-
209031 x 209013
1.643
-3.103
209031 x 209014
-1.643
3.103
S.E
1.868
0.959
S.E = standard error; *** significant at P<0.001 TABLE 4. Specific combining ability (SCA) effects
for RDM in F1 and F2 yam bean progenies evaluated at
Namulonge in Central Uganda High narrow sense heritability suggested
that the performance of the progenies could
be succinctly predicted from the parents and
confirmed potential for selection of superior 90 R. AGABA et al. 90
Figure 1. Storage root dry matter content of parents and crosses in F1 and F2 yam bean populations evaluated
at Namulonge in Central Uganda. RDM (%)
RDM (%)
Crosses
Parents
F1 RDM (%)
F2 RDM (%)
F1 RDM (%)
F2 RDM (%) R. AGABA et al. RDM (%)
Crosses
F1 RDM (%)
F2 RDM (%) Figure 1. Storage root dry matter content of parents and crosses in F1 and F2 yam bean populations evaluated
at Namulonge in Central Uganda. Genetic variability and inheritance of RDM. Conversely,
parents with negative significant GCA effects,
namely, 209013 and 209022 are less desirable
in the development of high dry matter yam
beans because such parents might be poor
combiners for RDM. parents; 209016 and 209018 had high GCA
effects which made them good combiners for
improvement of RDM in yam beans. The male
parent, 209014 had significant GCA effects
which identified it as the only good combiner
for improvement of RDM among the three P. tuberosus-chuins used. According to Kearsey
and Pooni (1998), the magnitudes of GCA and
SCA estimates shows the importance of
additive and non-additive gene effects in the
inheritance of quantitative traits and offer vital
genetic information about potential genetic
progress in breeding programmes. Therefore,
a breeder may use the current parents
(209014, 209016 and 209018) to improve
RDM in yam beans through interspecific
hybridisations. These parents might constitute
vital sources of high RDM genes for initiating
a breeding programme to improve RDM among
the yam bean germplasm in Uganda. The
results further identified the parents with
positive but non-significant GCA estimates
either in F1 or F2 (209015, 209004, and
209022) as suitable genotypes for inclusion in
yam bean breeding programmes. Conversely,
parents with negative significant GCA effects,
namely, 209013 and 209022 are less desirable
in the development of high dry matter yam
beans because such parents might be poor
combiners for RDM. in yam beans, and shows that progeny
performance can be predicted based on GCA
effects of parents. This observation is
supported by the argument that the ratio of
additive to non-additive variance in a population
gives an indication of relative importance of
GCA and SCA estimates in predicting progeny
performance (Owolade et al., 2009). High predictability ratios (σ2
GCA/σ2
SCA)
confirmed the prevalence of additive gene
effects in the inheritance of RDM. Bi et al. (2015) argued that a large ratio between GCA
and SCA effects reveals the relevance of
additive gene effects while a small value
signifies the presence of dominant and/or
epistatic gene effects. This finding rationalises
our earlier notion that additive gene effects are
more influential than non-additive gene effects
in the inheritance of RDM in yam beans. y
The presence of additive gene effects
indicates a high chance for predicting the
performance of progenies based on phenotypic
scores of the parents. Genetic variability and inheritance of RDM. RDM (%)
Crosses
Parents
F1 RDM (%)
F2 RDM (%) Parents Figure 1. Storage root dry matter content of parents and crosses in F1 and F2 yam bean populations evaluated
at Namulonge in Central Uganda. reported elsewhere (Sørensen, 1996; Agaba et
al., 2016). genotypes with significant response to
selection for RDM in early generations of yam
bean breeding. The broad sense heritability was
similarly high indicating the genetic potential
of the parents to largely pass on the RDM trait
to subsequent generations. This result is in
agreement with the findings by Grüneberg et
al. (2003) which demonstrated that
introgression of high RDM from the chuin into
the remaining cultivated yam bean gene pool
was possible resulting in feritle and vigorous
progenies. High heritability estimates for RDM
and associated traits in yam beans have been The high estimates for Baker’s ratio in F1
and F2 generations (Table 2) indicated the
importance of additive genetic effects in the
inheritance of RDM in yam bean which
suggests that the genes controlling RDM can
easily be fixed in advanced generations. A ratio
that is closer to unity enhances the argument
for predominance of additive genes in the
inheritance of the trait. High Baker’s ratio
reveals the relative importance of GCA to SCA
effects in the genetic determination of RDM Storage root dry matter in yam beans 91 parents; 209016 and 209018 had high GCA
effects which made them good combiners for
improvement of RDM in yam beans. The male
parent, 209014 had significant GCA effects
which identified it as the only good combiner
for improvement of RDM among the three P. tuberosus-chuins used. According to Kearsey
and Pooni (1998), the magnitudes of GCA and
SCA estimates shows the importance of
additive and non-additive gene effects in the
inheritance of quantitative traits and offer vital
genetic information about potential genetic
progress in breeding programmes. Therefore,
a breeder may use the current parents
(209014, 209016 and 209018) to improve
RDM in yam beans through interspecific
hybridisations. These parents might constitute
vital sources of high RDM genes for initiating
a breeding programme to improve RDM among
the yam bean germplasm in Uganda. The
results further identified the parents with
positive but non-significant GCA estimates
either in F1 or F2 (209015, 209004, and
209022) as suitable genotypes for inclusion in
yam bean breeding programmes. CONCLUSION Previously, significant GCA and SCA
effects and predominance of additive genes
for inheritance of root traits such as root length,
girth yield and earliness in yam beans were
reported by Jha and Singh (2014). Even though
Jha and Singh (2014) did not investigate the
dry matter trait, it could be hypothesised that
inheritance of RDM would not significantly
deviate from the other quantitative root traits
in yam beans. Dry matter in parents and progenies. The
mean RDM content of parents and crosses in
the evaluated yam bean germplasm (Fig. 1)
falls within the ranges reported elsewhere
(Sørensen, 1996; Grüneberg et al., 2003;
Zanklan et al., 2007). However, the yam bean
parents used in this study can be categorised
into the low RDM genotypes (P. erosus and P. ahipa) and high dry matter genotypes that
belong to P. tuberosus - chuin cultivar. The
chuin is the only high dry matter cultivar
among the four groups of P. tuberosus known
as Ashipa, Yushpe, and Jíquima cultivars
(Sørensen, 1996). Recent molecular
characterisation has shown a clear separation
between chuin and Ashipa cultivar groups
(Santayana et al., 2014). The use of the chuin
in this study demonstrated high transmissibility
of the RDM trait in yam beans, and the results
demonstrated high potential for crop
improvement and integration of the yam bean
into new farming systems in Africa as
envisaged by Zanklan et al., 2007 and Heider
et al., 2011. This finding revealed that the
evaluated yam bean germplasm has potential
for improvement of storage root dry matter
content (RDM) which could accelerate
adoption of the crop in Uganda where high
dry matter trait is preferred among root crops
for consumption and industrial applications. Genetic variability and inheritance of RDM. The action of additive
gene effects in the inheritance of RDM is
supported by the arguments of Grüneberg et
al. (2003), that improvement of root dry matter
content in the yam beans is possible through
inter- and intra- specific hybridisation. However, gene effects in the present study
might have been overestimated because
combining ability studies from one location
tend to have high genetic effects and capture
less environmental variance. Such results
cannot be generalised beyond the particular test
environment. Consequently, increasing sample
size, replication and testing of progenies
across multi-locations might improve genetic
parameter estimates for RDM in yam beans. Crosses with positive SCA effects in F1 and
F2 (Table 4) represent promising families to
select for high RDM genotypes in future yam
bean breeding programmes. It was observed
that crosses with positive and significant SCA
effects also gave high values of RDM
compared to the population means in F1 and
F2 generations. Such associations between SCA
effects and mean performance of the crosses
have been reported in yam beans (Jha and
Singh, 2014), suggesting that enviromental
factors suh as drought would influence RDM
of root crops. This findng is supported by the
obervations of Zanklan et al. (2007) which
reaveled high root dry matter content in yam
beans grown under water stressed conditions
at Niaouli compared to the irrigated location Combining abilities. Significant GCA effects
were observed for several parents in this study
(Table 3) suggesting the presence of additive
gene effects in the expression of RDM among
the parents. The positive GCA effects obtained
identified most of the current parents with
potential for use in breeding programmes to
develop high RDM yam bean varieties. Female 92 R. AGABA et al. ACKNOWLEDGEMENT The authors acknowledge the National Crops
Resources Research Institute (NaCRRI)-
Namulonge for provision of field and laboratory
resources. We thank Dr. Ruth Albertyn for the
critical reviews and tutorials in scholarly writing
offered to the first author during the drafting
of this manuscript. This research was funded
by the International Potato Center (CIP) with
a grant from the Belgium Development
Corporation (BTC). Additional funding was
obtained from the Regional Universities Forum
for Capacity Building in Africa (RUFORUM)
under the grant, “Training the next generation
of scientists” with funding from Carnegie
Cooperation of New York. CONCLUSION at Songhai in Benin. The crosses that failed to
produce F2 seed in this study demonstrated
the inherent low rate of successful crossings
and high rates of flower abortions associated
with interspecific hybridisation among
Pachyrhizus species (Grüneberg et al., 2003). Previously, significant GCA and SCA
effects and predominance of additive genes
for inheritance of root traits such as root length,
girth yield and earliness in yam beans were
reported by Jha and Singh (2014). Even though
Jha and Singh (2014) did not investigate the
dry matter trait, it could be hypothesised that
inheritance of RDM would not significantly
deviate from the other quantitative root traits
in yam beans. at Songhai in Benin. The crosses that failed to
produce F2 seed in this study demonstrated
the inherent low rate of successful crossings
and high rates of flower abortions associated
with interspecific hybridisation among
Pachyrhizus species (Grüneberg et al., 2003). at Songhai in Benin. The crosses that failed to
produce F2 seed in this study demonstrated
the inherent low rate of successful crossings
and high rates of flower abortions associated
with interspecific hybridisation among
Pachyrhizus species (Grüneberg et al., 2003). Previously, significant GCA and SCA
effects and predominance of additive genes
for inheritance of root traits such as root length,
girth yield and earliness in yam beans were
reported by Jha and Singh (2014). Even though
Jha and Singh (2014) did not investigate the
dry matter trait, it could be hypothesised that
inheritance of RDM would not significantly
deviate from the other quantitative root traits
in yam beans. This study demonstrates that the evaluated yam
bean germplasm has potential for improvement
of storage root dry matter content (RDM) in
yam beans. It is also clear that the inheritance
of RDM in yam beans is conditioned by both
additive and non-additive gene effects;
although additive gene action is predominant
and associated with high Baker’s ratios and
narrow-sense heritabilities across F1 and F2
generations. The predominance of GCA over
SCA variance suggest that stringent early
generation selection would be effective to
improve RDM in the current yam bean
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Zanklan, A.S., Ahouangonou, S., Becker, H.C.,
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Evaluation of the storage root-forming
legume yam bean (spp.) under West African
Conditions. Crop Science 47(5):1934-1946. REFERENCES A combining ability
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https://zenodo.org/records/229900/files/BDJ_article_11337.pdf
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English
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Long-term seasonal dominance of the wasp Trihapsis polita Townes (Hymenoptera, Ichneumonidae) in the Brazilian Atlantic Forest
|
Biodiversity data jurnal
| 2,017
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cc-by
| 4,139
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Biodiversity Data Journal 5: e11337
doi: 10.3897/BDJ.5.e11337
Data Paper
Long-term seasonal dominance of the wasp
Trihapsis polita Townes (Hymenoptera,
Ichneumonidae) in the Brazilian Atlantic Forest
Bernardo F Santos , Alexandre P Aguiar , Anazélia M Tedesco , Julio C R Fontenelle
‡ Division of Invertebrate Zoology, American Museum of Natural History, New York, United States of America
§ Departamento de Ciências Biológicas, Universidade Federal do Espírito Santo, Vitória, Brazil
| Secretaria de Estado de Meio Ambiente e Recursos Hídricos, Cariacica, Brazil
¶ Instituto Federal de Minas Gerais, Campus Ouro Preto, Ouro Preto, Brazil
Corresponding author:
Academic editor: Jose Fernandez-Triana
Received: 25 Nov 2016 | Accepted: 28 Dec 2016 | Published: 04 Jan 2017
Citation: Santos B, Aguiar A, Tedesco A, Fontenelle J (2017) Long-term seasonal dominance of the wasp
Trihapsis polita Townes (Hymenoptera, Ichneumonidae) in the Brazilian Atlantic Forest. Biodiversity Data Journal
5: e11337. https://doi.org/10.3897/BDJ.5.e11337
Abstract
‡
§
|
¶ Biodiversity Data Journal 5: e11337
doi: 10.3897/BDJ.5.e11337
Data Paper Background The temporal dynamics of insect populations in tropical environments is highly complex
and poorly known. Long-term seasonality studies are scarce, and particularly so for
ichneumonid wasps (Hymenoptera Ichneumonidae). This study represents an effort to
elucidate aspects of seasonality and forest succession in the Brazilian Atlantic Forest. Long-term seasonal dominance of the wasp
Trihapsis polita Townes (Hymenoptera,
Ichneumonidae) in the Brazilian Atlantic Forest ‡ Division of Invertebrate Zoology, American Museum of Natural History, New York, United States of America
§ Departamento de Ciências Biológicas, Universidade Federal do Espírito Santo, Vitória, Brazil
| Secretaria de Estado de Meio Ambiente e Recursos Hídricos, Cariacica, Brazil
¶ Instituto Federal de Minas Gerais, Campus Ouro Preto, Ouro Preto, Brazil Corresponding author:
Academic editor: Jose Fernandez-Triana
Received: 25 Nov 2016 | Accepted: 28 Dec 2016 | Published: 04 Jan 2017
Citation: Santos B, Aguiar A, Tedesco A, Fontenelle J (2017) Long-term seasonal dominance of the wasp
Trihapsis polita Townes (Hymenoptera, Ichneumonidae) in the Brazilian Atlantic Forest. Biodiversity Data Journal
5: e11337. https://doi.org/10.3897/BDJ.5.e11337 © Santos B et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY
4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are
credited. Keywords Malaise trap; parasitoid; phenology; outbreak; Trihapsis polita; secondary forest Santos B et al. Santos B et al. Cryptinae were collected, of which 6,655 (79.4%) belonged to T. polita. The occurrence of
T. polita species was heavily concentrated in the RS, with abundance 148× higher than
during the DS. Seasonal fluctuation was also detected for Cryptinae as a whole, but was
two orders of magnitude lower. Sampling efficiency also varied widely among areas, with
the peak of abundance at the tall secondary forest. The dominance of T. polita in
secondary vegetation might be of general interest, as this type of forest is currently on the
rise, due to unprecedented levels of human pressure. New information We report on the seasonal and successional dominance of the ichneumonid wasp
Trihapsis polita (Cryptinae). A long-term survey of Cryptinae was carried out in a protected
area of Brazilian Atlantic Forest, in primary, tall secondary and low secondary forest areas. Specimens were collected during rainy season (RS) and dry season (DS) between 2000
and 2008, with total sampling effort of 4,095 trap-days. A total of 8,385 specimens of 2 Sampling methods Sampling description: Fieldwork was conducted over the course of eight years (2000–
2008). In each year, one field excursion was conducted during the dry season (DS, July-
August), and one during the rainy season (RS, October-November). Three Malaise traps
(Townes 1972) were set in each site, aligned and 25 meters apart from each other. The
traps were left operating for 4–5 weeks; the content of each trap was collected every 7
days and treated as a sample. Samples from the dry season of 2006 could not be
accessed and were not considered. Total sampling effort was 4,095 trap-days, 1,827 in the DS and 2,268 in the RS. Sampling
efficiency is expressed as the ratio between number of specimens and number of trap-
days. The number of collected specimens and sampling efficiency for T. polita were
compared to that of all other Cryptinae combined in order to contextualize its relative
abundance and to compare the temporal dynamics of this particular species to the
“background” of closely related species in the area. All specimens are deposited in the
entomological collection of Universidade Federal do Espírito Santo (Brazil). The full dataset
is available as supplementary material (Suppl. material 1). Geographic coverage Description: The study was conducted at Parque Estadual do Rio Doce, a 36,000 ha
protected area in the Brazilian Atlantic Forest, state of Minas Gerais. Three sites within the
park were sampled (Fig. 2): (1) an area of primary forest (PF) with well-developed canopy,
overspread high trees, abundant bamboo trees and well developed understory; (2) tall
secondary forest (TSF), an elevated, steep area; (3) an area of low secondary forest (LSF),
dominated by bamboos. Coordinates: 19˚48’18” and 19˚29’24” Latitude; 42˚38’’30” and 42˚28’’8” Longitude. Introduction The temporal dynamics of insect populations in rainforests is highly complex and poorly
studied (Hamer et al. 2005;Wolda 1988). Long-term studies of insect seasonality in the
tropics are scarce, largely due the difficulty in maintaining long-term sampling and sorting
(Grimbacher and Stork 2009). For Ichneumonidae, one of the largest insect families and a
dominant component of tropical faunas, data are particularly scarce, deriving mostly from
species that are easy to collect and identify (Gauld 2006). For Cryptinae, its largest and
least studied subfamily, with 408 genera and over 4,500 species, there are no detailed
studies of seasonality for any Neotropical taxon. Cryptines occur in all terrestrial
environments and are mostly ectoparasitoids of pupae and prepupae of a wide variety of
hosts, including moths, butterflies, beetles, social and solitary wasps. Figure 1. Female Trihapsis polita Townes, habitus. Female Trihapsis polita Townes, habitus. Female Trihapsis polita Townes, habitus. Long-term seasonal dominance of the wasp Trihapsis polita Townes (Hymenoptera, ... 3 Herein we report the seasonal distribution of a dominant cryptine species, Trihapsis polita
Townes (Fig. 1). Trihapsis Townes is known from two Brazilian species, T. punctata
Townes, and T. polita Townes, both described from a few specimens (Townes 1970). The
genus seems to be restricted to the Brazilian Atlantic Forest, but is not frequently collected:
a single specimen was obtained after an extensive collecting program in that region (Aguiar
and Santos 2010). The hosts and other biological aspects of the genus are still unknown. Usage rights Use license: Creative Commons Public Domain Waiver (CC-Zero) 4 Santos B et al. 4 Figure 2. Map showing the study area. A, Brazilian Atlantic Forest (green) with the location of Parque
Estadual do Rio Doce highlighted (white triangle). B, Map of the study area, with the three
sampled sites highlighted. PF, primary forest; TSF, tall secondary forest; LSF, low secondary
forest. Map showing the study area. A, Brazilian Atlantic Forest (green) with the location of Parque
Estadual do Rio Doce highlighted (white triangle). B, Map of the study area, with the three
sampled sites highlighted. PF, primary forest; TSF, tall secondary forest; LSF, low secondary
forest. Data resources Data package title: Raw_data
Resource link: http://www.systaxon.ufes.br/pubs/Tpolita/
Number of data sets: 1
Data set name: Raw_data
Data format: Excel
Description: Original raw data: number of female and male specimens of Trihapsis
polita and other Cryptinae in each collected sample. Column label
Column description
Area
Sampled area by successional state. LSF, low secondary forest; HSF, high secondary forest;
PF, primary forest. Dates-Start
Starting day for that sample, corresponding to the day when the Malaise trap sample
corresponding was initially set. Dates-End
Ending day for that sample, corresponding to the day when the Malaise trap sample was
collected. Data package title: Raw_data Column label
Column description
Area
Sampled area by successional state. LSF, low secondary forest; HSF, high secondary forest;
PF, primary forest. Dates-Start
Starting day for that sample, corresponding to the day when the Malaise trap sample
corresponding was initially set. Dates-End
Ending day for that sample, corresponding to the day when the Malaise trap sample was
collected. Season
Categorical variable indicating whether sample was collected during the dry season (DR) or
rainy season (RS). Cryptinae
Number of specimens of Cryptinae (except T. polita) collected in that sample. Trihapsis polita-
Males
Number of male specimens of T. polita collected in that sample
Trihapsis polita-
Females
Number of female specimens of T. polita collected in that sample
Total
Total number of specimens of T. polita collected in that sample
Pluviosity (mm)
Ammount of precipitation recorded during the week at which the sampling took place. Long-term seasonal dominance of the wasp Trihapsis polita Townes (Hymenoptera, ... 5 Long-term seasonal dominance of the wasp Trihapsis polita Townes (Hymenoptera, ... 5 Results and Discussion A total of 8,384 specimens of Cryptinae were collected, of which 6,657 (79.4%) belonged
to T. polita, and 1,727 (20.6%) to all other Cryptinae combined (Table 1). The overall
sampling efficiency was 2.04. For T. polita alone, sampling efficiency was 1.62, and for all
the other Cryptinae combined, 0.42. This quantitative superiority, however, was not uniform
(Fig. 3). During the dry season (DS), T. polita represented only about 7% of the total
Cryptinae collected. This situation was then drastically reversed in the rainy season (RS),
when this ratio jumped to nearly 85%. The differences between dry and rainy seasons
remain evident if the weekly totals are independently compared and are again clear if each
week (Fig. 4) is compared separately, year by year. Specimens
%
PF
TSF
LSF
Total
DS
RS
PF
TSF
LSF
Total
Trihapsis polita
1956
4691
10
6657
36
6621
69.2
86.9
6.4
79.4
other Cryptinae
871
710
146
1727
416
1275
30.8
13.1
93.6
20.6
Total
2827
5401
156
8384
452
7896
33.7
64.4
1.9
100.0
Table 1. Total number of specimens collected for T. polita vs. other Cryptinae in all areas (PF, primary forest;
TSF, tall secondary forest; LSF, low secondary forest) and seasons (DS, dry season; RS, rainy
season). Sampling effort was identical for all sampling areas, adding to 4,113 trap-days total, but
different between seasons (DS=1,836, RS=2,277). Total number of specimens collected for T. polita vs. other Cryptinae in all areas (PF, primary forest;
TSF, tall secondary forest; LSF, low secondary forest) and seasons (DS, dry season; RS, rainy
season). Sampling effort was identical for all sampling areas, adding to 4,113 trap-days total, but
different between seasons (DS=1,836, RS=2,277). Specimens
%
PF
TSF
LSF
Total
DS
RS
PF
TSF
LSF
Total
Trihapsis polita
1956
4691
10
6657
36
6621
69.2
86.9
6.4
79.4
other Cryptinae
871
710
146
1727
416
1275
30.8
13.1
93.6
20.6
Total
2827
5401
156
8384
452
7896
33.7
64.4
1.9
100.0 Figure 3. Mean number of specimens per sample (total of 3 traps; 195 samples/8 years), with standard
error bars, comparing T. polita (Tp) with other Cryptinae (Cr) in three successional areas,
primary forest (PF), tall secondary forest (TSF), and low secondary forest (LSF) (left chart),
and in the dry season (DS) vs. rainy season (RS) (right chart). PERD. Figure 4. Results and Discussion Total number of specimens collected in each sampling week (=each black & white bar pair,
equivalent to 3 areas x 3 traps) for T. polita (white, first plane) vs. other Cryptinae (black,
behind), along the sampled seasons (Day number of year, where DS=183–215, RS=286–322),
discriminated for each year (0–1 = 2000–2008). Left wall (YZ axes) shows daily records of
precipitation (cumulative values within each month), for all years (2893 records total); right wall
(YX axes) shows average of precipitation for each Day number of year (each day = average of
all years). Scale for number of specimens and precipitation (Y-axis) are identical. 6
Santos B et al. Santos B et al. 6 6 Figure 3. Mean number of specimens per sample (total of 3 traps; 195 samples/8 years), with standard
error bars, comparing T. polita (Tp) with other Cryptinae (Cr) in three successional areas,
primary forest (PF), tall secondary forest (TSF), and low secondary forest (LSF) (left chart),
and in the dry season (DS) vs. rainy season (RS) (right chart). PERD. Figure 3. Mean number of specimens per sample (total of 3 traps; 195 samples/8 years), with standard
error bars, comparing T. polita (Tp) with other Cryptinae (Cr) in three successional areas,
primary forest (PF), tall secondary forest (TSF), and low secondary forest (LSF) (left chart),
and in the dry season (DS) vs. rainy season (RS) (right chart). PERD. Mean number of specimens per sample (total of 3 traps; 195 samples/8 years), with standard
error bars, comparing T. polita (Tp) with other Cryptinae (Cr) in three successional areas,
primary forest (PF), tall secondary forest (TSF), and low secondary forest (LSF) (left chart),
and in the dry season (DS) vs. rainy season (RS) (right chart). PERD. Mean number of specimens per sample (total of 3 traps; 195 samples/8 years), with standard
error bars, comparing T. polita (Tp) with other Cryptinae (Cr) in three successional areas,
primary forest (PF), tall secondary forest (TSF), and low secondary forest (LSF) (left chart),
and in the dry season (DS) vs. rainy season (RS) (right chart). PERD. Figure 4. Total number of specimens collected in each sampling week (=each black & white bar pair,
equivalent to 3 areas x 3 traps) for T. polita (white, first plane) vs. other Cryptinae (black,
behind), along the sampled seasons (Day number of year, where DS=183–215, RS=286–322),
discriminated for each year (0–1 = 2000–2008). Results and Discussion Left wall (YZ axes) shows daily records of
precipitation (cumulative values within each month), for all years (2893 records total); right wall
(YX axes) shows average of precipitation for each Day number of year (each day = average of
all years). Scale for number of specimens and precipitation (Y-axis) are identical. Table 2. Comparison of sampling efficiency during the dry season (DS) vs. rainy season (RS) in the three
sampled successional areas. RS/DS, how many times larger was the sampling efficiency in the
rainy season in relation to the dry season. Sampling effort was identical for all areas, adding to
4,113 trap-days total, but different between seasons (DS=1,836, RS=2,277). %, percentage of
specimens. T. polita
other Cryptinae
Area
%
DS
RS
RS/DS
%
DS
RS
RS/DS
Primary forest (PF)
29.4
0.03
2.55
82.2
50.5
0.37
0.85
2.3
Tall secondary forest (TSF)
70.5
0.02
6.16
251.4
41.1
0.26
0.72
2.8
Low secondary forest (LSF)
0.2
0.00
0.01
3.2
8.4
0.10
0.11
1.1
PERD Total
79.4
0.02
2.91
148.3
20.6
0.25
0.56
2.3 The seasonal dominance of T. polita in the park was not a happenstance: it occurred
repeatedly through the years (Fig. 4), as evidenced by its nearly uninterrupted numerical
superiority in the samples (Table 3), and by its presence in 47.8% of the individual trap
samples from the RS, against only 9.2% for the DS. The seasonal dominance of T. polita in the park was not a happenstance: it occurred
repeatedly through the years (Fig. 4), as evidenced by its nearly uninterrupted numerical
superiority in the samples (Table 3), and by its presence in 47.8% of the individual trap
samples from the RS, against only 9.2% for the DS. Number of females and males of T. polita collected, as opposed to all other Cryptinae combined. %
♀, the percentage of specimens of T. polita that are females; % T. polita, the percentage of the total
specimens that correspond to T. polita Number of females and males of T. polita collected, as opposed to all other Cryptinae combined. %
♀, the percentage of specimens of T. polita that are females; % T. polita, the percentage of the total
specimens that correspond to T. polita Season, Year
T. polita ♀
T. polita ♂
other Cryptinae
% ♀
% T. Figure 4. Total number of specimens collected in each sampling week (=each black & white bar pair,
equivalent to 3 areas x 3 traps) for T. polita (white, first plane) vs. other Cryptinae (black,
behind), along the sampled seasons (Day number of year, where DS=183–215, RS=286–322),
discriminated for each year (0–1 = 2000–2008). Left wall (YZ axes) shows daily records of
precipitation (cumulative values within each month), for all years (2893 records total); right wall
(YX axes) shows average of precipitation for each Day number of year (each day = average of
all years). Scale for number of specimens and precipitation (Y-axis) are identical. Total number of specimens collected in each sampling week (=each black & white bar pair,
equivalent to 3 areas x 3 traps) for T. polita (white, first plane) vs. other Cryptinae (black,
behind), along the sampled seasons (Day number of year, where DS=183–215, RS=286–322),
discriminated for each year (0–1 = 2000–2008). Left wall (YZ axes) shows daily records of
precipitation (cumulative values within each month), for all years (2893 records total); right wall
(YX axes) shows average of precipitation for each Day number of year (each day = average of
all years). Scale for number of specimens and precipitation (Y-axis) are identical. For Cryptinae as a whole, sampling efficiency during the RS was 0.56, versus 0.25 in the
DS, that is, 2.25× higher in the rainy season. The increase matches the observations of
Kumagai (2002) for the Ichneumonidae in a preserved area in the Atlantic Forest, which
tripled in number in the traps in the RS. This indicates that a two to threefold increase
during the rainy season could be “normal” for Ichneumonidae as a whole. For T. polita,
however, seasonal influence was higher by two orders of magnitude, with sampling
efficiency rising from 0.02 in the DS to 2.92 in the RS – a 146× increase (Table 2). Long-term seasonal dominance of the wasp Trihapsis polita Townes (Hymenoptera, ... 7 Table 2. polita
RS, 2000
695
648
173
51.75%
88.59
DS, 2001
0
3
64
0.0%
4.48%
RS, 2001
370
280
135
56.92%
82.80%
DS, 2002
6
6
129
50.00%
8.51%
RS, 2002
639
758
382
45.74%
78.53%
DS, 2003
3
6
82
33.33%
9.89%
RS, 2003
137
637
110
17.70%
87.56%
DS, 2004
2
3
34
40.00%
12.82%
RS, 2004
1084
606
150
64.14%
91.85%
DS, 2005
1
1
27
50.00%
6.90%
RS, 2005
131
49
142
72.78%
55.90%
RS, 2006
23
45
62
33.82%
52.31%
DS, 2007
1
1
60
50.00%
3.23%
RS, 2007
364
155
121
70.13%
81.09%
DS, 2008
1
0
59
100%
1.67%
Overall DS
14
20
455
41.18%
6.95% Santos B et al. 8 Overall RS
3443
3178
1275
52.00%
83.85%
Total
3457
3198
1730
51.95%
79.37% There was a distinctive quantitative difference among the three successional areas for the
occurrence of both T. polita and other Cryptinae (Table 1). All differences discussed next
are statistically significant (see confidence interval bars in Fig. 3). For other Cryptinae, the
primary forest (PF) area had the highest abundance (871 specimens, 50.4% of the total),
closely followed by the tall secondary forest (TSF) (710; 41.1%) and low secondary forest
(LSF) (146; 8.5%). For T. polita, however, the TSF supported a much higher number of
specimens (4,691; 70.5%), followed by the PF (1,956; 29.4%). The LSF was clearly
avoided by this species, with only 10 specimens collected there throughout the study
(0.15%). Parasitoid wasps in general are most abundant in the rainy season, as compared to dry
periods (Gauld 1991). However, this relationship remains insufficiently discussed and
documented in the literature. The high level of dominance documented for T. polita has
never been reported for any species of Ichneumonidae in natural areas, representing a
noteworthy fact by itself. Previous faunistic studies of ichneumonid wasps in the Neotropics
(e.g. Aguiar and Santos 2010; Guerra and Penteado-Dias 2002; Kumagai and Graf 2000;
Kumagai and Graf 2000; Saaksjarvi et al. 2004) have never report any species as
particularly dominating the collected samples. Considering that studies at the species level
are scant for Ichneumonidae, the case for T. polita becomes of further interest, both
because it is a rare study and because it signals that there is much still to be gained from
studies on ichneumonid ecology. The notorious success of T. Table 2. polita in the tall secondary forest, as well as its distinct
population dynamics in relation to other Cryptinae, suggest it may naturally act as a large-
scale parasitoid in intermediate successional stages. This might also reveal to be of more
general interest, now or in the future: this type of forest is predicted to increase in the 21st
century due to industrialization and urbanization (Thomlinson et al. 1996), and yet, "there is
still a tremendous need to understand and further refine our knowledge of ecological
processes involved in secondary succession" (Guariguata and Ostertag 2001). Long-term seasonal dominance of the wasp Trihapsis polita Townes (Hymenoptera, ... Long-term seasonal dominance of the wasp Trihapsis polita Townes (Hymenoptera, ... 9 References •
Aguiar A, Santos B (2010) Discovery of potent, unsuspected sampling disparities for
Malaise and Möricke traps, as shown for Neotropical Cryptini (Hymenoptera,
Ichneumonidae). Journal of Insect Conservation 14 (2): 199‑206. https:// •
Aguiar A, Santos B (2010) Discovery of potent, unsuspected sampling disparities for
Malaise and Möricke traps, as shown for Neotropical Cryptini (Hymenoptera,
Ichneumonidae). Journal of Insect Conservation 14 (2): 199‑206. https://
doi org/10 1007/s10841 009 9246 x •
Gauld I (1991) The Ichneumonidae of Costa Rica, 1. Memoirs of the American
Entomological Institute 47: 1‑589. •
Gauld I (2006) Familia Ichneumonidae. in: Hanson, P.E, Gauld, I.D (Eds.). Hymenoptera
de la Región Neotropical. Memoirs of the American Entomological Institute 77: 446‑486. Grimbacher P Stork N (2009) Seasonality of a Diverse Beetle Assemblage Inhabiting •
Gauld I (2006) Familia Ichneumonidae. in: Hanson, P.E, Gauld, I.D (Eds.). Hymenoptera
de la Región Neotropical. Memoirs of the American Entomological Institute 77: 446‑486. •
Grimbacher P, Stork N (2009) Seasonality of a Diverse Beetle Assemblage Inhabiting
Lowland Tropical Rain Forest in Australia. Biotropica 41 (3): 328‑337. https://
doi.org/10.1111/j.1744-7429.2008.00477.x de la Región Neotropical. Memoirs of the American Entomological Institute 77: 446 486. •
Grimbacher P, Stork N (2009) Seasonality of a Diverse Beetle Assemblage Inhabiting
Lowland Tropical Rain Forest in Australia. Biotropica 41 (3): 328‑337. https://
doi.org/10.1111/j.1744-7429.2008.00477.x •
Guariguata MR, Ostertag R (2001) Neotropical secondary forest succession: changes
in structural and functional characteristics. Forest Ecology and Management 148:
185‑206. https://doi.org/10.1016/s0378-1127(00)00535-1 •
Guariguata MR, Ostertag R (2001) Neotropical secondary forest succession: changes
in structural and functional characteristics. Forest Ecology and Management 148:
185‑206. https://doi.org/10.1016/s0378-1127(00)00535-1 •
Guerra TM, Penteado-Dias A (2002) Abundância de Ichneumonidae (Hymenoptera) em
área de mata em São Carlos, Estado de São Paulo, Brasil. Acta Scientiarum 24:
363‑368. •
Hamer KC, Hill JK, Mustaffa N, Benedick S, Sherratt TN, Chey VK, Maryati M (2005)
Temporal variation in abundance and diversity of butterflies in Bornean rain forests:
opposite impacts of logging recorded in different seasons. Journal of Tropical Ecology
21 (4): 417‑425. https://doi.org/10.1017/s0266467405002361 •
Kumagai AF (2002) Os Ichneumonidae (Hymenoptera) da Estação Ecológica da
Universidade Federal de Minas Gerais, Belo Horizonte, com ênfase nas espécies de
Pimplinae . Revista Brasileira de Entomologia 46 (2): 189‑194. https://doi.org/10.1590/
s0085-56262002000200011 •
Kumagai AF, Graf V (2000) Ichneumonidae (Hymenoptera) de áreas urbana e rural de
Curitiba, Paraná, Brasil. Acknowledgements Rogério P. Martins (UFMG, Belo Horizonte) received BFS and AMT in a scientific visit and
donated the material used in this study. Maria Célia L. Carreiro kindly helped to count the
specimens. This project benefited from funding by the Conselho Nacional de Pesquisa e
Desenvolvimento/CNPq (Processes 520031/98-9 and 926.656.810), Brazil. References Acta Biológica Paranaense 29 https://doi.org/10.5380/
abpr.v29i0.588 •
Kumagai AF, Graf V (2000) Ichneumonidae (Hymenoptera) de áreas urbana e rural de
Curitiba, Paraná, Brasil. Acta Biológica Paranaense 29 https://doi.org/10.5380/
abpr.v29i0.588 •
Saaksjarvi I, Haataja S, Neuvonen S, Gauld I, Jussila R, Salo J, Burgos AM (2004)
High local species richness of parasitic wasps (Hymenoptera: Ichneumonidae;
Pimplinae and Rhyssinae) from the lowland rainforests of Peruvian Amazonia. Ecological Entomology 29 (6): 735‑743. https://doi.org/10.1111/
j.0307-6946.2004.00656.x •
Thomlinson J, Serrano M, M. Lopez Td, Aide TM, Zimmerman J (1996) Land-Use
Dynamics in a Post-Agricultural Puerto Rican Landscape (1936-1988). Biotropica 28
(4): 525. https://doi.org/10.2307/2389094 •
Townes H (1970) The Genera of Ichneumonidae, Part 2. Memoirs of the American
Entomological Institute 12: 1‑537. •
Townes H (1972) A light-weight Malaise trap. Entomological News 83: 239‑247. •
Wolda H (1988) Insect Seasonality: Why? Annual Review of Ecology and Systematics
19 (1): 1‑18. https://doi.org/10.1146/annurev.es.19.110188.000245 Santos B et al. 10 Authors: B.F.Santos, A.P.Aguiar & A.M.Tedesco
Data type: collecting records
Brief description: Original data, discriminated by individual trap-samples (585 records),
presented as Python lists (compatible with R, and most other computer languages). All variables
contain 585 items, with data corresponding according to position (index).
Filename: TpolitaRawData.txt - Download file (13.38 kb) Suppl. material 1: Raw data Authors: B.F.Santos, A.P.Aguiar & A.M.Tedesco
Data type: collecting records
Brief description: Original data, discriminated by individual trap-samples (585 records),
presented as Python lists (compatible with R, and most other computer languages). All variables
contain 585 items, with data corresponding according to position (index). Filename: TpolitaRawData.txt - Download file (13.38 kb)
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Single-cell analyses uncover granularity of muscle stem cells
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F1000Research
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F1000Research 2020, 9(F1000 Faculty Rev):31 Last updated: 22 JAN 2020 Open Peer Review
F1000 Faculty Reviews are written by members of
the prestigious
. They are
F1000 Faculty
commissioned and are peer reviewed before
publication to ensure that the final, published version
is comprehensive and accessible The reviewers
REVIEW
Single-cell analyses uncover granularity of muscle stem cells
[version 1; peer review: 3 approved]
John Saber
,
Alexander Y.T. Lin
, Michael A. Rudnicki
1,2
Sprott Centre for Stem Cell Research, Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, K1H 8L6, Canada
Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ottawa, ON, K1H 8M5, Canada
Equal contributors
Abstract
Satellite cells are the main muscle-resident cells responsible for muscle
regeneration. Much research has described this population as being
heterogeneous, but little is known about the different roles each
subpopulation plays. Recent advances in the field have utilized the power
of single-cell analysis to better describe and functionally characterize
subpopulations of satellite cells as well as other cell groups comprising the
muscle tissue. Furthermore, emerging technologies are opening the door to
answering as-yet-unresolved questions pertaining to satellite cell
heterogeneity and cell fate decisions. Keywords
satellite cells, heterogeneity, cell fate, single-cell analysis
1,2*
1,2*
1,2
1
2
*
Reviewer Status
Invited Reviewers
version 1
21 Jan 2020
1
2
3
21 Jan 2020, (F1000 Faculty Rev):31 (
First published:
9
)
https://doi.org/10.12688/f1000research.20856.1
21 Jan 2020, (F1000 Faculty Rev):31 (
Latest published:
9
)
https://doi.org/10.12688/f1000research.20856.1
v1 Equal contributors
* Open Peer Review
Reviewer Status
Invited Reviewers
version 1
21 Jan 2020
1
2
3 21 Jan 2020, (F1000 Faculty Rev):31 (
First published:
9
)
https://doi.org/10.12688/f1000research.20856.1
21 Jan 2020, (F1000 Faculty Rev):31 (
Latest published:
9
)
https://doi.org/10.12688/f1000research.20856.1
v1 Abstract Satellite cells are the main muscle-resident cells responsible for muscle
regeneration. Much research has described this population as being
heterogeneous, but little is known about the different roles each
subpopulation plays. Recent advances in the field have utilized the power
of single-cell analysis to better describe and functionally characterize
subpopulations of satellite cells as well as other cell groups comprising the
muscle tissue. Furthermore, emerging technologies are opening the door to
answering as-yet-unresolved questions pertaining to satellite cell
heterogeneity and cell fate decisions. F1000 Faculty Reviews are written by members of
the prestigious
. They are
F1000 Faculty
commissioned and are peer reviewed before
publication to ensure that the final, published version
is comprehensive and accessible. The reviewers
who approved the final version are listed with their
names and affiliations. Keywords satellite cells, heterogeneity, cell fate, single-cell analysis Any comments on the article can be found at the
end of the article. , Stanford University School of
Helen Blau
Medicine, Stanford, USA
1
, National Institutes of Health,
Vittorio Sartorelli
Bethesda, USA
2
, Kumamoto University, Kumamoto,
Yusuke Ono
Japan
3 , National Institutes of Health,
Vittorio Sartorelli
Bethesda, USA
2 , Kumamoto University, Kumamoto,
Yusuke Ono
Japan
3 Any comments on the article can be found at the
end of the article. Page 1 of 10 F1000Research 2020, 9(F1000 Faculty Rev):31 Last updated: 22 JAN 2020 Michael A. Rudnicki (
)
Corresponding author:
mrudnicki@ohri.ca
: Conceptualization, Writing – Original Draft Preparation, Writing – Review & Editing;
: Conceptualization, Writing –
Author roles: Saber J
Lin AYT
Original Draft Preparation, Writing – Review & Editing;
: Conceptualization, Resources, Supervision, Writing – Original Draft
Rudnicki MA
Preparation, Writing – Review & Editing
No competing interests were disclosed. Competing interests:
J.S. is supported by a doctoral fellowship from the Canadian Institutes of Health Research. A.Y.T.L. is supported by a
Grant information:
Postdoctoral Fellowship from the Ontario Institute for Regenerative Medicine. M.A.R. holds the Canada Research Chair in Molecular Genetics. These studies were carried out with the support of grants to M.A.R. from the US National Institutes for Health (R01AR044031), the Canadian
Institutes of Health Research (FDN-148387), the Muscular Dystrophy Association (USA), the Stem Cell Network, and the Jesse's Journey
Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. © 2020 Saber J
. This is an open access article distributed under the terms of the
, which
Copyright:
et al
Creative Commons Attribution License
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Saber J, Lin AYT and Rudnicki MA. How to cite this article:
Single-cell analyses uncover granularity of muscle stem cells [version 1; peer
F1000Research 2020, (F1000 Faculty Rev):31 (
)
review: 3 approved]
9
https://doi.org/10.12688/f1000research.20856.1
21 Jan 2020, (F1000 Faculty Rev):31 (
)
First published:
9
https://doi.org/10.12688/f1000research.20856.1 Michael A. Keywords Rudnicki (
)
Corresponding author:
mrudnicki@ohri.ca
: Conceptualization, Writing – Original Draft Preparation, Writing – Review & Editing;
: Conceptualization, Writing –
Author roles: Saber J
Lin AYT
Original Draft Preparation, Writing – Review & Editing;
: Conceptualization, Resources, Supervision, Writing – Original Draft
Rudnicki MA
Preparation, Writing – Review & Editing
No competing interests were disclosed. Competing interests:
J.S. is supported by a doctoral fellowship from the Canadian Institutes of Health Research. A.Y.T.L. is supported by a
Grant information:
Postdoctoral Fellowship from the Ontario Institute for Regenerative Medicine. M.A.R. holds the Canada Research Chair in Molecular Genetics. These studies were carried out with the support of grants to M.A.R. from the US National Institutes for Health (R01AR044031), the Canadian
Institutes of Health Research (FDN-148387), the Muscular Dystrophy Association (USA), the Stem Cell Network, and the Jesse's Journey
Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. © 2020 Saber J
. This is an open access article distributed under the terms of the
, which
Copyright:
et al
Creative Commons Attribution License
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Saber J, Lin AYT and Rudnicki MA. How to cite this article:
Single-cell analyses uncover granularity of muscle stem cells [version 1; peer
F1000Research 2020, (F1000 Faculty Rev):31 (
)
review: 3 approved]
9
https://doi.org/10.12688/f1000research.20856.1
21 Jan 2020, (F1000 Faculty Rev):31 (
)
First published:
9
https://doi.org/10.12688/f1000research.20856.1 Michael A. Rudnicki (
)
Corresponding author:
mrudnicki@ohri.ca Michael A. Rudnicki (
)
Corresponding author:
mrudnicki@ohri.ca : Conceptualization, Writing – Original Draft Preparation, Writing – Review & Editing;
: Conceptualization, Writing –
Author roles: Saber J
Lin AYT
Original Draft Preparation, Writing – Review & Editing;
: Conceptualization, Resources, Supervision, Writing – Original Draft
Rudnicki MA
Preparation, Writing – Review & Editing Page 2 of 10 F1000Research 2020, 9(F1000 Faculty Rev):31 Last updated: 22 JAN 2020 F1000Research 2020, 9(F1000 Faculty Rev):31 Last updated: 22 JAN 2020 Known heterogeneity in the satellite cell population Known heterogeneity in the satellite cell population
Satellite cells are the main powerhouse for skeletal muscle
regeneration. How satellite cells achieve this remarkable regen-
erative ability of generating progenitors yet maintain and balance
self-renewal capacity can be posited as either stochastic fate
acquisition or a hierarchical organization of asymmetric divi-
sions with determined cell fates12. Over the past decade, studies
have demonstrated that satellite cells are organized hierarchically,
whereby heterogeneity exists within this seemingly homogene-
ous Pax7+ population with functional subpopulations. Firstly, sat-
ellite cells have bi-potential activity and can generate both brown
fat and muscle, which alludes to multiple differentiation tra-
jectories4,13. In addition, subsets of Pax7-expressing satellite
cells have differing functional potential (Figure 1, Bottom). Two
such subpopulations are 1) Pax7+/Myf5– cells and 2) a Pax7Hi Figure 1. Single-cell analysis allows the identification of cell types residing in skeletal muscle. Top: Tools such as single-cell RNA
sequencing (scRNA-Seq) and cytometry time of flight (CyToF) have been used to determine the identity of numerous cell types residing in
skeletal muscle based on their transcriptional and protein signature. Notably, in addition to muscle fibers, muscle comprises satellite cells,
immune cells, fibroblasts, endothelial cells, and Schwann cells. Bottom: The heterogeneity within the satellite cell population can be analyzed
based on known markers associated with the stem cell state (Pax7+/Myf5–, Pax7Hi, and H2B-GFP+ label-retaining cells). Furthermore, different
cell states for satellite cells can be inferred using pseudotime analysis of existing transcriptional and proteomic datasets. Cells along the
gray-to-red gradient represent the known cascade of transcription factor expression following the differentiation of satellite cells into
myocytes. The diverging blue gradient illustrates potential unknown cell fates. FAP, fibro-adipogenic progenitor; SMMC, smooth muscle and
mesenchymal cell. Page 3 of 10 Figure 1. Single-cell analysis allows the identification of cell types residing in skeletal muscle. Top: Tools such as single-cell RNA
sequencing (scRNA-Seq) and cytometry time of flight (CyToF) have been used to determine the identity of numerous cell types residing in
skeletal muscle based on their transcriptional and protein signature. Notably, in addition to muscle fibers, muscle comprises satellite cells,
immune cells, fibroblasts, endothelial cells, and Schwann cells. Bottom: The heterogeneity within the satellite cell population can be analyzed
based on known markers associated with the stem cell state (Pax7+/Myf5–, Pax7Hi, and H2B-GFP+ label-retaining cells). Furthermore, different
cell states for satellite cells can be inferred using pseudotime analysis of existing transcriptional and proteomic datasets. Introduction impacted in disease backgrounds, such as Duchenne’s muscular
dystrophy and aging11. In this review, we will discuss recent
insights into the molecular control of satellite cell function and
how the emergence of single-cell technologies is impacting the
discovery of novel cellular functions and cell fates. Over the past decade, developmental biology has furthered our
understanding of the origins of satellite cells (or muscle stem
cells) and the mechanisms that govern their quiescence, activa-
tion, and differentiation1. In adult homeostasis, satellite cells are
quiescent and express the nodal transcription factor Pax72–4. Upon
physiological insult, such as an injury or exercise, satellite
cells become activated, enter the cell cycle, and generate myo-
genic progenitors. The activation of satellite cells has long been
believed to be linear, with a sequential cascade of transcription
factor expression5. Activated satellite cells become committed
progenitors, also known as myoblasts, that express Myf5 and
MyoD as well as Myogenin upon entering the differentiation
program to become post-mitotic myocytes6. Myocytes will fuse
to form myotubes that ultimately create the scaffold for muscle
tissue7. Moreover, satellite cells are able to self-renew, allow-
ing the long-term maintenance of the stem cell pool. Outside of
these intrinsic molecules that determine satellite cell states,
extrinsic cues also influence division kinetics and satellite cell
commitment and differentiation8–10. Both intrinsic and extrin-
sic factors are crucial for satellite cell function, and both are Known heterogeneity in the satellite cell population Using the Tabula Muris
Consortium dataset, one group was able to validate a Twist2+
population that expressed Nrp1 to allow for selective fusion
and generation of type IIB fibers22,24,25. The first thorough study
captured and performed scRNA-Seq on over 12,000 mononu-
clear cells from adult hindlimb muscle with complementary pro-
filing using CyTOF26. The combination of an X-shift clustering
algorithm based on CyTOF data and scRNA-Seq clustering
identified 10 major populations: B and T cells, macrophages,
endothelial cells, fibro-adipogenic progenitors (FAPs), neu-
trophils, muscle stem (satellite) cells, integrin-α7+/VCAM1– cells,
Schwann cells, and an uncharacterized population negative for
canonical markers (Figure 1, Top). Interestingly, these undis-
covered integrin-α7+/VCAM1– cells display myogenic potential
in vitro, though they are auxiliary to satellite cells during regen-
eration, as they enhanced satellite cell transplantation potential
but failed to transplant as an isolated population. These
integrin-α7+/VCAM1– cells have a transcriptomic signature more
akin to mesenchymal cells and have been aptly named smooth
muscle and mesenchymal cells (SMMCs). This study also found
an interstitial Scleraxis+ population expressing tenocyte markers,
which broadly falls under the fibroblast classification and may
cooperate with FAPs to promote muscle regeneration. Prob-
ing resident muscle cells with two powerful single-cell tech-
nologies, CyTOF and scRNA-Seq, resulted in the identification
of two uncharacterized populations. Recent advances in muscle regeneration using
single-cell technologies The question of how complex tissues are formed from the basic
individual units of cells has been advanced with the recent
explosion in single-cell biology. Tissues are heterogeneous in
composition, and studying cells at single-cell resolution facili-
tates insights into understanding individual cellular functions
and their lineage relationships. These two concepts of function
and lineage relationships have propelled many recent studies to
discover new cell types and states, refined differentiation hier-
archies during development and regeneration, and identified
populations that are aberrant in disease contexts. Here, we will
primarily focus on the two most mature and commercially
available single-cell technologies: single-cell RNA-sequencing
(scRNA-Seq) and single-cell proteomics with cytometry by
time of flight (CyTOF). Both of these two technologies enable
researchers to stratify heterogeneous populations within the total
tissue and extrapolate trajectories with pseudotime algorithms
to infer dynamic changes from static snapshots of specific
populations. The power of single-cell analyses can also refine differentiation
trajectories and capture cell state transitions (Figure 1, Bottom). A study from the Blau laboratory used CyTOF as a discovery tool
and conducted a screen for new cell surface markers expressed
in satellite cells and cultured myoblasts27. Using the classical
myogenic
transcription
factors—Pax7,
Myf5,
Myod,
and
Myogenin—to infer their clustering and population trajecto-
ries, they identified two progenitor populations: P1 and P2. P1 is
Pax7Lo, Myf5Hi, MyoDLo, and MyogeninHi and is marked by
CD9, while the P2 population is Pax7Lo, Myf5Hi, MyoDHi, and
MyogeninHi and is distinguished by the presence of both CD9
and CD104 (or integrin beta 4). Interestingly, the P1 population
seems to appear before the P2 population, despite seemingly turn-
ing off the expression of MyoD. The presence of Myogenin and
absence of MyoD would suggest that the P1 population is fur-
ther along the differentiation pathway, contrary to the proposed
dynamics. However, both populations seem to exist at later
stages of the myogenic lineage, since they both lack IdU incor-
poration following injury, confirming they do not enter the cell
cycle and may both represent variations of post-mitotic myocytes. Known heterogeneity in the satellite cell population Cells along the
gray-to-red gradient represent the known cascade of transcription factor expression following the differentiation of satellite cells into
myocytes. The diverging blue gradient illustrates potential unknown cell fates. FAP, fibro-adipogenic progenitor; SMMC, smooth muscle and
mesenchymal cell. Figure 1. Single-cell analysis allows the identification of cell types residing in skeletal muscle. Top: Tools such as single-cell RNA
sequencing (scRNA-Seq) and cytometry time of flight (CyToF) have been used to determine the identity of numerous cell types residing in
skeletal muscle based on their transcriptional and protein signature. Notably, in addition to muscle fibers, muscle comprises satellite cells,
immune cells, fibroblasts, endothelial cells, and Schwann cells. Bottom: The heterogeneity within the satellite cell population can be analyzed
based on known markers associated with the stem cell state (Pax7+/Myf5–, Pax7Hi, and H2B-GFP+ label-retaining cells). Furthermore, different
cell states for satellite cells can be inferred using pseudotime analysis of existing transcriptional and proteomic datasets. Cells along the
gray-to-red gradient represent the known cascade of transcription factor expression following the differentiation of satellite cells into
myocytes. The diverging blue gradient illustrates potential unknown cell fates. FAP, fibro-adipogenic progenitor; SMMC, smooth muscle and
mesenchymal cell. Page 3 of 10 F1000Research 2020, 9(F1000 Faculty Rev):31 Last updated: 22 JAN 2020 F1000Research 2020, 9(F1000 Faculty Rev):31 Last updated: 22 JAN 2020 group observed upon taking the top ~10% of GFP+ cells from
Pax7-nGFP mice, both of which show greater stemness upon
transplantation, as determined by their ability to repopulate the
stem cell pool14,15. It remains unknown whether other functional
subpopulations that differ in self-renewal capacity exist in the
total Pax7+ population. Furthermore, some satellite cells have
long-term label retention based on H2B-GFP16, while another
rare subpopulation is Pax3+ and is resistant to radiation and to
genotoxic stress17,18. All these findings suggest a functional hier-
archical classification of satellite cells, which was reaffirmed by
using the Pax7-CreER;R26RBrainbow2.1 mouse, where clonal
dominance arose after repetitive injuries but clonal diversity
was retained in homeostasis19. However, it has been difficult to
assess the functional subpopulations of satellite cells and their
relationship to one another. The application of single-cell
technologies should clarify these conundrums. Two large-scale studies profiled a multitude of adult organs
and tissues using a microwell-based method or droplet-based
scRNA-Seq. Each group identified a cluster of muscle cells; how-
ever, neither study presented novel stratification or discovery
of the muscle stem cell populations22,23. Current application of single-cell technologies on
satellite cells The recent application of single-cell technologies to study
satellite cells, and by association and more broadly speaking mus-
cle tissue, makes it possible to functionally delineate hierarchies
and identify molecular programs governing sequential cell fates. Though studying single satellite cells is not a new idea, previ-
ous experimental designs and methods were not high throughput
and limited the possibility of highly quantitative results. The
first findings pertaining to single-cell analysis of satellite cells
captured single cells from a muscle fiber using a micropi-
pette, which is an extremely tedious and low-throughput
endeavor20. A contemporary paper isolated Tomato+ cells from
Pax7-CreER;R26RLSL-tdTomato mice and captured them on the
Fluidigm C1 platform21. However, their platform captured only
21 cells which did not cluster the population into any meaning-
ful subpopulations. As the authors suggested, and where more
recent studies have explored, the use of higher-throughput cap-
ture methods, primarily using droplet-based systems to capture
single cells including Dropseq or 10X Genomics platforms, has
been invaluable in identifying these subpopulations. Complementary approaches to the experiments described above
have been conducted to infer activation and differentiation
hierarchies with scRNA-Seq. A few recent studies started by
profiling the total muscle over a regeneration time course to
assess populations that temporally change28,29. Both studies saw
cell-type-specific clusters emerge and disappear throughout
the progress of regeneration, reinforcing the idea that muscle Page 4 of 10 F1000Research 2020, 9(F1000 Faculty Rev):31 Last updated: 22 JAN 2020 F1000Research 2020, 9(F1000 Faculty Rev):31 Last updated: 22 JAN 2020 changes in cell dynamics are relative rather than absolute. Fur-
thermore, they miss certain cell types such as tenocytes, possibly
because they are grouped together with the FAP population as
fibroblasts26. Two groups then focused on satellite cell activa-
tion and used pseudotime algorithms to reconstruct differen-
tiation trajectories. The study from the Sartorelli lab profiled
homeostatic and 60-hour post-notexin-injured satellite cells and
cultured myoblasts32; their analyses reaffirmed that metabolic regeneration is a dynamic process (Figure 2). However, these
results seem to contrast with previous publications regarding cell
dynamics, possibly owing to the different injury models or to
limitations in inferring cell types based solely on transcriptional
profiling30. For example, FAPs have been shown to increase pro-
portionally to myogenic progenitors following an injury rather
than decrease31. However, this discrepancy might be due to
scRNA-Seq being heavily skewed by other cells, whereby e 2. Dynamics of muscle-resident cells during regeneration. Current application of single-cell technologies on
satellite cells It would be
interesting to probe for the molecules enriched in these “reserve
cells”, since they may elucidate pathways satellite cells undergo
to return to quiescence once activated. Another study was
able to parse the heterogeneity of the Syndecan family (1–4)
and their differential expression among quiescent, activated,
cycling, and committed satellite and their progenitor cells28. Notably, the authors show that quiescent satellite cells can be
divided into subpopulations that express Sdc2 and Sdc3 and those
that don’t. Finally, another focused on an aging context and dem-
onstrated that aged T-cells and satellite cells are transcriptomically
more similar to their respective young and injured cell type, allud-
ing to the possibility that these predispositions in aged contexts
could be causative of poor regenerative outcomes29. These descrip-
tive studies using scRNA-Seq predominantly reiterate previous
known molecules and paradigms in muscle regeneration36. from another. Currently, only the study by Giordani et al. has
scratched the surface in muscle by profiling resident muscle cells
using scRNA-Seq and CyToF26. However, other techniques currently being used in other fields
can shed light onto the next steps of multimodal research in sat-
ellite cells and muscle regeneration. Single-cell analysis for
RNA and protein (CITE-seq) has been described, allowing
the simultaneous quantification of RNA transcripts and pro-
tein products in a single cell38. This relies on the detection of
oligonucleotide-labeled antibodies for the identification of pro-
teins using similar workflow to scRNA-Seq. However, this tech-
nique allows the detection of cell surface proteins only, which
limits its use for investigating differences in gene regulation. Another study by Genshaft et al. used proximity extension assays
(PEAs, similar to proximity ligation assay) to evaluate intracellu-
lar protein levels by measuring the generation of a DNA reporter
following the interaction of two antibodies targeting the same
protein39. This allows the simultaneous detection of proteins and
RNA from single cells. However, this technique is limited to a
small panel of proteins. Nevertheless, performing similar experi-
ments in satellite cells can help identify some of the molecular
differences between different subpopulations. For example,
one can test the stemness of the Myf5Lo or Pax7Hi populations
by simultaneously investigating the expression of many genes
involved in cellular quiescence. More interestingly, one study combined scRNA-Seq with sub-
sequent functional validation using single-cell time lapse
imaging and provided insights contrary to convention37. Current application of single-cell technologies on
satellite cells The
authors focused on the differences between young and aged sat-
ellite cells and the transition from quiescence to activation. One
noteworthy observation was that Pax7 does not monotonically
decrease upon activation, whereas quiescence-associated Spry1
monotonically decreases. With the use of single-cell imaging
and immunostaining, they found that the most motile and acti-
vated cells were actually enriched for Pax7. Thus, by conducting
orthogonal studies on single cells, the authors drew on a strength
and were able to associate specific molecular features with behav-
ior. Another interesting finding was that both label-retaining
cells (LRCs) and nonLRCs occupy the same transcriptional
space, such that the two populations do not form distinct
clusters based on their label retention function. Surprisingly,
the activated cell cluster largely comprised LRCs, suggesting
their increased ability to enter the cell cycle, which was recon-
firmed with EdU experiments. How and why LRCs are able to
activate quicker yet maintain label retention is an interesting
and unresolved question. Finally, the study posits whether aged
and young satellite cells have different trajectories and states
or merely arrive at the same state albeit at different rates. Both
aged and young samples overlapped in their trajectories, suggest-
ing that cell state transitions were similar but the progression or
rate of activation was slower in aged satellite cells. Chromatin accessibility at the single-cell level can also
complement scRNA-Seq data in identifying regulators of cell
fate. In addition to being present at transcription start sites, it is
known that chromatin accessibility determined by DNase hyper-
sensitivity sites is also localized to distal regions, suggesting a
regulatory role in gene transcription rather than simply a direct
effect on gene transcription40. Thus, obtaining relevant single-cell
accessibility information is relevant for deconvoluting the
epigenetic mechanisms governing gene transcription in satellite
cells, whether it be for understanding heterogeneity or determin-
ing modulators of cell fate. So far, no such experiments have
been conducted in muscle, but other areas of research have put
such techniques to the test. Single-cell assay for transposase-
accessible chromatin using sequencing (ATAC-Seq) coupled with
scRNA-Seq have allowed the identification of gene expression
and chromatin accessibility from the same cell41. Moreover, single-cell chromatin immunoprecipitation coupled
with sequencing (scChIP-Seq) will be invaluable to complete the
picture. One group has used scChIP-Seq to compare H3K27me3
patterns in cells originating from breast cancer tumors42. Current application of single-cell technologies on
satellite cells Single-cell RNA sequencing has been used to describe
ltiple muscle-resident cells through regeneration following injury28,29. Plotted are the relative proportion of each cell type
ulk cell population at each stage. Some cells, such as satellite cells and T cells, make up a higher proportion of the popul
Others, like B cells and fibro-adipogenic progenitors (FAPs), seem to be comparatively reduced in numbers. It is
ytes react during muscle regeneration. However, they may follow the same trend as FAPs owing to them not being disc
in the fibroblast compartment. Figure 2. Dynamics of muscle-resident cells during regeneration. Single-cell RNA sequencing has been used to describe the dynamics
of multiple muscle-resident cells through regeneration following injury28,29. Plotted are the relative proportion of each cell type compared to
the bulk cell population at each stage. Some cells, such as satellite cells and T cells, make up a higher proportion of the population following
injury. Others, like B cells and fibro-adipogenic progenitors (FAPs), seem to be comparatively reduced in numbers. It is unknown how
tenocytes react during muscle regeneration. However, they may follow the same trend as FAPs owing to them not being discriminated from
FAPs in the fibroblast compartment. Figure 2. Dynamics of muscle-resident cells during regeneration. Single-cell RNA sequencing has been used to describe the dynamics
of multiple muscle-resident cells through regeneration following injury28,29. Plotted are the relative proportion of each cell type compared to
the bulk cell population at each stage. Some cells, such as satellite cells and T cells, make up a higher proportion of the population following
injury. Others, like B cells and fibro-adipogenic progenitors (FAPs), seem to be comparatively reduced in numbers. It is unknown how
tenocytes react during muscle regeneration. However, they may follow the same trend as FAPs owing to them not being discriminated from
FAPs in the fibroblast compartment. F1000Research 2020, 9(F1000 Faculty Rev):31 Last updated: 22 JAN 2020 F1000Research 2020, 9(F1000 Faculty Rev):31 Last updated: 22 JAN 2020 F1000Research 2020, 9(F1000 Faculty Rev):31 Last updated: 22 JAN 2020 changes drive the activation of satellite cells33,34. However, one
interesting finding was a split in their trajectory for cultured pri-
mary myoblasts, with one branch representing differentiation
categorized by Myogenin expression, while the other branch
retained cyclinD1 and D2. The authors suggest that this latter
branch represents the previously identified “reserve cells” that
may represent a self-renewing population in vitro35. Current application of single-cell technologies on
satellite cells Briefly,
they found that a subset of cells from untreated tumors had a
decrease in H3K27me3 levels, a pattern similar to cells from
tumors that have developed drug resistance. This led to an
increase in the expression of genes that are normally repressed. This study is a good example of the potential of scChIP-Seq in
identifying cell heterogeneity. However, these current methods do
not allow the simultaneous measurement of enough variables
to obtain a full understanding from within the same cell. Therefore,
future
work
bringing
together
scRNA-Seq,
ChIP-Seq, and ATAC-Seq would be invaluable in painting a Emerging technologies However,
given the importance of the niche in the function of many cells,
it is important to investigate the function of satellite cells while
maintaining the niche. Few techniques allow this. One approach
gaining popularity is the use of antibodies conjugated to
oligonucleotides. This allows the specific detection of proteins
while permitting multiplexing that would be impossible to
achieve using conventional microscopy techniques. The first tech-
nique of its kind is CODEX, which uses tagged antibodies with
double-stranded DNA containing specific overhangs45. By
carefully selecting the nucleotide sequence, this technique allows
the iterative detection of antibodies—and thus proteins—based
on the incorporation of a fluorophore-tagged nucleotide. Using
this technique, the authors described a striking impact that the
niche has on immune cell receptor expression from the spleen. An evolution of this technique is Immuno-SABER, whereby
fluorophore-tagged imagers bind in a controlled manner to the
target DNA sequence conjugated to an antibody46. This tech-
nique has the advantage of not requiring in situ amplification of
DNA and would also allow, in theory, the simultaneous detection
of multiple proteins. Imaging mass cytometry is also a powerful
tool to allow the acquisition of images of cells from bulk tissues
without perturbation of the niche47. Imaging mass cytometry has
already been used to describe cancer cell subpopulations and
cell–cell interactions, information which would be lost using
sorting strategies47. Coupled with the recently described muscle-
resident cells, this would allow the imaging of dozens of markers
per cell, and many different types of cells, to further eluci-
date the niche requirement in the function of satellite cells
and better understand the dynamics of muscle regeneration26. For example, do FAPs or macrophages modulate satellite cell
function through direct interaction or paracrine signaling? Although current approaches do allow the investigation of
such questions, they are incapable of multiplexing, an essen-
tial factor for considering the niche in its entirety given the
complexity of cell types and the difficulty in inferring cell
types solely based on gene transcription. Moreover, maintain-
ing the niche is important for accurately studying satellite
cells, since its disruption has a rapid, profound effect on their
transcriptome48–50. Single-cell analyses still have a lot of utility in answering ques-
tions about muscle regeneration. For instance, the full story of
satellite cell heterogeneity remains largely unclear. Functional
differences have been documented14,15,17,18, yet only one study has
been able to capture subpopulations of Pax7Hi and Pax7Lo cells32. Emerging technologies To fully describe satellite cells and other cells residing in mus-
cle, as well as their overall function, current approaches based
predominantly on scRNA-Seq are insufficient. Multimodal
approaches, where multiple facets of the cell are considered simul-
taneously, will be needed to better understand the relationship
among DNA structure, its impact on transcription, and the
resulting proteins being formed that discriminate one cell Page 6 of 10 F1000Research 2020, 9(F1000 Faculty Rev):31 Last updated: 22 JAN 2020 F1000Research 2020, 9(F1000 Faculty Rev):31 Last updated: 22 JAN 2020 complete picture of the epigenetic landscape and its functional
consequence on satellite cell gene expression. their isolation but also reaffirm concepts and decades of func-
tional regenerative myogenesis work without major paradigm-
shifting outcomes. The discovery of these new cell types and
states allows for further investigation into their ontogeny, such as
CD9+ myogenic progenitors or SMMCs26,27. These single-cell
studies also highlight that discovery-based tools are predomi-
nantly descriptive in nature and have thus far generated only tissue
atlases. Trajectories and cell states determined by algorithms are
only inferences and will require functional validation, suggest-
ing that classical lineage tracing is not replaceable. Moreover,
one of the pitfalls of transcriptomic trajectories is that true cel-
lular lineages are mitotic cells and their descendants, such that
scRNA-Seq may miss certain dynamisms. For instance, trajec-
tory analysis is a forward pathway, yet self-renewing muscle stem
cells must also move backwards—or loop—to return to quies-
cence, which currently has not been captured in these projections. Similarly, generating descendants from self-renewing mitotic
stem cells at different times or accounting for proliferative history
may also be lost in pseudotime analyses. Despite this, progress is
being made in merging the inferred lineages using single-cell
transcriptomics with single-cell genetic lineage tracing, poten-
tially alleviating some of the issues with the inferences while still
allowing high-throughput analysis51. Additionally, new imaging techniques are quickly gaining
popularity for the investigation of single-cell function. Spatial-
omics techniques are now able to capture gene expression at
the single-cell level in relation to spatial information (MERFISH
and Seurat)43,44. MERFISH and Seurat allow the integration of
RNA-FISH data with scRNA-Seq, allowing the quantification
of RNA with subcellular localization. Lastly, many omics techniques rely on the isolation of cells
of interest from the niche prior to data acquisitions. Emerging technologies Other documented factors of heterogeneity have not been
reflected in current single-cell approaches. Perhaps profiling
large purified populations of satellite cells in combination with
multiplexing or epitope tagging would provide more resolution. Accordingly, further investigation using CyTOF and markers
associated with asymmetric cell divisions or intracellular signal-
ing and phospho-proteins, such as Notch components or the PAR
complexes, could further stratify homeostatic and injury-induced
satellite cells. Other unresolved questions pertain to the regula-
tion of multipotential specification, which has yet to be investi-
gated using single-cell approaches, and characterizing cells in a
disease state such as Duchenne’s muscular dystrophy. As previ-
ously mentioned, many new multimodal techniques also allow for
simultaneous recordings of omics that can also be combined with
spatial information. As these technologies keep maturing and
become applicable and easily translatable to muscle stem cell
biology, it will be an exciting time to keep refining the ideas in
a context-dependent manner ranging from development to adult
homeostasis, regeneration, and disease. Abbreviations
ATAC-Seq, assay for transposase-accessible chromatin using
sequencing; CyTOF, cytometry time of flight; FAP, fibro-
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single-cell chromatin immunoprecipitation coupled with sequenc-
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46. Saka SK, Wang Y, Kishi JY, et al.: Immuno-SABER enables highly
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37(9): 1080–90. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation
47. Giesen C, Wang HA, Schapiro D, et al.: Highly multiplexed imaging of tumor
tissues with subcellular resolution by mass cytometry. Nat Methods. 2014;
11(4): 417–22. PubMed Abstract | Publisher Full Text | F1000 Recommendation
48. Machado L, Esteves de Lima J, Fabre O, et al.: In Situ Fixation Redefines
Quiescence and Early Activation of Skeletal Muscle Stem Cells. Cell Rep. 2017;
21(7): 1982–93. PubMed Abstract | Publisher Full Text | F1000 Recommendation
49. van den Brink SC, Sage F, Vértesy Á, et al.: Single-cell sequencing reveals
dissociation-induced gene expression in tissue subpopulations. Nat Methods. 2017; 14(10): 935–6. PubMed Abstract | Publisher Full Text | F1000 Recommendation
50. van Velthoven CTJ, de Morree A, Egner IM, et al.: Transcriptional Profiling of
Quiescent Muscle Stem Cells In Vivo. Cell Rep. 2017; 21(7): 1994–2004. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation
51. Kester L, van Oudenaarden A: Single-Cell Transcriptomics Meets Lineage
Tracing. Cell Stem Cell. 2018; 23(2): 166–79. PubMed Abstract | Publisher Full Text | F1000 Recommendation 48. Machado L, Esteves de Lima J, Fabre O, et al.: In Situ Fixation Redefines
Quiescence and Early Activation of Skeletal Muscle Stem Cells. Cell Rep. 2017;
21(7): 1982–93. PubMed Abstract | Publisher Full Text | F1000 Recommendation
49. van den Brink SC, Sage F, Vértesy Á, et al.: Single-cell sequencing reveals
dissociation-induced gene expression in tissue subpopulations. Nat Methods. 2017; 14(10): 935–6. PubMed Abstract | Publisher Full Text | F1000 Recommendation
50. ;
( )
PubMed Abstract | Publisher Full Text | F1000 Recommendation 21. Cho DS, Doles JD: Single cell transcriptome analysis of muscle satellite cells
reveals widespread transcriptional heterogeneity. Gene. 2017; 636: 54–63. PubMed Abstract | Publisher Full Text | Free Full Text 43. Chen KH, Boettiger AN, Moffitt JR, et al.: RNA imaging. Spatially resolved,
highly multiplexed RNA profiling in single cells. Science. 2015; 348(6233):
aaa6090. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation 43. Chen KH, Boettiger AN, Moffitt JR, et al.: RNA imaging. Spatially resolved,
highly multiplexed RNA profiling in single cells. Science. 2015; 348(6233):
aaa6090. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation 22. Tabula Muris Consortium, Overall coordination, Logistical coordination, et al.:
Single-cell transcriptomics of 20 mouse organs creates a Tabula Muris. Nature. Page 8 of 10 F1000Research 2020, 9(F1000 Faculty Rev):31 Last updated: 22 JAN 2020 F1000Research 2020, 9(F1000 Faculty Rev):31 Last updated: 22 JAN 2020 44. Satija R, Farrell JA, Gennert D, et al.: Spatial reconstruction of single-cell
gene expression data. Nat Biotechnol. 2015; 33(5): 495–502. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation
45. Goltsev Y, Samusik N, Kennedy-Darling J, et al.: Deep Profiling of Mouse Splenic
Architecture with CODEX Multiplexed Imaging. Cell. 2018; 174(4): 968–981.e15. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation
46. Saka SK, Wang Y, Kishi JY, et al.: Immuno-SABER enables highly
multiplexed and amplified protein imaging in tissues. Nat Biotechnol. 2019;
37(9): 1080–90. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation
47. Giesen C, Wang HA, Schapiro D, et al.: Highly multiplexed imaging of tumor
tissues with subcellular resolution by mass cytometry. Nat Methods. 2014;
11(4): 417–22. PubMed Abstract | Publisher Full Text | F1000 Recommendation
48. Machado L, Esteves de Lima J, Fabre O, et al.: In Situ Fixation Redefines
Quiescence and Early Activation of Skeletal Muscle Stem Cells. Cell Rep. 2017;
21(7): 1982–93. PubMed Abstract | Publisher Full Text | F1000 Recommendation
49. van den Brink SC, Sage F, Vértesy Á, et al.: Single-cell sequencing reveals
dissociation-induced gene expression in tissue subpopulations. Nat Methods. 2017; 14(10): 935–6. PubMed Abstract | Publisher Full Text | F1000 Recommendation
50. van Velthoven CTJ, de Morree A, Egner IM, et al.: Transcriptional Profiling of
Quiescent Muscle Stem Cells In Vivo. Cell Rep. 2017; 21(7): 1994–2004. Editorial Note on the Review Process are written by members of the prestigious
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Yusuke Ono
Department of Muscle Development and Regeneration, Institute of Molecular Embryology and Genetics
(IMEG), Kumamoto University, Kumamoto, Japan
No competing interests were disclosed. Competing Interests:
1
Vittorio Sartorelli
Laboratory of Muscle Stem Cells and Gene Regulation, National Institute of Arthritis, Musculoskeletal and
Skin Diseases (NIAMS), National Institutes of Health, Bethesda, MD, USA
No competing interests were disclosed. Competing Interests:
2
Helen Blau
Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, Institute for Stem Cell
Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA
No competing interests were disclosed. Competing Interests:
3
Page 10 of 10 ;
( )
PubMed Abstract | Publisher Full Text | F1000 Recommendation van Velthoven CTJ, de Morree A, Egner IM, et al.: Transcriptional Profiling of
Quiescent Muscle Stem Cells In Vivo. Cell Rep. 2017; 21(7): 1994–2004. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation
51. Kester L, van Oudenaarden A: Single-Cell Transcriptomics Meets Lineage
Tracing. Cell Stem Cell. 2018; 23(2): 166–79. PubMed Abstract | Publisher Full Text | F1000 Recommendation 44. Satija R, Farrell JA, Gennert D, et al.: Spatial reconstruction of single-cell
gene expression data. Nat Biotechnol. 2015; 33(5): 495–502. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation
45. Goltsev Y, Samusik N, Kennedy-Darling J, et al.: Deep Profiling of Mouse Splenic
Architecture with CODEX Multiplexed Imaging. Cell. 2018; 174(4): 968–981.e15. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation
46. Saka SK, Wang Y, Kishi JY, et al.: Immuno-SABER enables highly
multiplexed and amplified protein imaging in tissues. Nat Biotechnol. 2019;
37(9): 1080–90. PubMed Abstract | Publisher Full Text | Free Full Text | F1000 Recommendation
47. Giesen C, Wang HA, Schapiro D, et al.: Highly multiplexed imaging of tumor
tissues with subcellular resolution by mass cytometry. Nat Methods. 2014;
11(4): 417–22. PubMed Abstract | Publisher Full Text | F1000 Recommendation Page 9 of 10 Page 9 of 10 F1000Research 2020, 9(F1000 Faculty Rev):31 Last updated: 22 JAN 2020 Current Peer Review Status: Current Peer Review Status: Editorial Note on the Review Process Version 1 Yusuke Ono
Department of Muscle Development and Regeneration, Institute of Molecular Embryology and Genetics
(IMEG), Kumamoto University, Kumamoto, Japan
No competing interests were disclosed. Competing Interests:
1 Yusuke Ono
Department of Muscle Development and Regeneration, Institute of Molecular Embryology and Genetics
(IMEG), Kumamoto University, Kumamoto, Japan
No competing interests were disclosed. Competing Interests:
1 Vittorio Sartorelli
Laboratory of Muscle Stem Cells and Gene Regulation, National Institute of Arthritis, Musculoskeletal and
Skin Diseases (NIAMS), National Institutes of Health, Bethesda, MD, USA
No competing interests were disclosed. Competing Interests:
2 Skin Diseases (NIAMS), National Institutes of Health, Bethesda, MD, USA
No competing interests were disclosed. Competing Interests:
Helen Blau
Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, Institute for Stem Cell
Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA
No competing interests were disclosed. Competing Interests:
3 Helen Blau
Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, Institute for Stem Cell
Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USA
No competing interests were disclosed. Competing Interests:
3 Page 10 of 10
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https://academicjournals.org/journal/AJB/article-full-text-pdf/1BF8AAB9301.pdf
|
English
| null |
Characterization of bacteriocin produced by Lactobacillus plantarum F1 and Lactobacillus brevis OG1
|
African journal of biotechnology
| 2,003
|
cc-by
| 6,283
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Full Length Research Paper Accepted 9 July 2003 Lactobacillus plantarum F1 and L. brevis OG1 isolated from Nigerian fermented food products,
produced bacteriocins that had broad spectrum of inhibition against both pathogenic, food spoilage
organisms and various lactic acid bacteria. The test organisms exhibited activities of 6400 and 3200
AU/ml respectively against Escherichia coli NCTC10418 and Enterococcus faecalis EF1, but did not
inhibit Candida albicans ATCC10231 and Klebsiella sp. UCH15. Comparison of the antimicrobial
spectra and characterization of the two bacteriocins were not identical. Bacteriocin produced by L. brevis OG1 was the most heat stable at 121oC for 60 min, while that of L. plantarum F1 was stable at
121oC for 10 min. The bacteriocins produced by the test isolates maintained full stability after storage
for 60 days at – 200C; partial stability after storage for 120 days at 40C; while activity was not detected
after storage for 80 to 120 days at 370C. Bacteriocin produced by L. brevis OG1 was stable at pH range
of 2.0 to 8.0 while, that of L. plantarum F1 was found to be stable at pH 2.0 to 6.0. Their active principle
was proteinaceous in nature since the bacteriocins were inactivated by proteolytic enzymes, but not
by other non–proteolytic enzymes. mitomycin C and uv light did not affect the activity of the
bacteriocins, while chloroform extraction completely destroyed their activity. Exposure to surfactant
resulted in an increase in the bacteriocin titre, except Nonidet P-40, which led to total loss of
bacteriocin activity. The bacteriocins were able to pass through cellulose membranes with 100,000
KDa and 1,000,000 KDa but could not pass through one with a 10,000 KDa and 1,000 KDa molecular
weight cut off. The paper concluded that the ability of bacteriocins produced by the test isolates in
inhibiting a wide-range of bacteria, is of potential interest for food safety and may have future
applications as food preservative. Key words: Bacteriocins, lactic acid bacteria, indicator organisms, fermented foods, antagonistic activity. *Corresponding author: E-mail: topzybanwo@yahoo.com African Journal of Biotechnology Vol. 2 (8), pp. 219-227, August 2003
Available online at http://www.academicjournals.org/AJB
ISSN 1684–5315 © 2003 Academic Journals African Journal of Biotechnology Vol. 2 (8), pp. 219-227, August 2003
Available online at http://www.academicjournals.org/AJB
ISSN 1684–5315 © 2003 Academic Journals Production of crude bacteriocin samples Production of crude bacteriocin samples Lactobacillus species were propagated in 1000 ml MRS broth (pH
7.0, glucose, 0.25% w/v, peptone, 0.5% w/v) for 72 h at 300C
anaerobically (Oxoid Gas Generating Kit) in triplicate. For extraction
of bacteriocin, a cell-free solution was obtained by centrifuging
(10,000 rpm for 20 min. at 40C with Beckman L5050B) the culture
and was adjusted to pH 7.0 by means of 1M NaOH to exclude the
antimicrobial effect of organic acid, followed by filtration of the
supernatant through a 0.2 m pore-size cellulose acetate filter. The
supernatant was dialysed for 24 h at 40C (Schillinger and Lucke,
1989). Inhibitory activity from hydrogen peroxide was eliminated by
the addition of 5 mg/ml catalase (C-100 bovine liver, Sigma) (Daba
et al., 1991). Sample collection Cassava (Manihot esculenta Crantz) tubers and maize (Zea mays)
grains used in this study were obtained from retail market in South-
Western Nigeria. Purification of bacteriocin samples Ammonium Sulphate Precipitation: The crude bacteriocin samples
produced
were
treated
with
solid
ammonium
sulphate
(Mallinckrodth Chemical, Inc., Paris, KY, USA) to 0, 30, 35, 40, 45,
50, 55 and 60% saturation. The mixtures were stirred for 2 h at 4ºC
and later centrifuged at 20,000 rpm for 1 h (4ºC). The precipitates
were re-suspended in 25 ml of 0.05 M potassium phosphate buffer
(pH 7.0). Dialysis was followed in a tubular cellulose membrane
(Specrapor, 1000 dalton MWco, Fisher Scientific Pittsburgh, PA
USA) against 2 litres of the same buffer for 18 h in spectrapor No. 4
dialysis tubing. Assay of the bacteriocin activity was carried out and
titer was determined in both the precipitate and supernatant to
know which one actually contain the bacteriocin (Fraction 1)
(Jimenez-Diaz et al., 1993). Traditional Fermentation of Cassava Cassava tubers (1 kg) was peeled, cut into pieces and washed
several times in water followed by soaking in water (submerged
fermentation) for period of 3 days at ambient temperature (26C
1C). The softened pulpy mass of the fermented cassava samples
was disintegrated and passed through a clean coarse sieve to
remove lumps and fibers after which the mass was allowed to
sediment (Oyewole and Odunfa, 1990). Bacterial strains and cultures was also found to produce a bacteriocin- like inhibitory
compound against the lactic acid bacteria of wines
(Yurdugul and Bozoglu, 2002). Lactic acid bacterial strains were isolated from cassava retting and
traditional prepared ogi (Table 1). For all samples, 10 g were
added to 90 ml of sterile diluent’s containing 0.1% peptone water
and homogenized for 30 s. From appropriate 10-fold dilutions;
isolation of bacteria was carried out on MRS agar and incubated
anaerobically at 300C for 48 h. The cultures were purified by
repeated streaking. Strains were characterized using the AP1
50CH strips and AP1 50 CHL medium (AP1 Systems, Biomerieux
Sa, France). The food spoilage and pathogenic bacteria used as
indicator organisms were obtained from the culture collection of
Medical Microbiology, Laboratory, University College Hospital,
Ibadan, Nigeria. Several types of bacteriocins from food-associated
lactic
acid
bacteria
have
been
identified
and
characterized, of which the important ones are nisin,
diplococcin, acidophilin, bulgarican, helveticins, lactacins,
and plantaricins (Nettles and Barefoot, 1993) Of these,
bacteriocin and nisin produced by Lactococcus lactis ssp. lactis, has been the most extensively characterized
(Buchman et al., 1988; Liu and Hansen, 1990). At
present, nisin is the only bacteriocin commercially
available
and
marketed
(Balasubramanyam
and
Varadara, 1998). It has been reported that nisin is more
active against Gram-positive bacteria, particularly the
spore-formers
(Delves-Broughton,
1990). Other
bacteriocins of Lactobacilli have been reported to be
effective against closely related species of mesophilic
Lactobacillus and therefore considered as potential
natural food preservatives (Daeschel 1993; De Vugst and
Vandamme, 1994). Table 1. Lactic acid bacteria isolated from fermented ogi and
cassava retting. Table 1. Lactic acid bacteria isolated from fermented ogi and
cassava retting. Table 1. Lactic acid bacteria isolated from fermented ogi and
cassava retting. Name of product
Associated lactic acid bacteria
Ogi
L. reuteri, L. leichmani, L. plantarum, L. casei, L. fermentum, L. brevis, L. alimentarius, L. buchneri and
L. jensenii. Cassava retting
L. plantarum, L. brevis, L. fermentum, L. delbrueckii, L. jenseni, L. casei and L. mesenteroides. However, studies relating to the antibacterial properties
of these organisms have been limited and not fully
exploited for use (Reddy et al., 1984; Abdel-Bar et al.,
1987). Three of the most important aspects in the study
of bacteriocins are their production, characterization and
purification. Therefore, this paper reports the bacteriocin
of two lactic acid bacteria isolates from Nigeria fermented
foods. INTRODUCTION Lactobacilli are important organisms recognized for their
fermentative ability as well as their health and nutritional
benefits
(Gilliand,
1990). They
produce
various
compounds such as organic acids, diacetyl, hydrogen
peroxide, and bacteriocin or bactericidal proteins during
lactic fermentations (Lindgren and Dobrogosz, 1990). Bacteriocins are proteinaceous antibacterial compounds
and exhibit bactericidal activity against species closely
related to the producer strain (De Vugst and Vandamme,
1994) many bacteriocins are active against food-borne
pathogens especially against Listeria monocytogenes
(Vignolo et al., 1996; De Martins and Franco, 1998;
Bredholt et al., 1999). Leuconostoc mesenteroides L124
and L. curvatus L442 isolated from dry fermented
sausages, produce bacteriocin antagonistic towards
closely related species and pathogens (Mataragas et al,
2002). An isolate of L. mesenterioides sub sp. cremoris *Corresponding author: E-mail: topzybanwo@yahoo.com Afr. J. Biotechnol. 220 Ogunbanwo et al. U/mg), in 0.05 M Tris hydrochloride (pH 8.0); lysozyme (Serva,
20600 U/mg), in 1N NaOH (pH 6.5); -amylase (type IX, 1000
U/mg), in 1N NaOH (pH 6.5); dextranase in 0.2 M citrate (pH 6.1)
and proteinase K (11.5 U/mg) in 1N NaOH (pH 6.5). Samples of
bacteriocin (500 l) were incubated with 500g of each enzyme per
ml for 60 min at 37ºC except for samples containing trypsin, -
chymotrypsin and catalase, which were incubated at 25ºC. Prior to
being assayed for bacteriocin activity, preparations containing
papain were adjusted to pH 6.0 and those containing trypsin and
chymotrypsin were treated with trypsin-chymotrypsin inhibitor
(Sigma) according to the manufacturer’s instructions (Wanda and
Bonita, 1991). protein (bacteriocin). The mixture was centrifuged at 13,000 rpm
for 10 minutes after which the supernatant was decanted. The
resulting pellet was dissolved in 2 ml of potassium phosphate buffer
(Fraction 2). Ultrafiltration studies: The Fraction 2 (Trichloroacetic acid
precipitation) bacteriocin sample was resuspended to 1/30 volume in
potassium phosphate buffer (50mM, pH 7.0). Several aliquots (1ml)
were ultrafiltered through various filtron membranes (Filtron
Technology Corp; Northborough, Mass), including 1,000,000,
100,000, 10,000 and 1,000 KDa. molecular exclusion sizes. Bacteriocin activity was determined in retained and eluted fractions
(Jimenez-Diaz et al., 1993) and protein concentration of the
fractions were determined by the Bradford method (Bradford,
1976). Stability of bacteriocin during storage: Purified bacteriocin was
stored at –20, 4 and 37ºC. At different time intervals, samples were
taken from the stored material (ten Brink et al., 1994) to determine
bacteriocin activity. Determination of bacteriocin activity: A well diffusion assay
procedure was used (Schillinger and Lucke, 1989; Takahiro et al.,
1991). Aliquots of 50 l from each bacteriocin dilution (see
determination of bacteriocin titire) were placed in wells in plates
seeded with the bioassay strain. The plates were incubated
overnight at 30ºC for lactic acid bacteria indicators (anaerobically)
and at 37ºC for non-lactic acid bacteria indicators (aerobically), and
the diameters of the inhibition zone were taken (Rammelsberg and
Radler, 1990). Extraction of bacteriocin with organic solvents: Various organic
solvents
including
iso–amylalcohol,
chloroform,
n–propanol,
hexane, Di – ethyl ether, petroleum ether were added to purified
bacteriocin in 1:1 ratio. After thorough mixing, phase separation
was achieved by centrifugation (10 min at 5000 rpm). When
propanol was used for the extraction, 50 g/l NaCl was added to the
mixture in order to obtain phase separation. Ogunbanwo et al. The organic phase and
the aqueous phase were collected and solvent removed by
evaporation at 45ºC. The residue from the organic phase was
resuspended in an amount of saline (8.5 g/l NaCl) equal to the
starting volume of the original supernatant fluid (ten Brink et al.,
1994). Bacteriocin activity of both preparations was then
determined. Determination of bacteriocin titre: The titres of bacteriocin
produced were quantified by two fold serial dilutions of bacteriocin
in saline solution and aliquots of 50 l from each dilution were
placed in wells in plates seeded with the bioassay strain. These
plates were incubated anaerobically at 300C for lactic acid bacteria
indicators and aerobically at 370C for non-lactic acid bacteria
indicators for 18-24 h and examined for the presence of 2 mm or
larger clear zones of inhibition around the wells. The antimicrobial
activity of the bacteriocin was defined as the reciprocal of the
highest dilution showing inhibition of the indicator lawn and was
expressed in activity units per ml (AU ml-1) (Graciela et al., 1995). Effect of mitomycin C on bacteriocin activity: Mitomycin C was
added at a final concentration of 1.0 g/ml to purified bacteriocin
and incubation was carried out at 30ºC. Samples were removed at
60, 120, 240 and 360 min. and analysed by the well diffusion
method (Wanda and Bonita, 1991). Effect of uv light on bacteriocin activity: A 10 ml aliquot of
purified bacteriocin was placed in a sterile petri dish and exposed to
short – wave uv light from a 15 – W General Electric germicidal bulb
at a distance of 30 cm. Times of exposure ranged from 0 to 5 min. (Wanda and Bonita, 1991). After each time interval, bacteriocin
activity was analysed by the well diffusion method. Characterization of bacteriocin The purified bacteriocin samples (Fraction 2) were characterized
with respect to thermal and pH stability, susceptibility to
denaturation by enzymes, stability during storage, extraction with
organic solvent, treatment with dissociating agents and mitomycin C
and uv light induction. Effect of surfactant on bacteriocin activity: This was carried out
by incorporating non-ionic (triton X100, tween 20, tween 80, nonidet
P40), anionic (sodium dodecyl sulphate, deoxycholic acid) and
dipolar ionic (N–hexadecyl–N,N–dimethyl-3-ammonio–1–propane
sulphonate, N–lauroylsarcosine) surfactants. The surfactants were
obtained from Sigma Chemical Co. and were added to purified
bacteriocin at a concentration of 0.1 ml or 0.01 g of surfactant ml-1
of bacteriocin solutions. These preparations were incubated at 30ºC
for 60 min. (Kelly et al., 1996) and assayed for bacteriocin activity
against indicator organisms by using titre evaluation. Heat Resistance: Purified bacteriocin (400l) was exposed to
various heat treatments: 40, 60, 80, 100 and 121ºC. Aliquot
volumes of each Fraction were then removed after 0, 30, 60 or 90
min (ten Brink et al., 1994) and assayed for bacteriocin. pH Sensitivity: Purified bacteriocin (400 l) were adjusted to pH 2,
4, 6, 8, 10, and 12 with hydrochloric acid (HCl) and sodium
hydroxide (NaOH), incubated for 4 h at room temperature (ten Brink
et al., 1994) and similarly assayed. Enzyme Treatments: Purified bacteriocin was assessed for its
sensitivity to various enzymes. Enzymes (all obtained from Sigma)
and their respective buffers were lipase (type 1, 8.6 U/mg) in 0.05 M
Tris hydrochloride (pH 8.0), 0.01 M CaCl2; -chymotrypsin (type 11,
47 U/mg) in 0.05 M Tris hydrochloride (pH 8.0) 0.01 M CaCl2;
pronase E (type xxvi, 4.1 U/mg) in 0.01m sodium borate, 0.05 M
HCl, 5 mM CaCl2, 1 mM CaCl2 (pH 7.2); pepsin (3,2001 U/ml), in
0.2 M citrate (pH 6.0); catalase (2,000 U/mg), in 10 mM potassium
phosphate (pH 7.0); phospholipase C (type 1, 10 U/mg), in 0.05 M
Tris hydrochloride (pH 7.0), 0.01M CaCl2; trypsin (type x, 15000 Traditional preparation of ogi The cereal grain (Z. mays) were cleaned and steeped in water for 2
days in earthenware pot (or any suitable container). The water is
decanted and the grains wet-milled before sieving with muslin cloth
or fine wire-mesh. The pomace is discarded and the starch
suspension is allowed to sediment during which fermentation is
carried out for 2-3 days by the natural flora of the grains (Odunfa
and Adeyele, 1985). Trichloroacetic acid (TC) precipitation: Five percent (5%)
equivalent of TC was added to 25 ml of Fraction 1 to precipitate the 221 RESULTS The selected Lactobacillus strains (L. plantarum F1 and
L. brevis OG1) produced bacteriocin, which showed
inhibitory activity against one or more of the Gram-
positive and Gram-negative target strains (Table 2). The
two species of Lactobacillus had different profiles of
inhibition, and the profiles were strain specific. L. 222 Afr. J. Biotechnol. Table 2. Inhibition of various indicator organisms by bacteriocin produced by lactic acid bacteria. Indicator organisms
Strain No
Origin
L. plantarum (F1)
L. brevis (OG1)
Bacillus cereus
Bacillus stearothermophilus
Bacillus subtilis
Micrococcus luteus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus epidermidis
Staphylococcus faecalis
Staphylococcus pyogenes
Listeria denitrificans
Listeria monocytogenes
Candida albicans
Escherichia coli
Escherichia coli
Enterococcus faecalis
Aeromonas pobvia
Vibro cholerea
Shigella flexneri
Shigella dysentry
Salmonella typhimurium
Salmonella kentucky
Klebsiella spp
Clostridium sporagenes
Serratia marcescens
Helicobacter pylori
Streptococcus thermophilus
Lactobacillus acidophilus
Lactobacillus brevis
Lactobacillus plantarum
Lactobacillus reuteri
Lactobacillusdelbrueckii
Leuconostoc mesentaroides
ATCC9634
NCIB8222
ATCC6633
NCIB196
ATCCI4458
NCTC6571
NCTC5413
ATCC19433
ATCC19615
ATCC14870
587CHRL
ATCC10231
NCTC10418
K12
EFI
AP15534
AP23622
AP23498
AP22433
ATCC13311
AT1
UCH15
NCIB532
UI5
NCTC11637
IW4
U1
OGW1
F1
PW1
PT6
M8
Reference strain
“”
“”
“”
“”
“”
“”
“”
“”
“”
,,,
“”
“”
“”
“”
“”
“”
“”
“”
“”
Reference strain
Sputum
Reference strain
Soil
Reference strain
Iru
Ugba
Ogi
Foofoo
Palm wine
Pito
Meat
+(10mm)
+(8mm)
+(10mm)
+(12mm)
+(8mm)
+(8mm)
+(10mm)
+(6mm)
+(5mm)
+(7mm)
+(7mm)
-
+(8mm)
+(12mm)
+(10mm)
-
-
+(7mm)
+(6mm)
+(7mm)
+(8mm)
-
+(12mm)
+(8mm)
+(10mm)
+(5mm)
-
-
-
-
+(8mm)
+(6mm)
+(8mm)
+(6mm)
+(7mm)
+(10mm)
+(5mm)
+(6mm)
+(8mm)
-
-
+(10mm)
+(9mm)
-
+(6mm)
+(8mm)
+(12mm)
-
+(8mm)
+(5mm)
+(5mm)
+(6mm)
+(9mm)
-
+(8mm)
-
-
-
-
-
+(8mm)
+(6mm)
-
+(5mm)
Key: No inhibition = −; inhibition = + plantarum F1 and L. brevis OG1 were characterized by
the broad-spectrum inhibition of microorganisms. All the
bacteriocin produced by the test isolates inhibited E. coli
(NCTC 10418) and Enterococcus faecalis (EF1) but did
not inhibit Candida albicans. (ATCC 10231) and
Klebsiella spp (UCH15). The largest spectrum of
inhibition was showed by L. plantarum F1, which inhibited
24 out of 32 indicator strains. However, it was generally
observed that bacteriocin from the producer organism
had no inhibitory effect on the organism producing it. for 60 minutes, but declined thereafter (Figure 1), while
bacteriocin produced by L. plantarum F1 activity (6400
AU/ml) remained constant after heating at 121ºC for 10
followed by subsequent decline. RESULTS At 90 minutes there was
no detectable bacteriocin activity in L. plantarum F1
(Figure 1). (
g
)
Effect of time and temperature of storage on
bacteriocin activity was also carried out. It was observed
that all the bacteriocin produced by the test isolates
maintained full stability after storage for 60 days at –
20ºC; partial stability after storage for 120 days at 4ºC,
while no activity was detected after storage for 80 to 120
days at 37ºC (Figure 2). Effect of pH on activity of
bacteriocin was carried out. It was observed that
bacteriocin produced by L. brevis OG1was stable at pH 2
to 8, while for L. plantarum F1, it was found to be stable
at pH 2 to 6 (Table 3). The effects of heat, storage time, pH, enzymes and
surfactants on bacteriocin activity were determined using
E. faecalis (EF1) as indicator organism. The inhibitory
compound produced by the test isolates was considered
to be heat stable. Bacteriocin produced by L. brevis OG1
was considered to be the most heat stable, as the activity
(3200 AU/ml) remained constant after heating at 121ºC 223 Ogunbanwo et al. Figure 1. Effect of temperature on bacteriocin activity produced by the test isolates. 0
1000
2000
3000
4000
5000
6000
7000
40
60
80
100
121
40
60
80
100
121
Bacteriocin activity (AU/ml)
10
30
60
90
Temperature (0C)
L. plantarum F1
L. brevis OG1 Figure 1. Effect of temperature on bacteriocin activity produced by the test isolates. Figure 2. Effect of time and temperature of Storage on bacteriocin activity produced by the test isolates. 0
2
4
6
8
10
12
14
0
20
40
60
80
100 120
0
20
40
60
80
100 120
Time (min)
Zone of inhibition (mm)
-20
4
37
L. brevis OG1
L. plantarum F1 Figure 2. Effect of time and temperature of Storage on bacteriocin activity produced by the test isolates. Figure 2. Effect of time and temperature of Storage on bacteriocin activity produced by the test isolates Table 3. Effect of pH treatment on bacteriocin activity (AU/ml)
Produced by the test isolates. Table 3. Effect of pH treatment on bacteriocin activity (AU/ml)
Produced by the test isolates. Bacteriocin produced by the test isolates was tested for
their sensitivity (loss of activity) to various enzymes. RESULTS brevis OG1
Control
Nonidet P – 40
N – lauroyl sarcosine
N-dimethyl-3-ammonio-
1-propane sulphonate
Hexadeyltrimethyl
ammonium bromide
Tritox X – 100
Tween 20
Tween 80
Deoxycholic acid
Sodium dodeyl sulphate
6400
0
12800
6400
12800
12800
25600
25600
12800
12800
3200
0
3200
6400
6400
6400
6400
6400
3200
6400 exception of nonidet P-40, which led to total loss of
bacteriocin activity. N-lauroyl sarcosine had little or no
effect on bacteriocin activity produced by the test
isolates. Purification steps of the bacteriocin are summarized in
Table 7. The bacteriocin of L. plantarum F1 and L. brevis
OG1 were recovered following the 60% saturation of the
culture broths with ammonium sulphate with an increase
to specific activity of 9.4 and 5.2 AU/g protein
respectively (Fraction 1). The second step in the
purification protocol was Trichloroacetic acid precipitation
of Fraction 1. At this stage of purification, the recovery
was 18.3% for both isolates. The specific activity
increased to 85.3 and 44.4 AU/g protein for L. plantarum
F1 and L. brevis OG1 respectively (Fraction 2). Finally,
these fractions were subjected to ultrafiltration using
various filtron membranes. The eluted and retained
fractions were collected and assayed for bacteriocin
activity. At this stage, when filtered through membrane
with 1,000,000 KDa molecular weight cut off, the
purification factor was 7.5 and the recovery was 1.2% in
L. plantarum F1 while in L. brevis OG1, purification factor
reached 9.0 and the recovery was 1.0%. The bacteriocins
were able to pass through cellulose membranes with
100,000 KDa and 1,000,000 KDa, but filtration was not
achieved with 10,000 KDa and 1,000 KDa molecular
weight cut off. However, partial loss of bacteriocin activity
was observed during ultrafiltration (Table 8). Table 5. Influence of organic solvents in the extraction of
bacteriocin produced by the test isolates. Table 5. Influence of organic solvents in the extraction of
bacteriocin produced by the test isolates. L. plantarum F1
L. RESULTS The
antimicrobial activity was lost or unstable after treatment
with all the proteolytic enzymes, whereas treatment with
lipase, catalase, phospholipase C, lysozyme, -amylase,
dextranase, mitomycin and uv-light did not affect the
activity of bacteriocin produced by the test isolates. Bacteriocin produced by L. plantarum F1 showed
decreased in activity when treated with pronase E (Table
4). PH
L. plantarum F1
L. brevis OG1
2
6400
3200
4
6400
3200
6
6400
3200
8
3200
3200
10
1600
1600
12
400
400 Various organic solvents were tested for the extraction
of bacteriocin produced by the test isolates. It was Afr. J. Biotechnol. 224 Table 6. Effect of surfactants on activity of bacteriocin (AU/ml)
produced by the test isolates. Table 4. Effect of enzymes, mitomycins and uv light treatments on
activity (AU/ml) of bacteriocin produced by the test isolates. Table 4. Effect of enzymes, mitomycins and uv light treatments on
activity (AU/ml) of bacteriocin produced by the test isolates. Table 4. Effect of enzymes, mitomycins and uv light treatments on
activity (AU/ml) of bacteriocin produced by the test isolates. Treatments
L. plantarum F1
L. brevis OG1
Control
Lipase
-chymotrypsin
Pronase E
Pepsin
Catalase
Phospholipase C
Trypsin
Lysozyme
- Amylase
Dextranase
Proteinase K
Mitomycin C
UV light
6400
6400
nd
1600
nd
6400
6400
nd
6400
6400
6400
nd
6400
6400
3200
3200
nd
nd
nd
3200
3200
nd
3200
3200
3200
nd
3200
3200
nd = non-detectable
Table 5. Influence of organic solvents in the extraction of
bacteriocin produced by the test isolates. L. plantarum F1
L. brevis OG1
Organic solvent
Organic
phase
Aqueous
phase
Organic
phase
Aqueous
phase
Control
I – amylalcohol
Choloform
N – propanol
Hexane
Di – ethylether
-
6
-
8
-
-
10
6
-
5
8
10
-
8
-
8
-
-
12
8
-
4
6
10 Treatments
L. plantarum F1
L. brevis OG1
Control
Lipase
-chymotrypsin
Pronase E
Pepsin
Catalase
Phospholipase C
Trypsin
Lysozyme
- Amylase
Dextranase
Proteinase K
Mitomycin C
UV light
6400
6400
nd
1600
nd
6400
6400
nd
6400
6400
6400
nd
6400
6400
3200
3200
nd
nd
nd
3200
3200
nd
3200
3200
3200
nd
3200
3200
nd = non-detectable
Table 5. Influence of organic solvents in the extraction of
bacteriocin produced by the test isolates. Surfactants
L. plantarum F1
L. RESULTS brevis OG1
Organic solvent
Organic
phase
Aqueous
phase
Organic
phase
Aqueous
phase
Control
I – amylalcohol
Choloform
N – propanol
Hexane
Di – ethylether
Petroleum ether
-
6
-
8
-
-
-
10
6
-
5
8
10
7
-
8
-
8
-
-
-
12
8
-
4
6
10
5
Zone of inhibition (mm)
Control = bacteriocin without addition of organic solvent Control = bacteriocin without addition of organic solvent observed that extraction with polar solvents such as
hexane, di-ethyl ether, and petroleum ether did not result
in the removal bacteriocin produced at the aqueous
phase to the organic phase, while chloroform extraction
completely destroyed the bacteriocins activity. However,
when different alcohols such as n-propanol and Iso-amyl
alcohol
were
used
in
the
extraction
procedure,
bacteriocin was removed from the aqueous phase and
recovered from the organic phase (Table 5). DISCUSSION Bacteriocins produced by the test organisms have some
interesting characteristics that justify their study. The
most striking is that none of these bacteriocins is limited
by the extremely narrow antibacterial spectrum reported
for some bacteriocins of some lactic acid bacteria, for
example lactococcin A (Holo et al., 1991) and lactacin B
(Barefoot and Klaenhammer, 1983). The largest Table 6 showed the effect of dissociating agents on
bacteriocin activity. Exposure to surfactants resulted in
an increase in the bacteriocin titre (by at least one to two
fold dilutions) rather than any decrease in activity with the Ogunbanwo et al. 225 Table 7. Purification of bacteriocin produced by L. plantarum F1 and L. brevis OG1. Table 7. Purification of bacteriocin produced by L. plantarum F1 and L. brevis OG1. Organisms
Purification Stages
Volume
(ml)
Activity
(Auml-)
Total
activity
(AV)a
Protein
(gml-1)b
Specific
activityc
Purification
factord
Recovery
(%)e
L. plantarum
F1
Culture supernatant
Ammonium sulphate precipitation (Fraction 1)
Tricholoroacetic acid Precipitation (Fraction 2)
Ultrafiltration (1,000,000 KDa cut off) (Fraction 3)
1000
25
2
5
1600
3200
6400
3200
1600000
80000
12800
16000
410
340
75
5
3.9
9.4
85.3
640
1
2.4
9.1
7.5
100
82.9
18.3
1.2
L. brevis
OG1
Culture supernatant
Ammonium sulphate precipitation (Fraction 1)
Tricholoroacetic acid Precipitation (Fraction 2)
Ultrafiltration (1,000,000 KDa cut off) (Fraction 3)
1000
25
2
6
800
1600
3200
1600
800000
40000
6400
9600
394
310
72
4
2.0
5.2
44.4
400
1
2.6
8.5
9.0
100
78.7
18.3
1.0
aTotal activity was determined by the multiplication of volume by activity
bProtein concentration was determined by the Bradford method
cSpecific activity is the activity units divided by the protein concentration (AUg-1)
dPurification faction is the increase in the initial specific activity
eRecovery percentage is the remaining protein concentration as a percentage of the initial protein concentration Table 8. Ultra filtration study of bacteriocins. AU (%Initial Bacteriocin activity)b
aMembrane molecular
weight cut off (KDa)
L. plantarum F1
L. brevis OG1
Retentive (%)
Eluted fraction (%)
Retentive (%)
Eluted fraction (%)
1,000,000
400(6.25)
3200(50.0)
400(12.5)
1600(50.0)
100,000
3200(50.0)
1600(25.0)
1600(50.0)
800(25.0)
10,000
6400(100.0)
0(0.0)
3200(100.0)
0(0.0)
1,000
6400(100.0)
0(0.0)
3200(100.0)
0(0.0)
aFiltron membrane were used. bInitial bacteriocin activity from:
L. plantarum F1 = 6400 AU/ml
L. brevis OG1 = 3200 AU/ml Table 8. Ultra filtration study of bacteriocins. (Nettles and Barefoot, 1993), heat stability of L. brevis
OG1 at 121ºC for 60 min is novel. DISCUSSION Temperature stability is
important if the bacteriocins are to be used as a food
preservative,
because
many
procedures
of
food
preparation involve a heating step. spectrum of inhibition was exhibited by L. plantarum F1,
which inhibited 24 out of 32 indicator strains. Earlier
reports (Tagg et. al., 1976; Daeschel et al., 1985; Sanni
et al., 1999) have shown that some bacteriocins
produced by gram-positive bacteria have a broad
spectrum of activity. However, it was generally observed
that bacteriocin from the producer organism had no
inhibitory effect on the organism producing it. p
p
g
p
The activity of bacteriocin elaborated by the test
isolates was also pH dependent. The highest
antibacterial activity was exhibited in an acidic pH range
of 2 to 6, while inactivation occurred at pH 8 to 12. Two
bacteriocins, namely bulgarican and lactobulgarican,
isolated from L. bulgaricus, were shown to have the
highest activity and stability at pH 2.2 and 4.0
respectively, against a range of pathogenic and spoilage
bacteria (Reddy et. al., 1984; Abdel–Bar et. al., 1987). Bacteriocins produced by the test isolates remained fully
stable after storage for 60 days at -20C, but declined or
became non-detectable after storage for 80 to 120 days
at 37C, indicating that cold temperature may be the most The inhibitory compound (bacteriocins) produced by
the test isolates was heat stable. The bacteriocin
produced by L. brevis OG1 was considered to be the
most heat stable as there was no reduction in activity
after heating at 121ºC for 60 min, while that produced by
L. plantarum F1 was able to exhibit full activity after
heating at 121ºC for only 10 min. Andersson (1986) also
reported loss of activity after heat treatment at 121ºC for
15 min. Although heat stability of antibacterial substances
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Scissor-like Au4Cu2 Cluster with Phosphorescent Mechanochromism and Thermochromism
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Keywords: cluster; copper; gold; luminescence; mechanochromism; thermochromism Citation: Wu, X.-M.; Wang, J.-Y.;
Huang, Y.-Z.; Chen, Z.-N. Scissor-like
Au4Cu2 Cluster with Phosphorescent
Mechanochromism and
Thermochromism. Molecules 2023, 28,
3247. https://doi.org/
10.3390/molecules28073247 Citation: Wu, X.-M.; Wang, J.-Y.;
Huang, Y.-Z.; Chen, Z.-N. Scissor-like
Au4Cu2 Cluster with Phosphorescent
Mechanochromism and
Thermochromism. Molecules 2023, 28,
3247. https://doi.org/
10.3390/molecules28073247 Xue-Meng Wu 1,2,3, Jin-Yun Wang 1, Ya-Zi Huang 1 and Zhong-Ning Chen 1,2,3,4,* 1
State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter,
Chinese Academy of Sciences, Fuzhou 350002, China y
2
ShanghaiTech University, Pudong, Shanghai 201210, China y
y
j
g
4
Fujian Science & Technology Innovation Laboratory for Optoelectronic Information of China
Fuzhou 350108, China *
Correspondence: czn@fjirsm.ac.cn Abstract: Reaction of [Au(tht)2](ClO4) (tht = tetrahydrothiophene), [Cu(CH3CN)4](ClO4), 3,6-di-tert-
butyl-1,8-diethynyl-9H-carbazole (H3decz), and bis(2-diphenylphosphinophenyl)ether (POP) in the
presence of triethylamine (NEt3) gave the cluster complex Au4Cu2(decz)2(POP)2 as yellow crystals. As revealed by X-ray crystallography, the Au4Cu2 cluster exhibits scissor-like structure sustained
by two decz and two POP ligands and stabilized by Au-Cu and Au-Au interactions. The Au4Cu2
cluster shows bright yellow to orange photoluminescence upon irradiation at >300 nm, arising from
3[π (decz)→5d (Au)] 3LMCT (ligand-to-metal charge transfer) and 3[π→π* (decz)] 3IL (intraligand)
triplet states as revealed by theoretical and computational studies. When it is mechanically ground,
reversible phosphorescence conversion from yellow to red is observed owing to more compact
molecular packing and thus stronger intermetallic interaction. Variable-temperature luminescence
studies reveal that it displays distinct red-shifts of the emission whether the temperature is elevated
or lowered from ambient temperature, suggestive of exceptional thermochromic phosphorescence
characteristics. Citation: Wu, X.-M.; Wang, J.-Y.;
Huang, Y.-Z.; Chen, Z.-N. Scissor-like
Au4Cu2 Cluster with Phosphorescent
Mechanochromism and
Thermochromism. Molecules 2023, 28,
3247. https://doi.org/
10.3390/molecules28073247
Academic Editor: José Manuel
Gaspar Martinho
Received: 5 March 2023
Revised: 29 March 2023
Accepted: 3 April 2023
Published: 5 April 2023 molecules molecules Scissor-like Au4Cu2 Cluster with Phosphorescent
Mechanochromism and Thermochromism Xue-Meng Wu 1,2,3, Jin-Yun Wang 1, Ya-Zi Huang 1 and Zhong-Ning Chen 1,2,3,4,* 1. Introduction Stimuli-responsive luminescent compounds have been flourishingly developed as a
class of smart solid materials with potential applications in various fields such as sensing,
imaging, anti-counterfeiting, encryption, etc. [1–3]. Ligand-supported d10 metal cluster
complexes are particularly attractive because of the additional d10-d10 intermetallic contacts
that are extremely sensitive to external environments or stimuli and play a crucial role in
modulation of photophysical properties. A number of ligand-supported d10 metal clusters
have been found to display remarkable absorption and emission color changes upon
stimulation by external conditions such as light, heat, pressure, vapor, and so on [4–21],
associating mostly with the alteration of d10-d10 intermetallic contacts so that emissive
energy is thus perturbed. gy
p
Among various organic ligands, alkyl or aryl ethynyls are most frequently utilized
to sustain d10 metal cluster structures owing to the extraordinary binding capability and
coordination versatility of ethynyl moieties to d10 metal ions through σ/π bonding, as
well as diverse photophysical properties. Numerous alkyls or aryl acetylides linked d10
homometallic or heterometallic clusters have been acquired and characterized by X-ray
crystallography, showing intriguing luminescent properties and sensitive photophysical
responses to external stimulus [22–25]. Considering that bis(ethynyl) ligands are more
favorable for the formation of metal cluster architectures with folding and interpenetrating
topologies to afford better molecular rigidity and a stronger emissive characteristic, we are Copyright:
© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article
distributed
under
the
terms
and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/). https://www.mdpi.com/journal/molecules Molecules 2023, 28, 3247. https://doi.org/10.3390/molecules28073247 Molecules 2023, 28, 3247 2 of 11
a-
g
a-
g devoted to the fabrication of d10 metal cluster complexes using carbazole-functionalized
bis(ethynyl) ligands as rigid sustaining or protective ligands [26,27]. Since diphosphines
afford not only similar binding affinity to d10 metal ions as that of bis(ethynyl) ligands to
provide exceptional metal coordination architectures but also favor improving the solubility
in organic solvents, they are a class of excellent co-ligands with carbazole-functionalized
bis(ethynyl) compounds for the design of d10 metal cluster topologies [26,27]. are devoted to the fabrication of d metal cluster complexes using carbazole functional
ized bis(ethynyl) ligands as rigid sustaining or protective ligands [26,27]. Since diphos-
phines afford not only similar binding affinity to d10 metal ions as that of bis(ethynyl) lig-
ands to provide exceptional metal coordination architectures but also favor improving the
solubility in organic solvents, they are a class of excellent co-ligands with carbazole-func-
tionalized bis(ethynyl) compounds for the design of d10 metal cluster topologies [26,27]. With 3,6-di-tert-butyl-1,8-diethynyl-9H-carbazole (H3decz, Scheme 1) as a rigid t
p
g
ized bis(ethynyl) ligands as rigid sustaining or protective ligands [26,27]. Since diphos-
phines afford not only similar binding affinity to d10 metal ions as that of bis(ethynyl) lig-
ands to provide exceptional metal coordination architectures but also favor improving the
solubility in organic solvents, they are a class of excellent co-ligands with carbazole-func-
tionalized bis(ethynyl) compounds for the design of d10 metal cluster topologies [26,27]. With 3,6-di-tert-butyl-1,8-diethynyl-9H-carbazole (H3decz, Scheme 1) as a rigid t With 3,6-di-tert-butyl-1,8-diethynyl-9H-carbazole (H3decz, Scheme 1) as a rigid
bis(acetylide) ligand upon deprotonation, we are able to attain a series of heterometallic
cluster architectures with good molecular rigidity, impressive phosphorescent characteris-
tics, and excellent stimuli responsiveness [6,26–28]. In this work, we describe a scissor-like
Au4Cu2 cluster (Scheme 2) sustained by H3decz and bis(2-diphenylphosphinophenyl)ether
(POP). It is brilliantly phosphorescent in solution and solid state at ambient temperature,
originating from the 3LMCT (ligand-to-metal charge transfer) triplet state from decz to
Au as well as the 3IL (intraligand) transition of decz3−. Interestingly, it shows remark-
able phosphorescent mechanochromism and thermochromism, relevant to the variation of
d10-d10 Au-Cu/Au interactions exerted by mechanical and thermal stimulus. Particularly,
the observation of simultaneous red-shifts of the emission upon raising or lowering the
temperature manifests its exceptional thermochromic phosphorescence characteristics. bis(acetylide) ligand upon deprotonation, we are able to attain a series of heterometallic
cluster architectures with good molecular rigidity, impressive phosphorescent character-
istics, and excellent stimuli responsiveness [6,26–28]. In this work, we describe a scissor-
like Au4Cu2 cluster (Scheme 2) sustained by H3decz and bis(2-diphenylphosphinophe-
nyl)ether (POP). It is brilliantly phosphorescent in solution and solid state at ambient tem-
perature, originating from the 3LMCT (ligand-to-metal charge transfer) triplet state from
decz to Au as well as the 3IL (intraligand) transition of decz3−. Interestingly, it shows re-
markable phosphorescent mechanochromism and thermochromism, relevant to the vari-
ation of d10-d10 Au-Cu/Au interactions exerted by mechanical and thermal stimulus. Par-
ticularly, the observation of simultaneous red-shifts of the emission upon raising or low-
ering the temperature manifests its exceptional thermochromic phosphorescence charac-
teristics. bis(acetylide) ligand upon deprotonation, we are able to attain a series of heterometallic
cluster architectures with good molecular rigidity, impressive phosphorescent character-
istics, and excellent stimuli responsiveness [6,26–28]. In this work, we describe a scissor-
like Au4Cu2 cluster (Scheme 2) sustained by H3decz and bis(2-diphenylphosphinophe-
nyl)ether (POP). It is brilliantly phosphorescent in solution and solid state at ambient tem-
perature, originating from the 3LMCT (ligand-to-metal charge transfer) triplet state from
decz to Au as well as the 3IL (intraligand) transition of decz3−. Interestingly, it shows re-
markable phosphorescent mechanochromism and thermochromism, relevant to the vari-
ation of d10-d10 Au-Cu/Au interactions exerted by mechanical and thermal stimulus. Par-
ticularly, the observation of simultaneous red-shifts of the emission upon raising or low-
ering the temperature manifests its exceptional thermochromic phosphorescence charac-
teristics. Scheme 1. Synthetic route of ligand H3decz. Scheme 1. Synthetic route of ligand H3decz. Scheme 1. Synthetic route of ligand H3decz. Scheme 1. Synthetic route of ligand H3decz. Scheme 1. Synthetic route of ligand H3decz. Scheme 1. Synthetic route of ligand H3decz. Scheme 1. Synthetic route of ligand H3decz. Scheme 1. Synthetic route of ligand H3decz. Scheme 1. Synthetic route of ligand H3decz. N
H
But
But
+
H3decz
N
H
But
But
+
H3decz N
H
But
But
[Au(tht)2](ClO4)
POP
+
+
+
Au4Cu2(decz)2(POP)2
H3decz
O
PPh2
PPh2
[Cu(MeCN)4](ClO4)
NEt3
N
But
But
N
But
But
Au
Au Au
Au
Cu
Cu
PPh2
PPh2
Ph2P
Ph2P
O
O
1 : 2 : 1 : 1
Scheme 2. Synthetic route of Au4Cu2 cluster complex. N
H
But
But
[Au(tht)2](ClO4)
POP
+
+
+
Au4Cu2(decz)2(POP)2
H3decz
O
PPh2
PPh2
[Cu(MeCN)4](ClO4)
NEt3
N
But
But
N
But
But
Au
Au Au
Au
Cu
Cu
PPh2
PPh2
Ph2P
Ph2P
O
O
1 : 2 : 1 : 1
Scheme 2. Synthetic route of Au4Cu2 cluster complex. Scheme 2. Synthetic route of Au4Cu2 cluster complex. Au4Cu2(decz)2(POP)2
3
N
But
But
N
But
But
Au
Au Au
Au
Cu
Cu
PPh2
PPh2
Ph2P
Ph2P
O
O
1 : 2 : 1 : 1
Au4Cu2(decz)2(POP)2
N
But
But
N
But
But
Au
Au Au
Au
Cu
Cu
PPh2
PPh2
Ph2P
Ph2P
O
O Au4Cu2(decz)2(POP)2
Au4Cu2(decz)2(POP)2 Au4Cu2(decz)2(POP)2
Au4Cu2(decz)2(POP)2 Scheme 2. Synthetic route of Au4Cu2 cluster complex. Scheme 2. Synthetic route of Au4Cu2 cluster complex. Scheme 2. Synthetic route of Au4Cu2 cluster complex. Scheme 2. Synthetic route of Au4Cu2 cluster complex. Scheme 2. Synthetic route of Au4Cu2 cluster complex. Scheme 2. Synthetic route of Au4Cu2 cluster complex. 2.1. Synthesis and Characterization 2.1. Synthesis and Characterization Ligand H3decz was obtained by the synthetic route shown in Scheme 1. The
neutral Au4Cu2 cluster (Scheme 2) was prepared by the reactions of [Au(tht)2](ClO4),
[Cu(CH3CN)4](ClO4), H3decz, and POP in a 2:1:1:1 molar ratio with the assistance of NEt3
for deprotonation. Re-crystallization of the product by diffusion of diethyl ether into a
dimethylacetamide (DMAc) solution gave Au4Cu2(decz)2(POP)2·DMAc as yellow crystals
in 50% yield. Thermogravimetric analysis (Figure S4, Supplementary Materials) revealed Molecules 2023, 28, 3247 3 of 11
),
t3 that solvate DMAc could be removed at a temperature range of 170–250 ◦C. Noticeably,
the removal of solvated DMAc results in the disruption of the crystalline state to become a
non-crystalline morph. in 50% yield. Thermogravimetric analysis (Figure S4, Supplementary Materials) revealed
that solvate DMAc could be removed at a temperature range of 170–250 °C. Noticeably,
the removal of solvated DMAc results in the disruption of the crystalline state to become
a non crystalline morph y
p
The structure of Au4Cu2(decz)2(POP)2·DMAc was determined by single crystal X-ray
diffraction. As depicted in Figure 1, the Au4Cu2 cluster shows a scissor-like structure
consisting of four Au(I) and two Cu(I) atoms linked by two tri-anionic decz3−and two
POP ligands through Au–acetylide σ-coordination, Cu–acetylide π-coordination, and Cu–
N bonding. The Au4Cu2 cluster is further stabilized by significant d10-d10 intermetallic
interactions in view of the distinctly shorter Au–Cu (2.8501(19) and 3.039(2) Å) distances
than the sum of Au and Cu atoms (ca. 3.1 Å) as well as much smaller Au–Au (3.0370(7)
and 3.0632(7) Å) distances than the sum of two Au atoms (3.3 Å). The Au(I) center is
bound to CP donors from acetylide and POP with the C-Au-P angle of 170.7(4)–173.6(4)◦. The Cu(I) center is quasi-linearly bonded to the carbazole N atom and acetylide through
π-coordination. Trianionic decz3−acts as a µ4-bridging ligand bound to two Au(I) cen-
ters through Au–acetylide σ-coordination and two Cu(I) centers through Cu–acetylide
π-coordination and Cu–N bonding, respectively, strikingly different from the bonding
modes reported in Ag8, Ag16, and Ag29 silver(I) nanoclusters as well as Ag4Au6 and
Ag8Au10 heterometallic clusters sustained by H3decz [26,27]. a non-crystalline morph. The structure of Au4Cu2(decz)2(POP)2·DMAc was determined by single crystal X-ray
diffraction. As depicted in Figure 1, the Au4Cu2 cluster shows a scissor-like structure con-
sisting of four Au(I) and two Cu(I) atoms linked by two tri-anionic decz3− and two POP
ligands through Au–acetylide σ-coordination, Cu–acetylide π-coordination, and Cu–N
bonding. 2.1. Synthesis and Characterization The Au4Cu2 cluster is further stabilized by significant d10-d10 intermetallic inter-
actions in view of the distinctly shorter Au–Cu (2.8501(19) and 3.039(2) Å) distances than
the sum of Au and Cu atoms (ca. 3.1 Å) as well as much smaller Au–Au (3.0370(7) and
3.0632(7) Å) distances than the sum of two Au atoms (3.3 Å). The Au(I) center is bound to
CP donors from acetylide and POP with the C-Au-P angle of 170.7(4)–173.6(4)°. The Cu(I)
center is quasi-linearly bonded to the carbazole N atom and acetylide through π-coordi-
nation. Trianionic decz3− acts as a µ4-bridging ligand bound to two Au(I) centers through
Au–acetylide σ-coordination and two Cu(I) centers through Cu–acetylide π-coordination
and Cu–N bonding, respectively, strikingly different from the bonding modes reported in
Ag8, Ag16, and Ag29 silver(I) nanoclusters as well as Ag4Au6 and Ag8Au10 heterometallic
clusters sustained by H3decz [26,27]. Figure 1. (a) A view of the Au4Cu2 cluster complex. The hydrogen atoms and tert-butyl groups, to-
gether with the phenyl rings on phosphorous atoms, were omitted for clarity. (b) A view showing a
scissor-like structure of the Au4(decz)2(POP)2 framework formed by gold(I)–bis(acetylide) σ-coordi-
nation. Figure 1. (a) A view of the Au4Cu2 cluster complex. The hydrogen atoms and tert-butyl groups,
together with the phenyl rings on phosphorous atoms, were omitted for clarity. (b) A view show-
ing a scissor-like structure of the Au4(decz)2(POP)2 framework formed by gold(I)–bis(acetylide)
σ-coordination. Figure 1. (a) A view of the Au4Cu2 cluster complex. The hydrogen atoms and tert-butyl groups, to-
gether with the phenyl rings on phosphorous atoms, were omitted for clarity. (b) A view showing a
scissor-like structure of the Au4(decz)2(POP)2 framework formed by gold(I)–bis(acetylide) σ-coordi-
nation. Figure 1. (a) A view of the Au4Cu2 cluster complex. The hydrogen atoms and tert-butyl groups,
together with the phenyl rings on phosphorous atoms, were omitted for clarity. (b) A view show-
ing a scissor-like structure of the Au4(decz)2(POP)2 framework formed by gold(I)–bis(acetylide)
σ-coordination. The Au4Cu2 cluster was characterized by high-resolution mass spectrometry (HRMS)
and 1H and 31P NMR spectroscopy. In the positive ion HRMS, the observation of the mo-
lecular ion peak [Au4Cu2(decz)2(POP)2H]+ together with molecular fragments
[Au4Cu(Hdecz)2(POP)2]+, [Au4(Hdecz)2(POP)2H]+, and [Au3(decz)(POP)2H]+ suggests that
the cluster structure keeps in the gas ionization state. In the 31P NMR spectrum, two P
signals occur at 32.1 and 33.7 ppm, coinciding with the Au4Cu2 structure in a solid state. 2.1. Synthesis and Characterization The Au4Cu2 cluster was characterized by high-resolution mass spectrometry (HRMS)
and 1H and 31P NMR spectroscopy. In the positive ion HRMS, the observation of the
molecular ion peak [Au4Cu2(decz)2(POP)2H]+ together with molecular fragments
[Au4Cu(Hdecz)2(POP)2]+, [Au4(Hdecz)2(POP)2H]+, and [Au3(decz)(POP)2H]+ suggests
that the cluster structure keeps in the gas ionization state. In the 31P NMR spectrum, two P
signals occur at 32.1 and 33.7 ppm, coinciding with the Au4Cu2 structure in a solid state. 2.2. Photophysical Properties and Computational Studies λabs/nm (ε/M−1 cm−1)
λem (nm)/τem (µs)/Φem (%)
CH3Cl2
CH2Cl2 a
Solid b
PMMA Film c
290 (39680), 342 (17540),
400 (12200), 450 (11200)
576/2.6/3.2
565/14.5/50.2
580/6.0/1.2 (desolvated)
530/19.3/7.9
580/1.9/3.6
623/5.3/12.6 (ground)
529/16.7/5.6 (ground)
a Measured in degassed CH2Cl2 solutions upon excitation at 397 nm. b Excitation at 397 nm. c Consisting of 99%
PMMA and 1% Au4Cu2 complex upon excitation at 397 nm. Table 1. The UV-Vis absorption and luminescent data of Au4Cu2 cluster at ambient temper-
ature. λabs/nm (ε/M−1 cm−1)
λem (nm)/τem (μs)/Φem (%)
CH2Cl2
CH2Cl2 a
Solid b
PMMA Film c
290 (39680), 342 (17540),
400 (12200), 450 (11200)
576/2.6/3.2
565/14.5/50.2
580/6.0/1.2 (desolvated)
530/19.3/7.9
580/1.9/3.6
623/5.3/12.6 (ground)
529/16.7/5.6 (ground)
a Measured in degassed CH2Cl2 solutions upon excitation at 397 nm. b Excitation at 397 nm. c Con-
sisting of 99% PMMA and 1% Au4Cu2 complex upon excitation at 397 nm. Figure 2. The UV-Vis absorption (blue dash) and normalized emission (yellow solid) spectra of an
Au4Cu2 cluster in a CH2Cl2 solution at ambient temperature (excitation at 397 nm). Figure 2. The UV-Vis absorption (blue dash) and normalized emission (yellow solid) spectra of an
Au4Cu2 cluster in a CH2Cl2 solution at ambient temperature (excitation at 397 nm). To clarify the absorption and emission origins together with the excited state charac-
ter, we conducted a theoretical computational study using time-dependent density func-
tional theory (TD-DFT) at the PBE1PBE level. In S1-S5 states (Table S3), the holes (electron-
transferred moiety) focus on decz3− ligands, and the electrons (electron-accepting moiety)
are mostly populated at Au atoms and decz3− ligands. Thus, low-energy absorption origi-
nates largely from [π (decz)→5d (Au)] 1LMCT and [π→π* (decz)] 1IL transitions. For the
T1 state (Figure 3), since the hole and electron display a similar distribution as that in S1-
S5 singlet states, the phosphorescent emission is mostly contributed by 3[π (decz)→5d
(Au)] 3LMCT and 3[π→π* (decz3−)] 3IL triplet states, which accords with the emissive
origin of Ag4Au6 and Ag8Au10 heterometallic clusters [27] sustained by H3decz as well as
Au4Ag4 cluster complexes15 linked by 4,5-diethynylacridin-9-one. To clarify the absorption and emission origins together with the excited state character,
we conducted a theoretical computational study using time-dependent density functional
theory (TD-DFT) at the PBE1PBE level. In S1-S5 states (Table S3), the holes (electron-
transferred moiety) focus on decz3−ligands, and the electrons (electron-accepting moiety)
are mostly populated at Au atoms and decz3−ligands. 2.2. Photophysical Properties and Computational Studies The Au4Cu2 cluster (Table 1 and Figure 2) shows intense ligand-centered absorption
at <300 nm and a band at 340 nm due to metal-perturbed intraligand (IL) transitions of
decz3−ligands. The broad absorption in the low-energy region (380–500 nm), containing
two composite bands centered at 400 and 450 nm, is mainly ascribed to the admixture
of [π (decz)→5d (Au)] 1LMCT and [π→π* (decz)] 1IL states. Upon excitation at λex > 300 nm,
it exhibits bright yellow–orange luminescence peaked at 576 nm with a lifetime of 3.2 µs in
degassed CH2Cl2 solution at ambient temperature, which is typical of phosphorescence in
view of a large Stokes shift (Figure 2). Molecules 2023, 28, 3247 4 of 11
µs
ce Table 1. The UV-Vis absorption and luminescent data of Au4Cu2 cluster at ambient temperature. λabs/nm (ε/M−1 cm−1)
λem (nm)/τem (µs)/Φem (%)
CH3Cl2
CH2Cl2 a
Solid b
PMMA Film c
290 (39680), 342 (17540),
400 (12200), 450 (11200)
576/2.6/3.2
565/14.5/50.2
580/6.0/1.2 (desolvated)
530/19.3/7.9
580/1.9/3.6
623/5.3/12.6 (ground)
529/16.7/5.6 (ground)
a Measured in degassed CH2Cl2 solutions upon excitation at 397 nm. b Excitation at 397 nm. c Consisting of 99%
PMMA and 1% Au4Cu2 complex upon excitation at 397 nm. Table 1. The UV-Vis absorption and luminescent data of Au4Cu2 cluster at ambient temper-
ature. λabs/nm (ε/M−1 cm−1)
λem (nm)/τem (μs)/Φem (%)
CH2Cl2
CH2Cl2 a
Solid b
PMMA Film c
290 (39680), 342 (17540),
400 (12200), 450 (11200)
576/2.6/3.2
565/14.5/50.2
580/6.0/1.2 (desolvated)
530/19.3/7.9
580/1.9/3.6
623/5.3/12.6 (ground)
529/16.7/5.6 (ground)
a Measured in degassed CH2Cl2 solutions upon excitation at 397 nm. b Excitation at 397 nm. c Con-
sisting of 99% PMMA and 1% Au4Cu2 complex upon excitation at 397 nm. Figure 2. The UV-Vis absorption (blue dash) and normalized emission (yellow solid) spectra of an
Au4Cu2 cluster in a CH2Cl2 solution at ambient temperature (excitation at 397 nm). Figure 2. The UV-Vis absorption (blue dash) and normalized emission (yellow solid) spectra of an
Au4Cu2 cluster in a CH2Cl2 solution at ambient temperature (excitation at 397 nm). ble 1. The UV-Vis absorption and luminescent data of Au4Cu2 cluster at ambient temperature. ble 1. The UV-Vis absorption and luminescent data of Au4Cu2 cluster at ambient temper- Table 1. The UV-Vis absorption and luminescent data of Au4Cu2 cluster at ambient temperature. 2.2. Photophysical Properties and Computational Studies Thus, low-energy absorption
originates largely from [π (decz)→5d (Au)] 1LMCT and [π→π* (decz)] 1IL transitions. For
the T1 state (Figure 3), since the hole and electron display a similar distribution as that in
S1-S5 singlet states, the phosphorescent emission is mostly contributed by 3[π (decz)→5d
(Au)] 3LMCT and 3[π→π* (decz3−)] 3IL triplet states, which accords with the emissive
origin of Ag4Au6 and Ag8Au10 heterometallic clusters [27] sustained by H3decz as well as
Au4Ag4 cluster complexes15 linked by 4,5-diethynylacridin-9-one. IEW
5 of 11 Figure 3. Plots of the hole and electron involved in the lowest-energy emission transition of Au4Cu2
cluster by TD-DFT method at the PBE1PBE level. Figure 3. Plots of the hole and electron involved in the lowest-energy emission transition of Au4Cu2
cluster by TD-DFT method at the PBE1PBE level. Figure 3. Plots of the hole and electron involved in the lowest-energy emission transition of Au4Cu2
cluster by TD-DFT method at the PBE1PBE level. Figure 3. Plots of the hole and electron involved in the lowest-energy emission transition of Au4Cu2
cluster by TD-DFT method at the PBE1PBE level. Upon UV light irradiation
at
λex
> 300 nm,
the
crystalline species
Au4Cu2(decz)2(POP)2·DMAc (Figure 4) shows brilliant yellow emission peaked at 565 nm
ith
ho
ho e
e t effiie
y o e 50% Whe
ol ate DMA
a
e
o ed at 180 °C
UponUVlightirradiationatλex >300nm, thecrystallinespeciesAu4Cu2(decz)2(POP)2·DMAc
(Figure 4) shows brilliant yellow emission peaked at 565 nm with phosphorescent efficiency Molecules 2023, 28, 3247 5 of 11 5 of 11 over 50%. When solvate DMAc was removed at 180 ◦C under vacuum, the species color
deepened obviously, as indicated in the UV-Vis spectra (Figure 4a) and the photographs
(Figure 4b) under ambient light, companying with a conversion of the crystalline to the
amorphous state as revealed by X-ray diffraction. As depicted in Figure 4, the removal of
solvate DMAc resulted in the emission band centered at 565 nm red-shifting distinctly to
580 nm, in which intense yellow phosphorescence converted to weaker orange emitting
after desolvation. Undoubtedly, desolvation leads to more compact packing of Au4Cu2
clusters, thus lowering phosphorescent energy to some extent while enhancing thermally
induced non-radiative deactivation. with phosphorescent efficiency over 50%. 2.2. Photophysical Properties and Computational Studies When solvate DMAc was removed at 180 °C
under vacuum, the species color deepened obviously, as indicated in the UV-Vis spectra
(Figure 4a) and the photographs (Figure 4b) under ambient light, companying with a con-
version of the crystalline to the amorphous state as revealed by X-ray diffraction. As de-
picted in Figure 4, the removal of solvate DMAc resulted in the emission band centered at
565 nm red-shifting distinctly to 580 nm, in which intense yellow phosphorescence con-
verted to weaker orange emitting after desolvation. Undoubtedly, desolvation leads to
more compact packing of Au4Cu2 clusters, thus lowering phosphorescent energy to some
extent while enhancing thermally induced non-radiative deactivation. Figure 4. (a) The normalized UV-Vis absorption (dash) and emission (solid) spectra of Au4Cu2 clus-
ter before (yellow) and after removal of solvate DMAc (orange). (b) The images of Au4Cu2 cluster
complex under ambient light and UV light irradiation (365 nm) before and after desolvation. Figure 4. (a) The normalized UV-Vis absorption (dash) and emission (solid) spectra of Au4Cu2 cluster
before (yellow) and after removal of solvate DMAc (orange). (b) The images of Au4Cu2 cluster
complex under ambient light and UV light irradiation (365 nm) before and after desolvation. Figure 4. (a) The normalized UV-Vis absorption (dash) and emission (solid) spectra of Au4Cu2 clus-
ter before (yellow) and after removal of solvate DMAc (orange). (b) The images of Au4Cu2 cluster
complex under ambient light and UV light irradiation (365 nm) before and after desolvation. Figure 4. (a) The normalized UV-Vis absorption (dash) and emission (solid) spectra of Au4Cu2 cluster
before (yellow) and after removal of solvate DMAc (orange). (b) The images of Au4Cu2 cluster
complex under ambient light and UV light irradiation (365 nm) before and after desolvation. 2.3. Stimuli-Responsive Properties
2.3. Stimuli-Responsive Properties p
p
As shown in Figure 5, when pristine species of Au4Cu2 cluster was mechanically
ground, the UV-Vis absorption showed a drastic red-shift with yellow to orange transfor-
mation under ambient light. More interestingly, upon thoroughly ground, the phospho-
As shown in Figure 5, when pristine species of Au4Cu2 cluster was mechanically
ground, the UV-Vis absorption showed a drastic red-shift with yellow to orange transforma-
tion under ambient light. More interestingly, upon thoroughly ground, the phosphorescent
band peaked at 565 nm red-shifted dramatically to 623 nm, followed by the conversion of
bright yellow to deep red emission. Noticeably, drastic red-shift of the emission gave rise to
a dramatic broadening of the spectral band (Figure 5a), in which full width at half maxima
(FWHM) changed from 81 nm to 196 nm upon mechanical grinding, thus manifesting more
participation of metal cluster dominated triplet state. As demonstrated by TD-DFT studies,
as phosphorescent emission in Au4Cu2 cluster arises mostly from admixture 3LMCT/3IL
states, the dramatic broadening of the emission band after mechanical grinding is most
likely ascribed to more contribution from d10-d10 intermetallic interaction whereas less
component from 3IL state. On the other hand, when the ground species was exposed to
the vapor of DMAc or CH2Cl2, the color and emission could be perfectly reverted to the
original state, implying its excellent reversibility. g
p y
g
y
As shown in Figure 5c, mechanical grinding leads to the total disappearance of X-ray
diffraction patterns, signifying a transformation from a crystalline to a non-crystalline
state, as is observed in most metal coordination compounds with mechanochromic prop-
erties induced by intermolecular variations. When the ground species were exposed to
DMAc vapor, X-ray diffraction patterns could be reverted to their original state, further
demonstrating the reversibility of mechanochromism. It is known that stimuli-responsive
luminescent changes triggered by intermolecular interaction are highly dependent on the
doping concentration of the emitters, whereas those from an intramolecular effect are
insensitive to the doping [5,6]. In order to further address this issue, a 1% Au4Cu2 cluster
was doped in a PMMA matrix to explore possible machnochromism in the dilute matrix. 2.3. Stimuli-Responsive Properties
2.3. Stimuli-Responsive Properties In order to further address this issue, a 1% Au4Cu2 clust
was doped in a PMMA matrix to explore possible machnochromism in the dilute matri
As shown in Figure S7, the emission spectrum of 1% Au4Cu2 cluster in the PMMA matr
As depicted in Figure 6, pristine species of Au4Cu2(decz)2(POP)2·DMAc show phos-
phorescent emission centered at ca. 570 nm at an ambient temperature of 290 K. When
the temperature was step by step raised to 470 K, the emission band followed 570 nm
(290 K)→575 nm (320 K)→578 nm (350 K)→581 nm (380 K)→584 nm (410 K)→587 nm
(440 K)→590 nm (470 K) to show a gradual red-shift with a progressive reduction of the
intensity. The stepwise red-shift with temperature raising is ascribed to the gradual desol-
vation process as discussed above, whereas a progressive decrease in the emission intensity
is due to the increasingly increased thermally non-radiative deactivation process. On the
other hand, when the temperature is lowered to liquid nitrogen, the emission follows
570 nm (290 K)→580 nm (260 K)→593 nm (230 K)→595 nm (200 K) to show a gradual
red-shift, which is assignable to the stepwise shortening of intermetallic distances and the
successive increase in d10-d10 interaction so that phosphorescent energy is progressively
reduced. The consecutive emission enhancement is due to the step-by-step suppression of
thermal vibration and, thus, the reduced non-radiative deactivation with the temperature
progressively lowered to the liquid nitrogen range. As shown in Figure 5c, mechanical grinding leads to the total disappearance of X-ra
diffraction patterns, signifying a transformation from a crystalline to a non-crystallin
state, as is observed in most metal coordination compounds with mechanochromic prop
erties induced by intermolecular variations. When the ground species were exposed t
DMAc vapor, X-ray diffraction patterns could be reverted to their original state, furthe
demonstrating the reversibility of mechanochromism. It is known that stimuli-responsiv
luminescent changes triggered by intermolecular interaction are highly dependent on th
doping concentration of the emitters, whereas those from an intramolecular effect are in
sensitive to the doping [5,6]. In order to further address this issue, a 1% Au4Cu2 clust
was doped in a PMMA matrix to explore possible machnochromism in the dilute matri
As shown in Figure S7, the emission spectrum of 1% Au4Cu2 cluster in the PMMA matr
As depicted in Figure 6, pristine species of Au4Cu2(decz)2(POP)2·DMAc show phos-
phorescent emission centered at ca. 2.3. Stimuli-Responsive Properties
2.3. Stimuli-Responsive Properties It is known that stimuli-responsiv
luminescent changes triggered by intermolecular interaction are highly dependent on th
doping concentration of the emitters, whereas those from an intramolecular effect are in
sensitive to the doping [5,6]. In order to further address this issue, a 1% Au4Cu2 cluste
was doped in a PMMA matrix to explore possible machnochromism in the dilute matri
As shown in Figure S7, the emission spectrum of 1% Au4Cu2 cluster in the PMMA matr
As depicted in Figure 6, pristine species of Au4Cu2(decz)2(POP)2·DMAc show phos-
phorescent emission centered at ca. 570 nm at an ambient temperature of 290 K. When
the temperature was step by step raised to 470 K, the emission band followed 570 nm
(290 K)→575 nm (320 K)→578 nm (350 K)→581 nm (380 K)→584 nm (410 K)→587 nm
(440 K)→590 nm (470 K) to show a gradual red-shift with a progressive reduction of the
intensity. The stepwise red-shift with temperature raising is ascribed to the gradual desol-
vation process as discussed above, whereas a progressive decrease in the emission intensity
is due to the increasingly increased thermally non-radiative deactivation process. On the
other hand, when the temperature is lowered to liquid nitrogen, the emission follows
570 nm (290 K)→580 nm (260 K)→593 nm (230 K)→595 nm (200 K) to show a gradual
red-shift, which is assignable to the stepwise shortening of intermetallic distances and the
successive increase in d10-d10 interaction so that phosphorescent energy is progressively
reduced. The consecutive emission enhancement is due to the step-by-step suppression of
thermal vibration and, thus, the reduced non-radiative deactivation with the temperature
progressively lowered to the liquid nitrogen range. As shown in Figure 5c, mechanical grinding leads to the total disappearance of X-ra
diffraction patterns, signifying a transformation from a crystalline to a non-crystallin
state, as is observed in most metal coordination compounds with mechanochromic prop
erties induced by intermolecular variations. When the ground species were exposed
DMAc vapor, X-ray diffraction patterns could be reverted to their original state, furth
demonstrating the reversibility of mechanochromism. It is known that stimuli-responsiv
luminescent changes triggered by intermolecular interaction are highly dependent on th
doping concentration of the emitters, whereas those from an intramolecular effect are in
sensitive to the doping [5,6]. 2.3. Stimuli-Responsive Properties
2.3. Stimuli-Responsive Properties As shown in Figure S7, the emission spectrum of 1% Au4Cu2 cluster in the PMMA matrix Molecules 2023, 28, 3247 6 of 11
echanic did not display distinct variation upon mechanical grinding, thus confirming that the
phosphorescent mechanochromism of Au4Cu2 cluster results indeed from the variation of
intermolecular packing instead of intramolecular effect. p
,
g
p
exposed to the vapor of DMAc or CH2Cl2, the color and emission could be perfectly r
verted to the original state, implying its excellent reversibility. Figure 5. (a) The normalized UV-Vis absorption (dash) and emission (solid) spectra of Au4Cu2 clu
ter before and after mechanical grinding. (b) The images of Au4Cu2 cluster under ambient light an
UV light irradiation (365 nm) before and after mechanical grinding. (c) The simulated and measure
X-ray diffraction patterns of Au4Cu2 cluster upon mechanical grinding and DMAc vapor adsorptio
Figure 5. (a) The normalized UV-Vis absorption (dash) and emission (solid) spectra of Au4Cu2 cluster
before and after mechanical grinding. (b) The images of Au4Cu2 cluster under ambient light and
UV light irradiation (365 nm) before and after mechanical grinding. (c) The simulated and measured
X-ray diffraction patterns of Au4Cu2 cluster upon mechanical grinding and DMAc vapor adsorption. Figure 5. (a) The normalized UV-Vis absorption (dash) and emission (solid) spectra of Au4Cu2 clu
ter before and after mechanical grinding. (b) The images of Au4Cu2 cluster under ambient light an
UV light irradiation (365 nm) before and after mechanical grinding. (c) The simulated and measure
X-ray diffraction patterns of Au4Cu2 cluster upon mechanical grinding and DMAc vapor adsorptio
Figure 5. (a) The normalized UV-Vis absorption (dash) and emission (solid) spectra of Au4Cu2 cluster
before and after mechanical grinding. (b) The images of Au4Cu2 cluster under ambient light and
UV light irradiation (365 nm) before and after mechanical grinding. (c) The simulated and measured
X-ray diffraction patterns of Au4Cu2 cluster upon mechanical grinding and DMAc vapor adsorption. As shown in Figure 5c, mechanical grinding leads to the total disappearance of X-ra
diffraction patterns, signifying a transformation from a crystalline to a non-crystallin
state, as is observed in most metal coordination compounds with mechanochromic prop
erties induced by intermolecular variations. When the ground species were exposed t
DMAc vapor, X-ray diffraction patterns could be reverted to their original state, furthe
demonstrating the reversibility of mechanochromism. 3 1 Synthesis of Ligand H decz
3.1. Synthesis of Ligand H3decz 3.1. Synthesis of Ligand H3decz
H3decz was prepared by the modified procedures described in the literature [29]. Ad-
ditionally, 1,8-dibromo-3,6-di-tert-butylcarbazole (4.7 mmol, 2.0 g), Pd(PPh3)2Cl2 (0.3
mmol, 0.2 mg), CuI (0.3 mmol, 57.4 mg), ethynyltrimethylsilane (13 mmol, 5.0 g), and NEt3
(30 mL) were put into a high-pressure flask. After heating at 90 °C for 2 d, the black pre-
cipitate was removed through filtration. The filtrate was evaporated in a vacuum to give
H3decz was prepared by the modified procedures described in the literature [29]. Ad-
ditionally, 1,8-dibromo-3,6-di-tert-butylcarbazole (4.7 mmol, 2.0 g), Pd(PPh3)2Cl2 (0.3 mmol,
0.2 mg), CuI (0.3 mmol, 57.4 mg), ethynyltrimethylsilane (13 mmol, 5.0 g), and NEt3 (30 mL)
were put into a high-pressure flask. After heating at 90 ◦C for 2 d, the black precipitate
was removed through filtration. The filtrate was evaporated in a vacuum to give a brown
solid after the removal of the solvent. The product was then purified through silica gel
column chromatography using petroleum ether as an eluent, giving 3,6-di-tert-butyl-1,8-
bis((trimethylsilyl)ethynyl)-9H-carbazole as a yellow solid. Yield: 75%. y
y
y y
y
Then, 3,6-di-tert-butyl-1,8-bis((trimethylsilyl)ethynyl)-9H-carbazole (10 mmol, 4.3 g)
and K2CO3 (10 mmol, 1.4 g) were added to MeOH-CH2Cl2 (100 mL, v/v = 1:1). Upon
stirring at ambient temperature for 10 h, the solvents were removed under vacuum. The
crude product was chromatographed on a silica gel column using petroleum ether as an
eluent to give H3decz as a pale yellow solid. Yield: 80%. 1H NMR (CDCl3, ppm): 8.50 (s,
1H), 8.13 (d, 2H, J = 7.8 Hz), 7.68 (d, 2H, J = 8.1 Hz), 3.49 (s, 2H), and 1.48 (s, 18H). 13C NMR
(CDCl3, ppm): 142.71, 139.16, 127.49, 123.22, 117.95, 103.87, 81.37, 80.45, 34.69, and 31.87. 2.3. Stimuli-Responsive Properties
2.3. Stimuli-Responsive Properties 570 nm at an ambient temperature of 290 K. When
the temperature was step by step raised to 470 K, the emission band followed 570 nm
(290 K)→575 nm (320 K)→578 nm (350 K)→581 nm (380 K)→584 nm (410 K)→587 nm
(440 K)→590 nm (470 K) to show a gradual red-shift with a progressive reduction of the
intensity. The stepwise red-shift with temperature raising is ascribed to the gradual desol-
vation process as discussed above, whereas a progressive decrease in the emission intensity
is due to the increasingly increased thermally non-radiative deactivation process. On the
other hand, when the temperature is lowered to liquid nitrogen, the emission follows
570 nm (290 K)→580 nm (260 K)→593 nm (230 K)→595 nm (200 K) to show a gradual
red-shift, which is assignable to the stepwise shortening of intermetallic distances and the
successive increase in d10-d10 interaction so that phosphorescent energy is progressively
reduced. The consecutive emission enhancement is due to the step-by-step suppression of
thermal vibration and, thus, the reduced non-radiative deactivation with the temperature
progressively lowered to the liquid nitrogen range. 7 of 11
of
re Molecules 2023, 28, 3247 Figure 6. Temperature-dependent photoluminescent spectra of an Au4Cu2 cluster in solid state at
80–470 K upon excitation at 397 nm. 500
550
600
650
700
750
800
0.0
5.0x104
1.0x105
1.5x105
80 K
110 K
140 K
170 K
200 K
230 K
260 K
290 K
320 K
350 K
380 K
410 K
440 K
470 K
PL Intensity (a.u.)
Wavelength (nm)
Figure 6. Temperature-dependent photoluminescent spectra of an Au4Cu2 cluster in solid state at
80–470 K upon excitation at 397 nm. Figure 6. Temperature-dependent photoluminescent spectra of an Au4Cu2 cluster in solid state at
80–470 K upon excitation at 397 nm. Figure 6. Temperature-dependent photoluminescent spectra of an Au4Cu2 cluster in solid state at
80–470 K upon excitation at 397 nm. 3. Experimental
3. Experimental All operations were carried out under a dry argon atmosphere using Schlenk tech-
niques and vacuum-line systems. The solvents were dried, distilled, and degassed prior
to use. Spectroscopic-grade reagents for spectroscopic measurements were purchased
from commercial sources. Bis(2-diphenylphosphinophenyl)ether (POP), 1,8-dibromo-3,6-
di-tert-butylcarbazole, and other reagents were purchased from commercial sources with-
out further purification. Perchlorate salts are potentially explosive and should be used
carefully
All operations were carried out under a dry argon atmosphere using Schlenk tech-
niques and vacuum-line systems. The solvents were dried, distilled, and degassed prior
to use. Spectroscopic-grade reagents for spectroscopic measurements were purchased
from commercial sources. Bis(2-diphenylphosphinophenyl)ether (POP), 1,8-dibromo-3,6-di-
tert-butylcarbazole, and other reagents were purchased from commercial sources without
further purification. Perchlorate salts are potentially explosive and should be used carefully. 3.4. Crystal Structural Determination Crystals suitable for X-ray crystallographic measurement were grown by vapor diffus-
ing of diethyl ether to a DMAc solution. The single crystal X-ray diffraction was carried
out on a micro-focus metaljet diffractometer using Ga Kα radiation (λ = 1.3405 Å). Data
reduction was conducted with the CrysAlisPro package, and at last an analytical absorp-
tion correction was performed. The structure was determined using the Bruker SHELXTL
Software Package, a computer program for the automatic solution of crystal structures. The
structure was refined by the full-matrix least-square method with ShelXle Version 4.8.6, a
Qt graphical user interface for the SHELXL [30,31]. All non-hydrogen atoms were refined
anisotropically, whereas the hydrogen atoms were generated geometrically and refined
isotropically. CCDC 2245976 contains the supplementary crystallographic data for this
paper. These data can be obtained free of charge from the Cambridge Crystallographic
Data Centre via www.ccdc.cam.ac.uk/data_request/cif (accessed on 3 March 2023). 3.3. Physical Measurements The UV-Vis absorption spectra were recorded on a Perkin-Elmer Lambda 365 UV-Vis
spectrophotometer (Perkin-Elmer, Waltham, MA, USA). The infrared spectra (IR) were
conducted on a Bruker VERTEX 70 FT-IR spectrophotometer (Bruker, Mannheim, Germany)
with KBr pellets. The high-resolution mass spectrometry (HRMS) was performed on a Wa-
ters Xevo G2-Q-Tof mass spectrometer (Waters, Milford, MA, USA) using dichloromethane
and methanol mixtures as mobile phases. The 1H NMR spectra were recorded on a Bruker
Avance III (400 MHz) spectrometer (Bruker, Zurich, Switzerland) using SiMe4 as the inter-
nal reference, and 31P NMR spectra were measured using H3PO4 as the external reference. The excitation and emission spectra, together with emissive lifetimes in degassed solu-
tions, solid states, and films, were determined on an Edinburgh FLS1000 fluorescence
spectrometer (Edinburgh Instruments, Livingston, UK). Absolute quantum yields were
determined by the integrating sphere (142 mm in diameter) using the Edinburgh FLS1000
Spectrofuorophotometer (Edinburgh Instruments, Livingston, UK). The PMMA doping
was conducted by mixing 99% PMMA and 1% Au4Cu2 complex in CH2Cl2. The diluted
species obtained after the removal of the solvent were thus used for photophysical studies. 3.2. Synthesis of Au4Cu2(decz)2(POP)2 Freshly prepared [Au(tht)2](ClO4) (0.08 mmol, 37.8 mg) was obtained by mixing
an equivalent molar of Ag(tht)(ClO4) (0.08 mmol, 23.6 mg) and Au(tht)Cl (0.08 mmol,
18.5 mg) in 10 mL CH2Cl2 with stirring for 10 min. The turbid solution was treated by
centrifugation to remove the AgCl precipitate, giving a clear solution that was used directly
in the next step. To the filtrate was added [Cu(MeCN)4](ClO4) (0.04 mmol, 13.1 mg), POP (0.04 mmol,
21.6 mg), and H3decz (0.04 mmol, 13.1 mg). Upon stirring for 5 min, NEt3 (0.5 mL) was
added to the above solution, and the mixture immediately turned from yellow to dark
red. After the mixed solution was stirred overnight in the dark, the solvent was removed Molecules 2023, 28, 3247 8 of 11 under a vacuum to give a yellow solid. The solid was re-dissolved in 4 mL of dimethy-
lacetamide (DMAc). When the clear solution was exposed to diethyl ether for slow vapor
diffusion, yellow crystals were grown in one week. Yield: 50%. HRMS m/z (%): 2641.3815
(6) [Au4Cu2(decz)2(POP)2H]+ (Calcd 2641.4063), 2579.5228 (37) [Au4Cu(Hdecz)2(POP)2]+
(Calcd. 2579.4483), 2516.5594 (34) [Au4(Hdecz)2(POP)2H]+ (Calcd. 2516.5677), 1993.4868
(100) [Au3(decz)(POP)2H]+ (Calcd. 1993.4131). Anal. Calcd for C120H100Au4Cu2N2O2P4:
C, 54.57; H, 3.82; N, 1.06. Found: C, 54.39; H, 3.80; N, 0.97. 1H NMR (400 MHz, DMSO-d6,
δ): 8.26 (s, 1H), 8.24 (s, 1H), 8.23 (s, 1H), 8.21 (s, 1H), 8.12 (d, J = 4.2 Hz, 2H), 8.09 (s, 2H),
8.06 (s, 1H), 8.05 (s, 1H), 7.95 (s, 1H), 7.95 (s, 1H), 7.76 (d, J = 2.0 Hz, 4H), 7.74 (s, 4H), 7.70
(d, J = 2.3 Hz, 4H), 7.47–7.36 (m, 12H), 7.35 (s, 2H), 7.28 (dd, J = 8.2, 4.9 Hz, 3H), 7.14 (d,
J = 8.3 Hz, 8H), 6.86 (t, J = 7.5 Hz, 3H), 6.71 (d, J = 7.9 Hz, 2H), 6.69–6.66 (m, 2H), 6.50 (d,
J = 7.9 Hz, 3H), 6.06–6.00 (m, 3H), 5.32 (t, J = 4.9 Hz, 2H), 1.37 (s, 18H), 1.06 (s, 18H). 31P
NMR (162 MHz, DMSO-d6): 32.1 (2P), 33.7 (2P). IR (KBr, cm−1): 2105w (C≡C). 3.5. Computational Method The computational studies were carried out using the Gaussian 16 program pack-
age [32]. The geometrical structures in the ground state and the lowest-energy triplet state
were optimized respectively by the restricted and unrestricted density functional theory
(DFT) method by the use of the gradient-corrected correlation functional PBE1PBE [33,34]
and the D3 version of Grimme’s dispersion with the original D3 damping function [35]
(here abbreviated as PBE1PBE-GD3). The initial structure of the Au4Cu2 cluster was ob-
tained from crystal structural data. To decrease the computation time, the structure was
simplified by replacing the phenyl rings on phosphorous atoms with H atoms. To analyze Molecules 2023, 28, 3247 9 of 11 9 of 11 the absorption and emission transition properties, the calculations were conducted to give
60 singlet and 6 triplet excited states based on the optimized structures in the ground state
and lowest-energy triplet state, respectively, to determine the vertical excitation energies by
time-dependent density functional theory (TD-DFT) [36–38] at the PBE1PBE-GD3 level. The
polarizable continuum model method (PCM) [39] with CH2Cl2 as solvent was utilized for
the calculation of excited states. In these calculations, we used the Stuttgart-Dresden (SDD)
basis set with the f-type polarization functions (f-exponent is 1.050 for Au and 3.525 for Cu
atoms) and the effective core potentials (ECPs) to describe the Au and Cu atoms [40,41]. We
used the all-electron basis set of 6-31G** to describe other non-metal atoms, including P, O,
N, C, and H. We performed visualization of the hole and electron plots using GaussView,
and we analyzed the contributions of fragments to the hole and electron in the electronic
excitation process using the Multiwfn 3.8 program [42]. 4. Conclusions We obtain an Au4Cu2 cluster based on carbazole-bis(acetylide) as a supporting plat-
form, which shows a scissor-like structure as demonstrated by X-ray crystallography. The
Au4Cu2 cluster shows a yellow to red phosphorescence transformation upon mechanical
grinding, and red emission could be reversibly reverted to yellow emitting through DMAc
vapor adsorption. The observation of distinct red-shifts of the emission upon not only
raising but also lowering the temperature at ambient temperature reveals an exceptional
phosphorescent thermochromism due to different mechanisms. This work provides a vivid
example of stimuli-responsive modulation of phosphorescent properties in smart solid
materials taking advantage of d10-d10 intermetallic interactions. Supplementary Materials:
The following supporting information can be downloaded at:
https://www.mdpi.com/article/10.3390/molecules28073247/s1, Table S1: Crystallographic Data
for Au4Cu2 Cluster Complex; Table S2: Selective Interatomic Distances (Å) and Bonding Angles (◦)
of Au4Cu2 Cluster Complex; Table S3: The Absorption Transitions for Au4Cu2 Cluster Complex in
CH2Cl2 solution, Calculated by TD-DFT Method at the PBE1PBE Level (isovalue = 0.0004); Table S4:
The Emission Transitions for Au4Cu2 Cluster Complex in CH2Cl2 Solution, Calculated by TD-DFT
Method at the PBE1PBE Level (isovalue = 0.0004); Figure S1: The high-resolution mass spectrometry
of Au4Cu2 cluster complex; Figure S2: The 1H NMR spectrum of Au4Cu2 cluster complex in DMSO-
d6 solution at ambient temperature; Figure S3: The 31P NMR spectrum of Au4Cu2 cluster complex in
DMSO-d solution at ambient temperature; Figure S5: The measured (solid) and calculated (column
bar) UV-Vis absorption spectra for Au4Cu2 cluster complex by TD-DFT method at the PBE1PBE
level; Figure S6: Plots of the simulated and measured X-ray diffraction patterns of Au4Cu2 cluster
complex before and after desolvation; Figure S7: The normalized emission spectra of 1% Au4Cu2
cluster complex in PMMA matrix before and after mechanical grinding (excitation at 397 nm); Figure
S8: The normalized UV-Vis absorption (dash) and photoluminescent (solid) spectra of Au4Cu2 cluster
in as-prepared pristine state, desolvation and mechanical grinding. Author Contributions: Methodology, J.-Y.W.; software, J.-Y.W.; validation; Y.-Z.H.; formal analysis,
Y.-Z.H.; investigation, X.-M.W.; resources, X.-M.W.; data curation, X.-M.W. and Y.-Z.H.; writing—review
and editing, Z.-N.C.; visualization, J.-Y.W.; supervision, Z.-N.C.; project administration, Z.-N.C.; fund-
ing acquisition, Z.-N.C. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Natural Science Foundation of China (Grants
92061202 and U22A20387) and the Fujian Science and Technology Project (Grant 2020L3022). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. References 1. Sagara, Y.; Yamane, S.; Mitani, M.; Weder, C.; Kato, T. Mechanoresponsive Luminescent Molecular Assemblies: An Emerging
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14. Liu, Q.; Xie, M.; Chang, X.; Gao, Q.; Chen, Y.; Lu, W. Sample Availability: Samples of the compounds are available from the authors. Sample Availability: Samples of the compounds are available from the authors. 4. Conclusions Data Availability Statement: All data needed to support the conclusions of this manuscript are
included in the main text or the Supplementary Materials. Acknowledgments: We are grateful for financial support from the National Natural Science Foun-
dation of China (Grants 92061202 and U22A20387) and the Fujian Science and Technology Project
(Grant 2020L3022). Acknowledgments: We are grateful for financial support from the National Natural Science Foun-
dation of China (Grants 92061202 and U22A20387) and the Fujian Science and Technology Project
(Grant 2020L3022). 10 of 11 10 of 11 Molecules 2023, 28, 3247 Conflicts of Interest: The authors declare no conflict of interest. References Correlating thermochromic and mechanochromic phosphorescence with
polymorphs of a complex gold(I) double salt with infinite aurophilicity. Chem. Commun. 2018, 54, 12844. [CrossRef] 15. Xie, P.; Wang, J.-Y.; Huang, Y.-Z.; Wu, X.-M.; Chen, Z.-N. Heteroctanuclear Au4Ag4 Cluster Complexes of 4,5-Diethynylacridin-9-
One with Luminescent Mechanochromism. Molecules 2022, 27, 2127. [CrossRef] 16. Wang, X.-Y.; Yin, Y.; Yin, J.; Chen, Z.; Liu, S.H. Persistent room-temperature phosphorescence or high-contrast phosphorescent
mechanochromism: Polymorphism-dependent different emission characteristics from a single gold(I) complex. Dalton Trans. 2022, 50, 7744. [CrossRef] 17. Wang, Y.-W.; Yan, J.-J.; Hu, S.-N.; Young, D.J.; Li, H.-X.; Ren, Z.-G. A Photoluminescent Ag10Cu6 Cluster Stablized by a PNNP
Ligand and Phenylacetylides Selectively and Reversibly Senses Ammonia in Air and Water. Chem.-Asian J. 2021, 16, 2681. [CrossRef] 18. Tao, Y.-H.; Wang, Y.-W.; Hu, S.-N.; Young, D.J.; Lu, C.; Li, H.-X.; Ren, Z.-G. A photoluminescent Au(I)/Ag(I)/PNN coordination
complex for relatively rapid and reversible alcohol sensing. Dalton Trans. 2021, 50, 6773. [CrossRef] 19. Seki, T.; Tokodai, N.; Omagari, S.; Nakanishi, T.; Hasegawa, Y.; Iwasa, T.; Taketsugu, T.; Ito, H. Luminescent Mechanochromic
9-Anthryl Gold(I) Isocyanide Complex with an Emission Maximum at 900 nm after Mechanical Stimulation. J. Am. Chem. Soc. 2017, 139, 6514. [CrossRef] 20. Dong, Y.; Zhang, J.; Li, A.; Gong, J.; He, B.; Xu, S.; Yin, J.; Liu, S.H.; Tang, B.Z. Structure-tuned and thermodynamically controlled
mechanochromic self-recovery of AIE-active Au(I) complexes. J. Mater. Chem. C 2020, 8, 894. [CrossRef] 21. Yang, Z.C.; Song, K.Y.; Zhou, P.K.; Zong, L.L.; Li, H.H.; Chen, Z.R.; Jiang, R. Sensitive luminescence mechanochromism and
unique luminescence thermochromism tuned by bending the P-O-P skeleton in the diphosphonium/iodocuprate(I) hybrid. CrystEngComm. 2022, 24, 4940. [CrossRef] y
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22. Yam, V.W.-W.; Cheng, E.C.-C. Photochemistry and Photophysics of Coordination Compounds II. Photochemistry and Photophysics of
Coordination Compounds: Gold; Springer: Berlin/Heidelberg, Germany, 2007; Volume 281, pp. 269–309. p
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23. Pujadas, M.; Rodríguez, L. Luminescent Phosphine Gold(I) Alkynyl Complexes. Highlights from 2010 to 2018. Coord. Chem. Rev. 2020, 408, 213179. [CrossRef] 24. Zhang, Q.-C.; Xiao, H.; Zhang, X.; Xu, L.-J.; Chen, Z.-N. Luminescent oligonuclear metal complexes and the use in organic
light-emitting diodes. Coord. Chem. Rev. 2019, 378, 121. [CrossRef] 25. Xu, L.-J.; Wang, J.-Y.; Zhu, X.-F.; Zeng, X.-C.; Chen, Z.-N. Phosphorescent Cationic Au4Ag2 Alkynyl Cluster Complexes for
Efficient Solution-Processed Organic Light-Emitting Diodes. Adv. Funct. Mater. 2015, 25, 3033. [CrossRef] 11 of 11 11 of 11 Molecules 2023, 28, 3247 26. References Zhang, X.; Wang, J.-Y.; Huang, Y.-Z.; Yang, M.; Chen, Z.-N. Silver(I) nanoclusters of carbazole-1,8-bis(acetylide): From visible to
near-infrared emission. Chem. Commun. 2019, 55, 6281. [CrossRef] 27. Huang, Y.-Z.; Shi, L.-X.; Wang, J.-Y.; Su, H.-F.; Chen, Z.-N. Elaborate Design of Ag8Au10 Cluster [2]Catenane Phosphors for
High-Efficiency Light-Emitting Devices. ACS Appl. Mater. Interfaces 2020, 12, 57264. [CrossRef] g
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28. Xiao, H.; Zhang, X.; Wang, J.-Y.; Zhang, L.-Y.; Zhang, Q.-C.; Chen, Z.-N. Enhancing Phosphorescence through Rigidifying the
Conformation to Achieve High-Efficiency OLEDs by Modified PEDOT. ACS Appl. Mater. Interfaces 2019, 11, 45853. [CrossRef] 29. Bennington, M.S.; Feltham, H.L.C.; Buxton, Z.J.; Whitea, N.G.; Brooker, S. Tuneable reversible redox of cobalt(iii) carbazole
complexes. Dalton Trans. 2017, 46, 4696. [CrossRef] 30. Krause, L.; Herbst-Irmer, R.; Sheldrick, G.M.; Stalke, D. Comparison of silver and molybdenum microfocus X-ray sources for
single-crystal structure determination. J. Appl. Crystallogr. 2015, 48, 3. [CrossRef] 31. Hubschle, C.B.; Sheldrick, G.M.; Dittrich, B. ShelXle: A Qt graphical user interface for SHELXL. J. Appl. [CrossRef] 32. Frisch, M.J.; Trucks, G.W.; Schlegel, H.B.; Scuseria, G.E.; Robb, M.A.; Cheeseman, J.R.; Scalmani, G.; Barone, V.; Petersson, G.A.;
Nakatsuji, H.; et al. Gaussian 16, Revision, A.03; Gaussian Inc.: Wallingford, CT, USA, 2016. 33. Adamo, C.; Barone, V. Toward reliable density functional methods without adjustable parameters: The PBE0 model. J. Chem. Phys. 1999, 110, 6158. [CrossRef] 34. Ernzerhof, M.; Scuseria, G.E. Assessment of the Perdew-Burke-Ernzerhof exchange-correlation functional. J. Chem. Phys. 1999,
110, 5029. [CrossRef] 35. Grimme, S.; Antony, J.; Ehrlich, S.; Krieg, H. A consistent and accurate ab initio parameterization of density functional dispersion
correction (DFT-D) for the 94 elements H-Pu. J. Chem. Phys. 2010, 132, 154104. [CrossRef] 36. Bauernschmitt, R.; Ahlrichs, R. Treatment of Electronic Excitations within the Adiabatic Approxima
Density Functional Theory. Chem. Phys. Lett. 1996, 256, 454. [CrossRef] Ahlrichs, R. Treatment of Electronic Excitations within the Adiabatic Approximation of Time Dependent
Theory. Chem. Phys. Lett. 1996, 256, 454. [CrossRef] y
y
y
37. Casida, M.E.; Jamorski, C.; Casida, K.C.; Salahub, D.R. Molecular Excitation Energies to High-lying Bound States from Timedepen-
dent Density-functional Response Theory: Characterization and Correction of the Time-dependent Local Density Approximation
Ionization Threshold. J. Chem. Phys. 1998, 108, 4439. [CrossRef] y
38. Stratmann, R.E.; Scuseria, G.E.; Frisch, M.J. An Efficient Implementation of Time-dependent Density-f
Calculation of Excitation Energies of Large Molecules. J. Chem. Phys. 1998, 109, 8218. [CrossRef] 38. Stratmann, R.E.; Scuseria, G.E.; Frisch, M.J. References An Efficient Implementation of Time-dependent Density-functional Theory for the
Calculation of Excitation Energies of Large Molecules. J. Chem. Phys. 1998, 109, 8218. [CrossRef]
39. Scalmani, G.; Frisch, M.J. Continuous surface charge polarizable continuum models of solvation. I. General formalism. h, M.J. Continuous surface charge polarizable continuum models of solvation. I. General formalism
132, 114110. [CrossRef] 39. Scalmani, G.; Frisch, M.J. Continuous surface charge polarizable continuum models of solvation
J. Chem. Phys. 2010, 132, 114110. [CrossRef] 40. Andrae, D.; Häussermann, U.; Dolg, M.; Stoll, H.; Preuss, H. Energy-adjusted Ab initio Pseudopotentials for the Second and
Third Row Transition Elements. Theor. Chim. Acta 1990, 77, 123. [CrossRef] 41. Schwerdtfeger, P.; Dolg, M.; Schwarz, W.H.E.; Bowmaker, G.A.; Boyd, P.D.W. Relativistic effects in gold chemistry. I. Diatomic
gold compounds. J. Chem. Phys. 1989, 91, 1762. [CrossRef] 41. Schwerdtfeger, P.; Dolg, M.; Schwarz, W.H.E.; Bowmaker, G.A.; Boyd, P.D.W. Relativistic effects in gold chemistry. I. Diatomic
gold compounds. J. Chem. Phys. 1989, 91, 1762. [CrossRef] 42. Lu, T.; Chen, F. Multiwfn: A Multifunctional Wavefunction Analyzer. J. Comp. Chem. 2012, 33, 580. Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.
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English
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Circuit improvements for a voltage multiplier
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IEEE transactions on applied superconductivity
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550 550 Circuit Improvements for a Voltage Multiplier V.K. Semenov and Yu. A. Polyakov progress report on the development of our fundamentally
accurate digital-to-analog converter. Abstract-A
voltage (or flux) multiplier is a device which
multiplies applied SFQ pulses. Due to the quantum nature of an
SFQ pulse the device can be used as a voltage (or flux) amplifier
with a fundamentally accurate (integer) gain. Earlier we showed
that the device can be used for improvement of dc and ac
voltage standards. Recently we have found that a long voltage
multiplier can display complex dynamic behavior, which can
lead to undesirable collective oscillations. This report is devoted
to theoretical optimization and experimental investigation of
long voltage multipliers connected in series for dc current. Experimentally we have obtained accurate current steps with
about 0.1 V voltage drop for a 5 mm x 5 mm chip fabricated in
Nb technology and tested at 4 K. There are two competitive techniques to synthesize very
accurate waveforms and utilizing unique properties of
overdamped Josephson junctions. One of them is using only
voltage reproducers (or voltage multipliers with the gain
equals to one). These voltage reproducers are rather simple;
in fact, they are either two [3] or three [4] junction
interferometers. However, the simplicity of the technique has
evident back side: much larger parasitic capacitance between
the injectors and the reproducers, which dramatically shunts
high-frequency components of the synthesized waveforms. Reference
sinswave
signal
... 128prirWw
I
I
-
I x256 I I I
fie
AC Oulpuf
up lo 1 MH
Fig. 1. Block diagram of DAC based on processing SFQ pulses. Index
Terms-AC
Voltage Standard, Flux amplifier,
Quantum metrology, Programmable Voltage Standard. Index
Terms-AC
Voltage Standard, Flux amplifier,
Quantum metrology, Programmable Voltage Standard. Manuscript received September 18, 2000. Revised version received
December 15, 2000. This work was supported in part by DOC (NET at
Boulder) and directly by HYPRES, Inc.
V. K. Semenov is with Physics & Astronomy Department, State
University of New York, Stony Brook, NY 11794 USA. (e-mail:
Vasili.Semenov@sunysb.edu).
Yu. A. Polyakov is with Physics & Astronomy Department, State
University of New York, Stony Brook, NY 11794 USA, (e-mail:
polyakov@rsfq 1 .physics.sunysb.edu). y
Yu. A. Polyakov is with Physics & Astronomy Department, State
University of New York, Stony Brook, NY 11794 USA, (e-mail:
polyakov@rsfq 1 .physics.sunysb.edu). )
y y
,
V. K. Semenov is with Physics & Astronomy Department, State
University of New York, Stony Brook, NY 11794 USA. (e-mail:
Vasili.Semenov@sunysb.edu). Manuscript received September 18, 2000. Revised version received
December 15, 2000. This work was supported in part by DOC (NET at
Boulder) and directly by HYPRES, Inc. US. Government work not protected by U.S. copyright. 111. FREQUENCY
MULTIPLIER 111. FREQUENCY
MULTIPLIER Fig. 2. The structure of the pulse multiplier that effectively doubles
the frequency. IN
Fig. 3. Notations and equivalent circuits of the basic stages of the
voltage multiplier. The stages are situated between two peripherally
shorted superconductor films. These superconductor boxes arc
shown by dashed lines. IN
Fig. 3. Notations and equivalent circuits of the basic stages of the
voltage multiplier. The stages are situated between two peripherally
shorted superconductor films. These superconductor boxes arc
shown by dashed lines. An elementary frequency multiplier (Fig. 2) also utilizes
the Josephson frequency-to-voltage relationship. It is based
on the duplication of SFQ pulses generated by junction J1, It
is easy to show that the pulse repetition frequency is
accurately duplicated. A low-pass filter (right side in Fig.2)
should convert these SFQ pulses to dc voltage so that the
SFQ pulses generated by the junction in the next elementary
multiplier are separated by uniform time intervals. However,
because of thermal fluctuations SFQ pulses are generated by
junction J1 with significant time jitter. In other words, the
frequency multiplier probably has a significant phase noise. Underestimation of this noise was one of our misjudgments. A. Design Fig. 3 shows three main stages of our latest version of the
voltage multiplier. The injector (INJ) allows pulses to be
transfer or "injected" into a floating (galvanically isolated)
VM. The regular stage (REG) [3] operates as an elementary
voltage source with the voltage drop corresponding to the
frequency of passing SFQ pulses. It also transmits SFQ
pulses to the next stage. Both stages are situated between
superconductor films, which have several superconducting
connections near its edges. The third stage (CHK) uses two
galvanically isolated shields and behaves as two REG stages. Iv. VOLTAGE MULTIPLIERS external wave is not a sine wave but a train of very short
pulses. Each pulse causes 2n leaps in the every junction of
the array. The required variation of the output voltage is
provided by corresponding variation of the frequency of
these pulses. The known experimental results are rather
promising [5, 61. The major drawback of this technique (from
our point of view) is extremely high requirements for the
hardware that provide generation of short pulses with non-
uniform time intervals and their propagation along the
Josephson junction array. I. INTRODUCTIO A of
A our investigation of a prototype superconducting
Digital-to-Analog Converter (DAC) based on processing
Single Flux Quantum (SFQ) pulses [l, 21. Two unique
features make the device potentially attractive for metrology
applications: i) The generation of output voltage is based on
the fundamentally accurate Josephson frequency-to-voltage
relationship. ii) The device contains its own frequency
multipliers and therefore dramatically lower frequencies, a
few hundred megahertz rather than tens of gigahertz, can be
utilized as the external reference frequency. We expected that
the DAC would become a key component of novel standards
and calibrators of ac voltage with frequency up to 1 MHz. However, the implemented prototype had small (about 10
mV) output voltage which was far lower than the expected
1 V to 10 V target magnitude. More importantly, we found
that the generated voltage did not agree well with the
expected quantized values. At the time we believed that both
flaws could be fixed easily, but it took about two years to
find the origins of those deviations from the Josephson
frequency-to-voltage relationship. As a result we made
significant progress in the quality of the primary components
of our DAC. However, the whole system is still not ready for
testing at a metrology lab. So, this manuscript is a kind of a
few years ago we presented experimental results Reference
sinswave
signal Fig. 1. Block diagram of DAC based on processing SFQ pulses. The other technique originated from a conventional
voltage standard based on synchronization of an array of
Josephson junctions by an external wave, But now the US. Government work not protected by U.S. copyright. ss I Iv. VOLTAGE MULTIPLIER 11. DAC DESIGN The DAC based on processing SFQ pulses [7] consists of
(see Fig. 1) a frequency multiplier and a frequency divider
providing two local reference frequencies; a set of RSFQ
based [2] frequency synthesizers SD controlled by an
external input code; and a set of voltage amplifiers with
integer gains or Voltage Multipliers (VM) [7-91. Operation of
the DAC is based on the Josephson frequency-to-voltage
relationship. Namely, SFQ pulses generated by a synthesizer
with a variable frequency (that may be as high as 50 GHz for
our recent implementation based on 1 kA/cm2 fabrication
technology at HYPES, Inc.) and are converted to a
proportional variable voltage (that may be as high as 0.1 mV)
by the first Josephson junction of the VM. All the VMs,
which may have different gains and be fed by different
synthesizers, are ac coupled with the digital circuitry and
therefore can be connected in series for dc current, so the
output voltage produced by the DAC is the sum of the partial
voltages produced by the VMs. Fig. 4 shows a possible connection of the fragments in the
voltage multiplier as well as its major parasitic capacitances
and inductances. First, we would like to note that CHK cells
break the ground plane into islands to choke low frequency
parasitic oscillations [ 11. J7
'
J4 J7
'
J4
""4""
INJ
J4
J1
J12
IN
IN
Fig. 3. Notations and equivalent circuits of the basic stages of the
voltage multiplier. The stages are situated between two peripherally
shorted superconductor films. These superconductor boxes arc
shown b
d
h d lines 111. FREQUENCY
MULTIPLIER
LR
Low-
Pass
Filter
nn-
2v
+-
Fig. 2. The structure of the pulse multiplier that effectively doubles
the frequency. C. Experiments Most of our 5 x 5 mmZ chips contain an array of 24 voltage
multipliers each having gains 32 times. Maximum voltage
drop per stage is about 80 pV, which is achieved at 40 GHz
pulse repetition frequency. The total gain of the array is 768
which corresponds to an output voltage of about 60 mV. The
whole array structure occupies an area of about 5mm2 (or
20% of the chip area), giving a specific voltage drop of about
1.5 V/cm2. Chips are manufactured using 1 kA/cm2
HYPRES, Inc. fabrication technology. We tested about 20
designs with different variations of the parameters discussed
above. Current, mA
Fig. 5 . Detection of the phase noise of the reference signal in section
with lengths 96, 192, ... ,768. The effect has been observed only at
relatively low frequency, say, below 10 GHz. Current, mA F cJT
I
t
Ib Fig. 5 . Detection of the phase noise of the reference signal in section
with lengths 96, 192, ... ,768. The effect has been observed only at
relatively low frequency, say, below 10 GHz. JT cJ The parasitics shown in Fig. 4 are responsible for parasitic
resonances. SFQ pulses passing through voltage multipliers
can excite undesirable oscillations at these resonant
frequencies. As a result the multipliers are biased by ac as
well as dc currents causing the multipliers to operate
incorrectly. As we showed above, the total inductance of
multipliers depend on many parameters (including dc bias). All together, this leads to the appearance of a reproducible
fine structure along the current range of what should be a
horizontal voltage step. This structure was removed by
inserting resistive strips between the voltage multipliers. The
next effect is a self-detection of the phase noise. According to
Josephson frequency-to-voltage relationship the noise is
converted into a high-frequency voltage noise, which induces
ac currents flowing in particular via capacitors CJ (Fig. 4). This current increases if a larger number of voltage
multipliers is activated. The effect can be observed as a
rounding of current step, which is proportional to the number
of activated voltage multipliers as shown in Fig. 5. Fig. 4, Connection of two voltage multipliers activated by a
common source of SFQ pulses via terminals In. Both multipliers are
connected in series for dc bias Ib. A problem arises when Eq. (1) is applied to multi-stage
multipliers. 552
where f is t
current of the
is about half o
effective critic
~ ......... L_..l 5 552 where f is the frequency of SFQ pulses and Ib is the bias
current of the circuit. For a single VM stage, this inductance
is about half of the Josephson inductance associated with an
effective critical current of this stage: L,=(D,/Ic. these voltage multipliers. Note that conventional software
packages are not suitable for an electromagnetic analysis of
the system because of unequal values of mutual inductances
Mi,j and M j , i . ~ ......... L_..l
cJF cJT
I
t
Ib
Fig. 4, Connection of two voltage multipliers activated by a
common source of SFQ pulses via terminals In. Both multipliers are
connected in series for dc bias Ib. B. Theory The inductive parasitics of the voltage multipliers are
another reason for the improper operation of our first devices. In [lo] we pointed out that a variation of the delay T in
propagation of SFQ pulses along a circuit leads to the
variation of average magnetic flux. The inductance L
corresponding to this effect could be defined as the first
derivative of the magnetic flux over current: L = ( d a / dI) = (f . a*). ( d ~
/ dZb),
(1) (1) 52 v. PROGRAMMABLE VOLTAGE STANDARD AND DAC We
developed
room-temperature
interfaces
for
a
programmable voltage standard and for a DAC. The former
interface can hold up to twenty 5x5 mm2 chips. It has been
tested with two chips, while a 1 Volt demonstration would
require about 17 chips. q
The latter interface is fully assembled and passed all tests
that can be carried out without superconductor chips. VI. CONCLUSIO Current, mA In the frame of the project we encountered several
perplexing problems. One of them can be explained in terms
of a dynamic mutual inductance, which is developed in
voltage multipliers under influence of SFQ pulses. We think,
this effect has independent scientific interest, We found also
that the effective inductance of the voltage multiplier can be
controlled by the frequency of SFQ pulses. Such a variable
inductance can be used in future analog SFQ devices. Fig. 6. Accurate measurement of current steps, showing repeated
traces with random noise corresponding to an accuracy of - 100 nV. During the measurements we did not observe any deviation from a
quantized horizontal voltage step. Fig. 6. Accurate measurement of current steps, showing repeated
traces with random noise corresponding to an accuracy of - 100 nV. During the measurements we did not observe any deviation from a
quantized horizontal voltage step. 2700 ,
I
,
I
Wide Current Step
A I =.27 mA
2600 -
2400 I '
I
0.6
0.7
0.6
0.9
Current, mA 2700 ,
I
,
I
Wide Current Step
A I =.27 mA
2600 -
2400 I '
I
0.6
0.7
0.6
0.9
Current, mA
Fig. 7. The current range can be reasonably wide -*17%. It has
been shown experimentally that the range of the current step can be
increases if a few voltage multipliers are connected in parallel. Finally we have demonstrated that a DAC based on
processing SFQ pulses is a practical device, with near-term
application to a fast high-precision programmable ac voltage
standard. REFERENCES Fig. 7. The current range can be reasonably wide -*17%. It has
been shown experimentally that the range of the current step can be
increases if a few voltage multipliers are connected in parallel. [I]
V.K. Semenov, P.N. Shevchenko, and Yu. A. Polyakov, "Digital-to-
Analog Converter Based on Processing of SFQ pulses", Extended
Abstracts of ISEC97, vol. 2, pp. 320-322, Berlin, Germany, June,
1997. BO -
60 -
>
E
-
% 40-
-
d
3
20 -
0 -
0.5
0.6
0.7
0.8
0.9
1.0
1.1
Current, mA
Fig. 8. Output characteristics of voltage multiplier showing multiple
current steps for high voltages. The input voltage is changing from
0 to 100 p V in 5 p V steps. (A voltage of 60 m V corresponds to
about 1.5 Vkm2 specific voltage.) BO -
60 -
>
E
-
% 40-
-
d
3
20 -
0 -
0.5
0.6
0.7
0.8
0.9
1.0
1.1
Current, mA [2] V.K. Semenov,
P.N. Shevchenko, and
Yu. A. Polyakov,
"Superconductor Digital-to-Analog Converter Based on Processing of
SFQ pulses", CPEM98, Washington DC, USA, Conference Digest, pp. 647-648, June 1998. C.A. Hamilton, "Josephson Voltage Standard Based on Single-Flux-
Quantum Voltage Multipliers," IEEE Trans. Appl. Superconduct. vol. [3]
2, pp. 2637-42, 1992. F.Hirayama, H. Sasaki, S. Kuryo, M Maezawa, T. Kiknchi, A. Shqji,
"Fabrication and Tests of RSFQ-Logic-Based DIA Converter
Components," Applied Superconductivity 1999, Inst. Phys. Conf. Ser. pp
[4]
NO. 167, pp. 729-32,2000. S. Benz, Josephson "Arbitrary Waveform Synthesizer for AC & DC
Voltage Synthesis," ASC2000 Pre-conference booklet, p.84 Rep. #2EK04, Virginia Beach, Virginia, Sep. 17-22,2000. pp
[5] C.A. Hamilton, C.J. Burroughs, and R.L. Kautz, "Josephson DIA
Converter with Fundamental Accuracy,", IEEE Trans. Instrum. Meas. [6]
vol. IM-44, pp.266, 1999. Fig. 8. Output characteristics of voltage multiplier showing multiple
current steps for high voltages. The input voltage is changing from
0 to 100 p V in 5 p V steps. (A voltage of 60 m V corresponds to
about 1.5 Vkm2 specific voltage.) V.K. Semenov, "Digital to Analog Conversion Based on Processing of
the SFQ Pulses", IEEE Trans. on Appl. Supercond. vol. 3, p. 2637,
1993. pp
[7] V.K. Semenov, "Multi-Josephson Junction Structures for DC Voltage
Multiplication", in Extended Abstracts of ISEC'87, pp. 138-40,Tokyo,
Japan, 1987. [8] Fortunately most of these problems have been resolved by
reducing the phase noise of frequency multipliers and
damping resonant oscillations. C. Experiments For example, the second stage adds a further
delay to the already delayed SFQ pulses. This means that the
total delay for n multipliers is nz. A simple generalization of
this observation leads to the conclusion that despite the
physical identity of all stages, the inductance of the n-th stage
is n times larger than the first one: L, = n . L . The extra
inductance is induced by all previous stages. Thus if all
stages have inductance L, then each stage is magnetically
coupled to all foregoing stages, but it is not coupled with the
subsequent stages: q
g
Li = L,
Mi,j = L, Mj,i = 0, i > j . (2)
Thi
i
i di
h
l i d
i
i
f (2) Li = L,
Mi,j = L, Mj,i = 0, i > j . (2) This equation indicates the unusual inductive properties of 553 I I
2389.4 I
li 1
Current, mA
Fig. 6. Accurate measurement of current steps, showing repeated
traces with random noise corresponding to an accuracy of - 100 nV. During the measurements we did not observe any deviation from a
quantized horizontal voltage step. I I
2389.4 I
li 1
Current, mA ACKNOWLEDGMEN We would like to thank CA. Hamilton for initiation and
support of the project. We would like to thank also S. Benz,
A. Kirichenko, 0. Mukhanov and S. Sarwana for fruitful
discussions and technical assistance. REFERENCES The next 3 figures illustrate
correct operation of the whole structure: Voltage steps are
accurate (Fig. 6), wide (Fig. 7) and be located at high voltage
(Fig. 8). p
V.K. Semenov and M.A. Voronova, "DC Voltage Multipliers: a novel
Application of Synchronization in Josephson Junction Arrays", IEEE
Trans. on Magn., vol. 23, p p ~ 1476-79, March 1987. [9] p p
[lo] V.K. Semenov, "Comparison of Superconductor Analog-to-Digital
Converters (Theory)", Ext. Abstr. of ISEC'99, pp, 23-25, Jim, 1999.
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https://journal2.um.ac.id/index.php/gpji/article/download/2050/2015
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Indonesian
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PENGEMBANGAN MEDIA PELATIHAN SIGNAL-SIGNAL WASIT BOLAVOLI MENGGUNAKAN M-LEARNING
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Gelanggang Pendidikan Jasmani Indonesia/Gelanggang
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Alamat korespondensi:
E-mail: dona.sandy.fik@um.ac.id Abstract The purpose if this research and development to develop a training media volleyball
Referee signals that are packaged in the form of a digital book mobile learning. In
addition, media digital book mobile learning is expected to facilitate the exercise of the
activity of the participants to learn and practice the material volleyball Referee signals
independently.Research and development model using the conceptual model that is a
model thathas been validated to the operational in the procedure on a product. The
overall Group trials involving exercise activities 45 members. Products resulting from
this research and development is a media training volleyball Referee signals using M-
Learning. Data analysis of the results of trials of small group obtained the percentage of
89.33%. From the results of the analysis of the test data group obtained the percentage
of 92.81%. Conclusion the development of media training volleyball Referee signals
using M-Learning can be used as a medium is theright and properfor members of
activity Unit Activity in State University of Malang Volleyball. The purpose if this research and development to develop a training media volleyball
Referee signals that are packaged in the form of a digital book mobile learning. In
addition, media digital book mobile learning is expected to facilitate the exercise of the
activity of the participants to learn and practice the material volleyball Referee signals
independently.Research and development model using the conceptual model that is a
model thathas been validated to the operational in the procedure on a product. The
overall Group trials involving exercise activities 45 members. Products resulting from
this research and development is a media training volleyball Referee signals using M-
Learning. Data analysis of the results of trials of small group obtained the percentage of
89.33%. From the results of the analysis of the test data group obtained the percentage
of 92.81%. Conclusion the development of media training volleyball Referee signals
using M-Learning can be used as a medium is theright and properfor members of
activity Unit Activity in State University of Malang Volleyball. © 2017 Universitas Negeri Malang © 2017 Universitas Negeri Malang ISSN 0853-5043 (cetak) Alamat korespondensi:
E-mail: dona.sandy.fik@um.ac.id GPJI 1 (2) (2017) GPJI 1 (2) (2017) Abstrak _
Tujuan penelitian dan pengembangan ini untuk mengembangkan media pelatihan
signal-signal wasit bolavoli yang dikemas dalam bentuk buku digital mobile learning. Selain
itu, media buku digital mobile learning diharapkan dapat mempermudah peserta kegiatan
latihan untuk belajar dan mempraktekkan materi signal-signal wasit bolavoli secara
mandiri. Model penelitian ini menggunakan model konseptual. Uji coba kelompok
keseluruhan melibatkan 45 anggota kegiatan latihan. Produk yang dihasilkan dari
penelitian ini adalah media pelatihan signal-signal wasit bolavoli menggunakan M-
Learning. Dengan hasil analisis data uji coba kelompok kecil diperoleh persentase
89,33% dan hasil analisis data uji kelompok besar diperoleh persentase 92,81%. Kesimpulan pengembangan media pelatihan signal-signal wasit bolavoli menggunakan
M-Learning dapat digunakan sebagai media yang tepat dan layak untuk anggota kegiatan
Unit Aktivitas Bolavoli di Universitas Negeri Malang. PENGEMBANGAN MEDIA PELATIHAN SIGNAL-SIGNAL WASIT
BOLAVOLI MENGGUNAKAN M-LEARNING UNTUK MAHASISWA Info Artikel
____________________
Sejarah Artikel:
Diterima September 2017
Disetujui Oktober 2017
Dipublikasikan Desember 2017
____________________
Keywords:
Refereeing volleyball, Mobile
Learning
Abstrak
___________________________________________________________
_
Tujuan penelitian dan pengembangan ini untuk mengembangkan media pelatihan
signal-signal wasit bolavoli yang dikemas dalam bentuk buku digital mobile learning. Selain
itu, media buku digital mobile learning diharapkan dapat mempermudah peserta kegiatan
latihan untuk belajar dan mempraktekkan materi signal-signal wasit bolavoli secara
mandiri. Model penelitian ini menggunakan model konseptual. Uji coba kelompok
keseluruhan melibatkan 45 anggota kegiatan latihan. Produk yang dihasilkan dari
penelitian ini adalah media pelatihan signal-signal wasit bolavoli menggunakan M-
Learning. Dengan hasil analisis data uji coba kelompok kecil diperoleh persentase
89,33% dan hasil analisis data uji kelompok besar diperoleh persentase 92,81%. Kesimpulan pengembangan media pelatihan signal-signal wasit bolavoli menggunakan
M-Learning dapat digunakan sebagai media yang tepat dan layak untuk anggota kegiatan
Unit Aktivitas Bolavoli di Universitas Negeri Malang. Abstract
___________________________________________________________
_
The purpose if this research and development to develop a training media volleyball
Referee signals that are packaged in the form of a digital book mobile learning. In
addition, media digital book mobile learning is expected to facilitate the exercise of the
activity of the participants to learn and practice the material volleyball Referee signals
independently.Research and development model using the conceptual model that is a
model thathas been validated to the operational in the procedure on a product. The
overall Group trials involving exercise activities 45 members. Products resulting from
this research and development is a media training volleyball Referee signals using M-
Learning. Data analysis of the results of trials of small group obtained the percentage of
89.33%. From the results of the analysis of the test data group obtained the percentage
of 92.81%. Conclusion the development of media training volleyball Referee signals
using M-Learning can be used as a medium is theright and properfor members of
activity Unit Activity in State University of Malang Volleyball. © 2017 Universitas Negeri Malang
Alamat korespondensi:
E-mail: dona.sandy.fik@um.ac.id
ISSN 0853-5043 (cetak) PENDAHULUAN kemajuan suatu negara sangat besar, begitu
pula pendidikan yang baik akan menghasilkan
generasi
yang
bermutu
sehingga
dapat
memajukan
bangsa
dalam
berbagai
hal
terutama dalam mengikuti perkembangan Ilmu Pendidikan merupakan salah satu hal
yang sangat dibutuhkan oleh semua kalangan
di
negara
ini. Peran
pendidikan
dalam 238 Nutri Puspitasari, dkk / Gelanggang Pendidikan Jasmani Indonesia 1 (2) (2017) Permainan
bolavoli
juga
merupakan salah satu cabang olahraga yang
dipertandingkan secara resmi yang dinaungi
oleh Federation Internationale de Volleyball (FIVB)
sebagai
induk
organisasi
internasional,
sedangkan
di
Indonesia
dinaungi
oleh
Persatuan Bolavoli Seluruh Indonesia (PBVSI). Bolavoli
adalah
“olahraga
yang
dimainkan oleh dua tim dalam satu lapangan
yang dipisahkan oleh sebuah net” (PBVSI,
2015:1). Sedangkan menurut Permana (2008:7)
menyatakan bahwa, “bolavoli adalah olahraga
yang dimainkan oleh dua tim dan setiap tim
terdiri dari enam pemain, tim tersebut misalnya
tim A dan tim B. Untuk memulai permainan,
tim pertama yang akan melakukan serve dipilih
melalui “lempar koin”. Permainan bolavoli
merupakan permainan yang dilakukan dengan
kerja sama karena merupakan olahraga beregu. Setiap pemain harus mampu menguasai unsur
keterampilan dasar seperti passing, service, block,
dan smash. Keempat unsur tersebut sangat
berpengaruh dalam suatu permainan bolavoli
karena merupakan teknik dasar permainan
bolavoli. Menurut Sunardi dan Kardiyanto
(2013:15) teknik dasar permainan bolavoli
meliputi : (1) service (Underhand Serve dan
Overhead Serve), (2) passing (Pasing Bawah dan
Pasing Atas), (3) smash (Spike Open, Spike Quick Permainan bolavoli merupakan salah
satu cabang olahraga yang berkembang baik di
Indonesia, cabang olahraga yang populer dan
digemari oleh semua lapisan masyarakat ini
dilakukan
dari
berbagai
kalangan
bawah
sekalipun. Permainan ini banyak diminati
karena permainannya yang menarik dan dapat
memacu adrenalin mulai dari penonton sampai
permainan. Permainan
bolavoli
juga
merupakan salah satu cabang olahraga yang
dipertandingkan secara resmi yang dinaungi
oleh Federation Internationale de Volleyball (FIVB)
sebagai
induk
organisasi
internasional,
sedangkan
di
Indonesia
dinaungi
oleh
Persatuan Bolavoli Seluruh Indonesia (PBVSI). (
)
Bolavoli
adalah
“olahraga
yang
dimainkan oleh dua tim dalam satu lapangan
yang dipisahkan oleh sebuah net” (PBVSI,
2015:1). Sedangkan menurut Permana (2008:7)
menyatakan bahwa, “bolavoli adalah olahraga
yang dimainkan oleh dua tim dan setiap tim
terdiri dari enam pemain, tim tersebut misalnya
tim A dan tim B. Untuk memulai permainan,
tim pertama yang akan melakukan serve dipilih
melalui “lempar koin”. Permainan bolavoli
merupakan permainan yang dilakukan dengan
kerja sama karena merupakan olahraga beregu. Setiap pemain harus mampu menguasai unsur
keterampilan dasar seperti passing, service, block,
dan smash. Keempat unsur tersebut sangat
berpengaruh dalam suatu permainan bolavoli
karena merupakan teknik dasar permainan
bolavoli. Nutri Puspitasari, dkk / Gelanggang Pendidikan Jasmani Indonesia 1 (2) (2017) dan Spike dari Belakang), (4) bendungan block
(Side Step dan Cross Step). Pengetahuan dan Teknologi (IPTEK) yang
berkembang
dengan
pesat
di
masa
sekarang.Salah satu aspek yang mengalami
perkembangan pesat dalam dunia IPTEK
adalah dunia pendidikan yang di dalamnya
terdapat
suatu
proses
komunikasi
dan
penyampaian informasi dari seorang pengajar
kepada pebelajar, yang biasa kita dengar
dengan proses belajar mengajar. Permainan bolavoli telah berkembang
menjadi bagian dari olahraga pendidikan dan
olahraga prestasi. Dalam setiap pertandingan
bolavoli wajib dipimpin oleh wasit bolavoli
yang sudah memiliki sertifikat baik di tingkat
cabang, daerah, nasional, maupun tingkat
internasional. Pertandingan yang dipimpin oleh
wasit bolavoli profesional diharapkan agar
pertandingan berjalan dengan lancar dan sesuai
dengan
aturan-aturan
yang
sudah
ditetapkan.Perwasitan merupakan hal yang
sangat penting dalam permainan bolavoli agar
permainan berjalan dengan aturan yang sudah
ditetapkan. Dalam perwasitan bolavoli untuk
memimpin suatu pertandingan terdiri atas wasit
pertama, wasit kedua, pencatat, dan empat
hakim garis. Aspek
lain
yang
mengalami
perkembangan
pesat
dalam
dunia
Ilmu
Pengetahuan dan Teknologi (IPTEK) adalah
dunia pelatihan yang di dalamnya terdapat
suatu proses penyampaian informasi dan
komunikasi dari pelatih kepada peserta latihan. Seiring dengan kemajuan teknologi tersebut
peserta
latihan
semakin
mudah
untuk
mendapatkan informasi dengan cara yang
efektif dan efisien melalui media untuk
mendapatkan hasil latihan yang maksimal. g
Menurut Ahmadi dalam Purwalingga
(2014:14) wasit adalah orang yang memimpin
pertandingan dari awal hingga berakhirnya
permainan. Seorang wasit bertugas memimpin
dan mengatur pertandingan agar pertandingan
berjalan dengan lancar tanpa ada gangguan. Wasit harus tegas dalam mengambil keputusan,
bersikap jujur, adil dan tidak memihak salah
satu tim (netral). Hampir semua cabang
olahraga dalam setiap pertandingan dipimpin
oleh wasit, salah satunya yaitu cabang olahraga
bolavoli. Dalam setiap pertandingan resmi
bolavoli selalu dipimpin oleh wasit pertama
dan dibantu oleh wasit kedua. Wasit pertama
melakukan tugasnya sambil duduk atau berdiri
pada kursi wasit yang ditempatkan pada salah
satu ujung net (PBVSI, 2015:64). Sedangkan
“wasit kedua menjalankan tugasnya dengan
berdiri di luar lapangan permainan dekat
dengan tiang pada sisi berlawanan atau
berhadapan dengan wasit pertama” (PBVSI,
2015:65).Pengambilan keputusan antara wasit
pertama dan wasit kedua sangat diperlukan
kerjasama dan komunikasi yang baik agar
pertandingan berjalan dengan lancar. p
y
g
Permainan bolavoli merupakan salah
satu cabang olahraga yang berkembang baik di
Indonesia, cabang olahraga yang populer dan
digemari oleh semua lapisan masyarakat ini
dilakukan
dari
berbagai
kalangan
bawah
sekalipun. Permainan ini banyak diminati
karena permainannya yang menarik dan dapat
memacu adrenalin mulai dari penonton sampai
permainan. Nutri Puspitasari, dkk / Gelanggang Pendidikan Jasmani Indonesia 1 (2) (2017) Nutri Puspitasari, dkk / Gelanggang Pendidikan Jasmani Indonesia 1 (2) (2017) audio-visual,
Media
teknologi
berbasis
komputer,
dan
Media
teknologi
terpadu
(Dwiyogo, 2008:7-10).Media adalah segala
sesuatu yang digunakan untuk menyalurkan
informasi atau materi pada suatu kegiatan
tertentu
seperti
latihan,
sehingga
dapat
membantu dalam kegiatan pelaksanaan latihan
untuk mencapai tujuan latihan. Media yang
digunakan untuk latihan dapat berupa media
cetak
dan
non-cetak. Menurut
Dwiyogo
(2008:7) menyebutkan bahwa “media cetak
adalah cara-cara untuk memproduksi atau
menyebarkan materi, seperti buku dan materi
visual statis, yang pada umumnya dilakukan
melalui proses cetak mekanis atau fotografis”. harus didukung dengan media pelatihan yang
memadai sehingga anggota kegiatan di UABV
UM
dapat
dengan
mudah
belajar
dan
mempraktikkannya. Media pelatihan adalah segala sesuatu
yang digunakan untuk menyalurkan informasi
atau materi saat proses latihan, sehingga dapat
membantu
dalam
kegitan
latihan
untuk
mencapai tujuan latihan tersebut. Semakin
banyak media yang bisa digunakan akan
mempermudah
proses
latihan
tersebut,
sehingga
pelatih
tidak
kesulitan
untuk
memberikan materi latihan dan peserta latihan
lebih mudah untuk mencari materi latihan yang
disajikan. Menurut Dwiyogo (2010:229) “Media
adalah segala bentuk dan saluran yang dapat
digunakan dalam suatu proses penyajian
informasi. Media adalah alat atau cara yang
digunakan
sebagai
perantara
untuk
menyampaikan informasi (pesan) agar dapat
diterima oleh penerima informasi sepenuhnya”. Sadiman (2010:7) juga berpendapat bahwa
“Media adalah segala sesuatu yang dapat
digunakan untuk menyalurkan pesan dari
pengirim
ke
penerima
sehingga
dapat
merangsang pikiran, perasaan, perhatian dan
minat serta siswa sedemikian rupa sehingga
proses belajar terjadi”. Di antara beberapa jenis media yang
dijelaskan di atas, maka media bentuk digital
seperti mobile melalui smartphone menarik bagi
calon pengguna, dalam hal ini peserta kegiatan
latihan perwasitan bolavoli sangat berminat
dengan mobile bentuk smartphone akan berfungsi
untuk menyimpan materi tentang signal-signal
wasit bolavoli. Mobile Learning (M-Learning) merupakan
paradigma baru dalam perkembangan dunia
teknologi
informasi
dan
komunikasi. M-
Learning adalah media yang unik karena
pebelajar dapat mengakses materi pelatihan,
arahan dan aplikasi yang berkaitan dengan
pelatihan, kapanpun dan dimanapun. Oleh
karena itu dengan semakin berkembangnya
media pelatihan, m-learning dimaksudkan untuk
dapat memberikan kemudahan bagi anggota
kegiatan
Unit
Aktivitas
Bolavoli
untuk
mempelajari materi latihan perwasitan. Mobile
Learning
menurut
Quinn
(dalam
Hasan,
2010:42) sebagai: “The intersection of mobile
computing and e-learning: accessible resources
whereever you are, strong search capabilities, rich
interaction, powerful support for effective learning,
and
performance-based
assessment”. Dapat
disimpulkan dari definisi tersebut, m-learning
merupakan
model
pelatihan
yang
memanfaatkan
teknologi
informasi
dan
komunikasi. Nutri Puspitasari, dkk / Gelanggang Pendidikan Jasmani Indonesia 1 (2) (2017) Menurut Sunardi dan Kardiyanto
(2013:15) teknik dasar permainan bolavoli
meliputi : (1) service (Underhand Serve dan
Overhead Serve), (2) passing (Pasing Bawah dan
Pasing Atas), (3) smash (Spike Open, Spike Quick Pertandingan
yang
sedang
dipertandingkan dipimpin oleh wasit pertama,
wasit kedua, dan hakim garis yang bekerjasama
dengan
memberikan
isyarat
tangan
dan
bendera. Sebelum memberikan isyarat tangan
wasit pertama harus meniup peluit terlebih
dahulu sebaliknya sebelum meniup peluit wasit
kedua harus memberikan isyarat tangan sesuai
dengan kewenangan masing-masing. Di dalam
permainan bolavoli terdapat 26 macam gerakan
isyarat tangan yang resmi untuk wasit dan 5
macam gerakan isyarat bendera yang resmi
untuk hakim garis. Pemahaman mengenai
isyarat tangan dan isyarat bendera untuk wasit 239 Nutri Puspitasari, dkk / Gelanggang Pendidikan Jasmani Indonesia 1 (2) (2017) Mobile Learning juga mempermudah interaksi
antara
peserta
dengan
materi
pelajaran. Demikian juga interaksi antara peserta dengan
pelatih/pengajar maupun antara peserta dapat
berbagi informasi atau pendapat menyangkut
materi pelajaran. sehingga mengakibatkan anggota kesulitan
dalam mempelajari materi signal-signal wasit
bolavoli. Mobile Learning juga mempermudah interaksi
antara
peserta
dengan
materi
pelajaran. Demikian juga interaksi antara peserta dengan
pelatih/pengajar maupun antara peserta dapat
berbagi informasi atau pendapat menyangkut
materi pelajaran. Penelitian
terdahulu
yang
berkaitan
dengan pengembangan media signal-signal wasit
bolavoli yang pernah dilakukan oleh Nuha
(2012) mengembangkan pembelajaran perwa-
sitan bolavoli menggunakan Electronik Book
danterlihat kelemahan pada penayangan vidio
signal-signal wasitnya belum lengkap sesuai
dengan yang ada dibuku peraturan bolavoli. Selain itu, penelitian terdahulu dari Setyawan
(2013) yang mengembangkan Electronik Book
materi perwasitan bolavoli terlihat beberapa
kelemahan pada peraturan yang berkaitan
dengan signal-signal belum ditampilkan pada
slide.. Penelitian terdahulu yang dikembangkan
oleh
Purwalingga
(2014)
mengembangkan
media pembelajaran berbasis WEB tentang
materi perwasitan bolavoli terlihat beberapa
kelemahan pada gambar isyarat (signal) kurang
jelas dan gerakan isyarat (signal) belum
memenuhu standart. Media yang digunakan untuk keperluan
pelatihan
mempunyai
beberapa
kegunaan
untuk
membantu
peserta
latihan
dalam
mempelajari materi latihan, antara lain: (1)
Media
pelatihan
mampu
membangkitkan
motivasi dan minat peserta terhadap materi
latihan yang diberikan, (2) Media pelatihan
memudahkan peserta untuk belajar kapanpun
dan dimanapun, (3) Media pelatihan mampu
merangsang peserta untuk belajar dengan
semangat,
(4)
Media
pelatihan
dapat
membantu peserta untuk mengulangi materi
latihan yang sudah dijelaskan, dan sebagainya. Pengembangan media pelatihan berupa buku
digital (m-learning) diharapkan dapat menarik
perhatian pebelajar terhadap materi latihan
yang disajikan serta dapat berfungsi sebagai alat
perantara dalam menyampaikan materi yang
dapat memudahkan anggota UABV untuk
memahami konsep perwasitan bolavoli. Berdasarkan keberadaan hasil-hasil pene-
litian
terdahulu
tersebut,
maka
peneliti
melakukan pengkajian penelitian dan pe-
ngembangan yang berjudul “Pengembangan
Media Pelatihan Signal-Signal Wasit Bolavoli
Menggunakan
M-Learning
Untuk
Anggota
UABV di Universitas Negeri Malang”. Nutri Puspitasari, dkk / Gelanggang Pendidikan Jasmani Indonesia 1 (2) (2017) p p
Hasil analisis kebutuhan menggunakan
angket yang telah dilakukan pada tanggal 2
November 2016 terhadap 30 peserta kegiatan
dari 50 anggota aktif UABV Universitas Negeri
Malang yang dipilih secara acak, diperoleh data
sebagai berikut: 30 anggota (100%) pernah
mendapatkan materi tentang signal-signal wasit
bolavoli dalam kegiatan di UABV UM, 30
anggota (100%) menyatakan signal-signal wasit
memiliki peranan penting dalam permainan
bolavoli, 29 anggota (96,67%) ingin menjadi
wasit bolavoli, 30 anggota (100%) pernah
mengalami kesulitan dalam mempraktekkan
signal-signal wasit bolavoli, 17 anggota (56,67%)
mengalami kesulitan mencari media belajar
signal-signal wasit bolavoli, 26 anggota (86,67%)
menyatakan
perlu
dikembangkannya
multimedia handphone (mobile learning) dalam
kegiatan di UABV UM, 28 anggota (93,33%)
berfikir
perlu
ada
pengembangan
media
pelatihan mengenai signal-signal wasit dalam
permainan
bolavoli
dalam
bentuk
mobile
learning. Berdasarkan
masalah
yang
telah
diuraikan, maka penelitian dan pengembangan
ini bertujuan untuk mengembangkan dan
menghasilkan produk media pelatihan signal-
signal wasit bolavoli yang dikemas dalam
bentuk buku digital mobile learning. Nutri Puspitasari, dkk / Gelanggang Pendidikan Jasmani Indonesia 1 (2) (2017) Munadi (2010:8) menyatakan bahwa
media pembelajaran adalah “segala sesuatu
yang dapat menyampaikan dan menyalurkan
pesan dari sumber secara terencana sehingga
tercipta lingkungan belajar yang kondusif
dimana penerimanya dapat melakukan proses
belajar secara efisien dan efektif”. Sihkabuden
(2011:4) menjelaskan secara singkat bahwa
media
pembelajaran
dapat
dikemukakan
sebagai sesuatu (bisa alat, bahan, atau keadaan)
yang
dipergunakan
sebagai
perantara
komunikasi dalam kegiatan pembelajaran. Jadi
dapat disimpulkan media merupakan suatu
alat yang digunakan untuk menyampaikan
suatu pesan sehingga pesan tersebut dapat
diterima dengan baik oleh pembelajar sesuai
dengan yang diharapkan. McQuiggan, dkk., (2015:10) menjelaskan
beberapa manfaat M-Learning adalah “Ability to
learn on the go, Reach underserved children and
schools, Improve higher-order thinking skills, Support
alternative
learning
environments,
Enable
personalized learning, and Motivate students”. Maksud dari penjelasan manfaat diatas adalah
M-Learning mempunyai kemampuan untuk
belajar dimana saja, mampu melayani anak-
anak dan sekolah, meningkatkan keterampilan
berfikir yang tinggi, mendukung alternatif
lingkungan belajar, dan memungkingkan untuk
pembelajaran sendiri dan memotivasi siswa. Penggunaan
media
pada
waktu
pelaksanaan pelatihan sebaiknya mengikuti
perkembangan
dan
kemajuan
teknologi. Teknologi yang paling awal ditemukan yaitu
teknologi cetak, sedangkan teknologi yang
muncul
terakhir
adalah
teknologi
mikroprosesor yang melahirkan pemakaian
komputer dan kegiatan interaktif. Perkembangan
teknologi
tersebut,
mengembangan
media
yang
dapat
dikelompokkan ke dalam beberapa bagian,
yaitu: Media teknologi cetak, Media teknologi 240 Nutri Puspitasari, dkk / Gelanggang Pendidikan Jasmani Indonesia 1 (2) (2017) HASIL Data keenam yang disajikan melalui uji
coba kelompok besar maka didapatkan data
yaitu
kejelasan
media
sebanyak
95%,
kemenarikan media sebanyak 95%, kesesuaian
media sebanyak 95,83%, dan kemudahan
media sebanyak 92,4%. Total data yang
dihasilkan adalah skor
hasil 4455,
skor
maksimal 4800 dan persentase 92,81%. Data
tersebut dihasilkan dari 30 peserta latihan
UABV Universitas Negeri Malang.. Berdasarkan
evaluasi
ahli
terhadap
pengembangan buku digital (mobile learning)
tentang
signal-signal
wasit
bolavoli
yang
dilakukan oleh ahli media didapatkan data
yaitu
kejelasan
media
sebanyak
75%,
kemenarikan media sebanyak 100%, kesesuaian
media sebanyak 75%, dan kemudahan media
sebanyak 75%. Total data yang dihasilkan
adalah skor hasil 122, skor maksimal 160 dan
persentase 76,25% Data kedua berdasarkan evaluasi ahli
perwasitan bolavoli terhadap pengembangan
buku digital (mobile learning) tentang signal-signal
wasit bolavoli yang dilakukan oleh ahli
perwasitan bolavoli maka didapatkan data
yaitu
kejelasan
materi
sebanyak
100%,
kemenarikan
materi
sebanyak
100%,
kesesuaian
materi
sebanyak
88,63%,
kemudahan materi sebanyak 87,5%, dan
kelengkapan materi sebanyak 100%. Total data
yang dihasilkan adalah skor hasil 144, skor
maksimal 160 dan persentase 90%. Nutri Puspitasari, dkk / Gelanggang Pendidikan Jasmani Indonesia 1 (2) (2017) Analisis tugas, (5) Analisis insiden kritis, (6)
Analisis Tujuan, (7) Analisis masalah (8)
Analisis Media, (9) Analisis data yang masih
ada, (10) Analisis biaya. wasit bolavoli yang dilakukan oleh ahli
akademisi bolavoli didapatkan data yaitu
kejelasan
media
sebanyak
88,63%,
kemenarikan media sebanyak 100%, kesesuaian
media sebanyak 87,5%, kemudahan media
sebanyak 87,5%, dan kelengkapan materi
sebanyak 100%. Total data yang dihasilkan
adalah skor hasil 143, skor maksimal 160 dan
persentase 89,37%. Tahap-tahap penelitian ini antara lain
analisis,
desain,pengembangan,implementasi
dan evaluasi. Tahap analisis meliputi needs
assessment dan front-end analysis. Kegiatan yang
dilakukan dalam front-end analysis antara lain
audience analysis, technology analysis, media
analysis
dan
extant-data
analysis. Alasan
menggunakan model tersebut karena produk
pengembangan pada penelitian ini berbasis
computer yaitu multimedia interaktif Data keempat yang disajikan dari data
hasil validasi ahli materi bolavoli maka
didapatkan
data
yaitu
kejelasan
materi
sebanyak 75%, kemenarikan materi sebanyak
75%,
kesesuaian
materi
sebanyak
75%,
kemudahan
materi
sebanyak
75%,
dan
kelengkapan materi sebanyak 75%. Total data
yang dihasilkan adalah skor hasil 120, skor
maksimal 160 dan persentase 75%. Subjek yang terlibat dalam penelitian
dan pengembangan buku digital (mobile learning)
tentang signal-signal wasit bolavoli meliputi (a)
subjek ahli dan (b) subjek coba (c) subjek uji
lapangan. Instrumen
pengumpulan
data
penelitian dan pengembangan ini menggunakan
non tes antara lain : (1) observasi, (2)
wawancara, dan (3) kuisioner Dari data yang dijelaskan diatas, dapat
disimpulkan bahwa data hasil persentase dari
ahli media 76,25%, ahli perwasitan bolavoli
90%, ahli akademisi bolavoli 89,37%, dan ahli
materi bolavoli 75%. Teknik analisis data yang digunakan
dalam penelitian dan pengembangan ini adalah
teknik
analisis
deskriptif
kualitatif
dan
kuantitatif. Analisis kualitatif digunakan untuk
menganalisis hasil pengumpulan data dari
tinjauan para ahli. Analisis kuantitatif berupa
persentase dalam pemberian angket terhadap
peserta kegiatan latihan perwasitan. Data kelima yang disajikan merupakan
hasil uji coba kelompok kecil maka didapatkan
data yaitu kejelasan media sebanyak 85,83%,
kemenarikan
media
sebanyak
93,89%,
kesesuaian
media
sebanyak
93,33%,
dan
kemudahan media sebanyak 89,01%. Total data
yang dihasilkan adalah skor hasil 2144, skor
maksimal 2400 dan persentase 89,33%. Data
tersebut dihasilkan dari 15 peserta latihan
UABV Universitas Negeri Malang. ihan perwasitan. l
i
hli
t
h d METODE Metode
penelitian
yang
digunakan
adalah metode penelitian dan pengembangan. model penelitian dan pengembangan yang
digunakan dalam penelitian ini berupa model
konseptual berdasarkan operasionalisasi teori
atau prinsip-prinsip yang telah tervalidasi ke
dalam langkah-langkah atau prosedur yang
mengarah
suatu
produk
sebagai
wujud
pemecahan masalah. Penelitian ini meng-
gunakan pendekatan problem oriented karena
masalah yang diteliti berdasarkan kondisi nyata
di lapangan. langkah-langkah yang digunakan
dalam
penelitian
dan
pengembangan
ini
mengacu pada sebuah model pengembangan
untuk multimedia yang dikembangkan oleh Lee
& Owen (2004:28) tentang Multimedia-Based
Instructional Design yang terdiri dari sepuluh
jenis penelitian awal, yaitu:(1) Analisis audiens,
(2) Analisis teknologi, (3) Analisis situasi, (4) Data hasil analisis kebutuhan terhadap
30 anggota UABV Universitas Negeri Malang
yang dipilih secara acak menyatakan belum
pernah mendapatkan materi tentang signal-
signal wasit bolavoli melalui handphone (mobile
learning) pada kegiatan latihan perwasitan di
UABV, sehingga anggota kegiatan latihan di
UABV mengalami kesulitan mencari media
belajar
signal-signal
perwasitan
bolavoli, 241 Nutri Puspitasari, dkk / Gelanggang Pendidikan Jasmani Indonesia 1 (2) (2017) Nutri Puspitasari, dkk / Gelanggang Pendidikan Jasmani Indonesia 1 (2) (2017) Nutri Puspitasari, dkk / Gelanggang Pendidikan Jasmani Indonesia 1 (2) (2017) multimedia interaktif. Multimedia interaktif
sangat
berguna
untuk
proses
kelancaran
pelaksanaan
pelatihan
karena
dapat
menggantikan fungsi guru itu sebagai sumber
belajar Munadi (2012). Multimedia sangat
dibutuhkan agar anggota kegiatan UABV dapat
melakukan latihan perwasitan bolavoli dengan
lancar. dapat menimbulkan motivasi belajar yang lebih
baik serta dapat berinteraksi secara langsung
(Akbar, 2013). Hasil validasi ahli terdapat beberapa
saran dan masukan yang sifatnya kecil yang
diberikan oleh ahli perwasitan bolavoli bahwa
(1) ukuran vidio yang ditampilkan diperbesar
lagi, dan (2) untuk isi materi perlu dicermati
karena banyak kalimat yang susah dipahami. Saran dan masukan yang diberikan oleh ahli
perwasitan bolavoli telah diperbaiki oleh
peneliti pada produk pengembangan media
pelatihan
signal-signal
wasit
bolavoli
menggunakan
M-Learning
untuk
anggota
UABV di Universitas Negeri Malang. Multimedia interaktif sangat dibutuhkan
dalam proses latihan karena dapat memberikan
informasi dengan cepat, luar dan luwes agar
tidak menghambat proses pelatihan. Pelatihan
signal-signal wasit bolavoli anggota UABV di
Universitas
Negeri
Malang, menggunakan
multimedia interaktif bentuk buku digital mobile
learning sebagai produk dari penelitian dan
pengembangan sebagai salah satu alat bantu
pelatihan yang efektif dan efisien. Dalam
rangka mendukung kelancaran pelatihan pada
kegiatan
latihan
perwasitan,
maka
membutuhkan media yang kreatif dan inovatif
untuk menarik peserta didik mempelajari
sebuah materi. Materi dalam kegiatan latihan
perwasitan yang diberikan kepada peserta dapat
berupa dasar utama untuk menjadi wasit (Wati,
2010) Pada
produk
pengembangan
media
pelatihan
signal-signal
wasit
bolavoli
menggunakan
M-Learning
untuk
anggota
UABV di Universitas Negeri Malang ini sudah
divalidasi oleh ahli akademisi bolavoli agar
penyusunan isi materi yang terdapat didalam
produk ini mudah untuk dipahami.Dalam
angket evaluasi ahli akademisi bolavoli terdapat
40 pertanyaan dan terdiri dari 5 indikator yaitu
kejelasan
media,
kemenarikan
media,
kesesuaian media, kemudahan media dan
kelengkapan materi. Hasil penilaian seluruh
indikator diperoleh hasil rata-rata persentase
89,37% termasuk kriteria baik sekali (Arikunto
dan Jabar, 2010:35) dan dapat digunakan tanpa
revisi. Produk hasil pengembangan berupa
media pelatihan signal-signal wasit bolavoli ini
tepat
digunakan
untuk
menambah
kete-
rampilan perwasitan bolavoli untuk anggota
UABV di Universitas Negeri Malang. Produk
yang
dihasilkan
dari
pengembangan
ini
memuat materi tentang isyarat-isyarat tangan
wasit bolavoli dan bendera hakim garis
bolavoli. Perwasitan merupakan salah satu
materi dalam latihan bolavoli di UABV yang
menarik banyak minat peserta latihan untuk
lebih
mempelajari
keterampilan
dalam
perwasitan bolavoli. Belajar akan efektif dan
menyenangkan jika individu memiliki minat
terhadap materi yang akan dipelajari. PEMBAHASAN Peneliti
mengembangkan
media
pelatihan yang akan digunakan dalam kegiatan
latihan perwasitan bolavoli. (Rusman, dkk
2012) mengatakan bahwa media sebagai salah
satu alat komunikasi dalam penyampaian pesan
sangat bermanfaat jika diimplementasikan ke
dalam
pelaksanaan
proses
pembelajaran
termasuk aktivitas fisik yang lainnya seperti
aktivitas
latihan. Namun
demikian
agar
pelaksanaan kegiatan latihan wasit bolavoli
terjadi interaksi antara pelatih dan anggota
latihan, media yang digunakan dapat berbentuk Data ketiga berdasarkan evaluasi ahli
akademisi bolavoli terhadap pengembangan
buku digital (mobile learning) tentang signal-signal 242 Nutri Puspitasari, dkk / Gelanggang Pendidikan Jasmani Indonesia 1 (2) (2017) Nutri Puspitasari, dkk / Gelanggang Pendidikan Jasmani Indonesia 1 (2) (2017) Saran dan masukan yang diberikan oleh
ahli
akademisi
bolavoli
terhadap
produk
pengembangan media pelatihan signal-signal
wasit bolavoli menggunakan M-Learning untuk
anggota UABV di Universitas Negeri Malang
yaitu (1) urutan proses isyarat kurang urut, (2)
isyarat untuk tos/undian sebelum permainan
harus ada, (3) gunakan istilah pada signal
dipoint
tertentu
harus
sesuai,
dan
(4)
keterangan isyarat: wasit I sendiri, wasit II
sendiri dan hakim garis sendiri. Pada
kegiatan
pelatihan
tentunya
dibutuhkan
media
yang
tepat
untuk
meningkatkan kemampuan ataupun keteram-
pilan peserta latihan. Keterampilan dan latihan
dapat diperkaya dengan media yang efektif dan
efisien, maka dari itu media pelatihan yang
bervariasi sangat berkaitan dengan prinsip
latihan yang bertujuan untuk meningkatkan
keterampilan perserta latihan. Prinsip variasi
adalah untuk mengatasi kebosanan dalam
latihan
karena
latihan
yang
monoton
(Budiwanto, 2012). media yang digunakan
dalam pembelajaran bermanfaat memperjelas
penyajian pesan dan informasi. Materi yang
disajikan
dengan
menggunakan
media,
pembelajaran yang tepat dan menarik maka
akan memperjelas materi yang disajikan dan g
Pembelajaran atau pelatihan bolavoli
tentang materi signal-signal wasit bolavoli
merupakan bentuk pembelajaran atau pelatihan
yang bersifat motorik yaitu peserta latihan
melakukan berbagai macam gerak tubuh
sebagai signal dari perwasitan. Bahan ajar
signal akan dapat diikuti oleh peserta didik
secara
langsung
jika
tersedia
media
pembelajaran
atau
pelatihan
yang
tepat. Munadi (2012) mengatakan bahwa jika tujuan
pembelajaran atau pelatihan bersifat motorik
seperti gerak dan aktivitas, maka media film
dan atau vidio tepat digunakan karena peserta
didik akan terlibat langsung menirukan atau
melakukan gerakan signal yang ditayangkan
oleh media. Oleh karena itu, jika berbagai 243 Nutri Puspitasari, dkk / Gelanggang Pendidikan Jasmani Indonesia 1 (2) (2017) Nutri Puspitasari, dkk / Gelanggang Pendidikan Jasmani Indonesia 1 (2) (2017) kegiatan pembelajaran atau pelatihan didukung
oleh media pembelajaran yang tepat misalnya
memanfaatkan media M-learning dalam proses
pembelajaran maka lingkungan belajar akan
lebih dinamis, tidak membosankan, benar-
benar menjadi pusat aktivitas belajar yang
maksimal
dan
memperlancar
peranannya
sebagai
pusat
dan
transformasi
materi
pembelajaran atau pelatihan. KESIMPULAN Hasil
penelitian
dan
pengembangan
dapat
disimpulkan
sebagai
berikut:
(1)
berdasarkan hasil dari evaluasi para ahli bahwa
produk pengembangan media pelatihan signal-
signal wasit bolavoli menggunakan M-Learning
untuk anggota UABV di Universitas Negeri
Malang dinyatakan memiliki ketepatan dengan
orang coba, kemenarikan dan kegunaan media
dan memiliki kesesuaian materi yang diajarkan,
(2) berdasarkan hasil evaluasi uji produk
kelompok besar dapat disimpulkan bahwa
produk pengembangan media pelatihan signal-
signal wasit bolavoli menggunakan M-Learning
untuk anggota UABV di Universitas Negeri
Malang dinyatakan memiliki kemenarikan &
kegunaan media dan kejelasan & pemahaman
materi yang diajarkan. p
j
p
Pada pengembangan media pelatihan
signal-signal wasit bolavoli menggunakan M-
Learning untuk anggota UABV di Universitas
Negeri Malang ini mengacu pada 3 penelitian
terdahulu, dengan merujuk pada kekurangan
dan
kelebihan
yang
dimiliki
produk
sebelumnya, berikut adalah kesimpulan dari 3
penelitian
terdahulu. Pertama,
penelitian
terdahulu dari Nuha, (2012) dapat ditarik
kesimpulan bahwa dari segi kekurangan produk
yang
dibuat
memiliki
kekurangan
pada
penayangan vidio signal-signal wasitnya belum
lengkap sesuai dengan yang ada dibuku
peraturan bolavoli. Kedua, penelitian terdahulu
dari Setyawan (2013) dapat ditari kesimpulan
bahwa dari segi kekurangan produk yang
dibuat memiliki kekurangan dalam peraturan
yang berkaitan dengan signal-signal belum
ditampilkan pada slide. Ketiga, penelitian
terdahulu dari Purwalingga (2014) dapat ditari
kesimpulan bahwa dari segi kekurangan produk
yang
dibuat
memiliki
kekurangan
untuk
gambar isyarat (signal) kurang jelas dan gerakan
isyarat (signal) belum memenuhi standart. DAFTAR PUSTAKA Akbar,
S. 2013. Instrumen
Perangkat
Pembelajaran. Bandung:
PT
Remaja
Rosdakarya Offset. Arikunto, S & Jabar, C. 2010. Evaluasi Program
Pendidikan. Jakarta: PT Bumi Aksara. Budiwanto, S. 2012. Metodologi
Latihan
Olahraga. Malang: Universitas
Negeri
Malang (UM PRESS). y
( g
)
Hasil
produk
pengembangan
media
pelatihan
signal-signal
wasit
bolavoli
menggunakan
M-Learning
untuk
anggota
UABV di Universitas Negeri Malang telah
melalui beberapa tahapan revisi dan memiliki
kelebihan yang dimiliki yaitu adalah (1) pada
produk pengembangan media pelatihan signal-
signal wasit bolavoli menggunakan M-Learning
untuk anggota UABV di Universitas Negeri
Malang berisi teks dan vidio yang menarik, (2)
produk media buku digital mobile learning ini
menampilkan menu pilihan yang menarik dan
memudahkan pengguna untuk mengaksesnya,
(3)
produk
dapat
diakses
menggunakan
perangkat tanpa terhubung dengan internet
(offline), seperti: handphone, smartphone, tablet,
notebook, komputer dan sebagainya sehingga
mempunyai efisiensi yang tinggi, (4) produk
media buku digital mobile learning menyajikan isi
materi 26 isyarat tangan wasit bolavoli dan 5
isyarat bendera hakim garis bolavoli yang
dilengkapi dengan vidio pada setiap materi
yang disajikan. Dwiyogo, W. D. 2008. Aplikasi Teknologi
Pembelajaran
Media
Pembelajaran
Penjas & Olahraga. Malang: Universitas
Negeri Malang. Dwiyogo,
W. D. 2010. DimensiTeknologi
Pembelajaran
Pendidikan Jasmani &
Olahraga. Malang:
Wineka
Media. Hasan, M. 2010. Pengembangan Model Mobile
Learning Sebagai Strategi Pengembangan
Elearning,
(Online).(http://www.mediapendidikan. net/index.php?option=com_content&vie
w=category&id=29&Iternid=37),
diakses tanggal 4 juli 2014. Lee,
William
&
Owens,
Diana. 2004
Multimedia-Based
Istructional
Design Computer-Based
Training Web-
Based Training
Distance
Broadcast
Training Performance-Based
Solutions Second Edition. San
Francisco: Pfeiffer. 244 Nutri Puspitasari, dkk / Gelanggang Pendidikan Jasmani Indonesia 1 (2) (2017) McQuiggan, S., Kosturki, L., McQuiggan, J.,
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Learners. New Jersey: Jonh Wiley &
Sons. Munadi, Y. 2012. Media PembelajaranSebuah
Pendekatan
Baru. Jakarta:
GaungPersada Press. PBVSI. 2015-2016. Peraturan
Permainan
Bolavoli. Jakarta: Pengurus PBVSI Pusat. Purwalingga, Y. 2014. Pengembangan Media
Pembelajaran
Berbasis
WEB
Tentang
Signal-signal
Perwasitan
bagi
Peserta
Ekstrakulikuler Bolavoli di SMK Negeri 11
Malang. FIK-UM Rusman, Kurniawan, D. & Riyana,
C. 2011. Pembelajaran
Berbasis
Teknologi Informasi
dan
Komunikasi. Jakarta: PT
Raja
Grafindo Persada. Rusman, Kurniawan, D. & Riyana,
C. 2011. Pembelajaran
Berbasis
Teknologi Informasi
dan
Komunikasi. Jakarta: PT
Raja
Grafindo Persada. C. ,
,
y
,
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Berbasis
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dan
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Raja
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dkk. 2010. Media
Pendidikan:
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Pengembangan,
dan
Pemanfaatannya. Jakarta:
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Mata
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106-22.pdf), diakses 6 April
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dan
Pemanfaatannya. Jakarta:
PT
Raja
Grafindo Persada. Sugiyono. 2013. Metode Penelitian Pendidikan. Bandung: ALFABETA. Wati,
Unik
Ambar. 2010. Pengembanga
Multimedia Pembelajaran untuk
Mata
Kuliah Pembelajaran Terpadu. Jurnal
Penelitian Ilmu
Pengetahuan. (Online),1(1),(http://www.ijpcbs.com/fi
les/2
106-22.pdf), diakses 6 April
2017. Wati,
Unik
Ambar. 2010. Pengembanga
Multimedia Pembelajaran untuk
Mata
Kuliah Pembelajaran Terpadu. Jurnal
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106-22.pdf), diakses 6 April
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English
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In Vivo Imaging Reveals Composite Coding for Diagonal Motion in the Drosophila Visual System
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PloS one
| 2,016
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cc-by
| 7,449
|
OPEN ACCESS Citation: Yue Y, Ke S, Zhou W, Chang J (2016) In
Vivo Imaging Reveals Composite Coding for
Diagonal Motion in the Drosophila Visual System. PLoS ONE 11(10): e0164020. doi:10.1371/journal. pone.0164020 Citation: Yue Y, Ke S, Zhou W, Chang J (2016) In
Vivo Imaging Reveals Composite Coding for
Diagonal Motion in the Drosophila Visual System. PLoS ONE 11(10): e0164020. doi:10.1371/journal. pone.0164020 Editor: Gregg Roman, University of Mississippi,
UNITED STATES Received: March 4, 2016
Accepted: September 19, 2016
Published: October 3, 2016 Copyright: © 2016 Yue et al. This is an open access
article distributed under the terms of the Creative
Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in
any medium, provided the original author and
source are credited. In Vivo Imaging Reveals Composite Coding
for Diagonal Motion in the Drosophila Visual
System Yuanlei Yue1,2☯, Shanshan Ke1,2☯, Wei Zhou1,2, Jin Chang1,2* 1 Britton Chance Center for Biomedical Photonics, Wuhan National Laboratory for Optoelectronics-
Huazhong University of Science and Technology, Wuhan 430074, China, 2 MoE Key Laboratory for
Biomedical Photonics, Department of Biomedical Engineering, Huazhong University of Science and
Technology, Wuhan 430074, China a1111 ☯These authors contributed equally to this work. * changjin@mail.hust.edu.cn Data Availability Statement: All relevant data are
within the paper and Supporting Information file. Data Availability Statement: All relevant data are
within the paper and Supporting Information file. Funding: This work was supported by the National
Basic Research Programme of China (Grant No. 2015CB352006), Science Fund for Creative
Research Group of China (Grant No. 61421064),
China Postdoctoral Science Foundation founded
project (2013M542010, 2015T80788),
Fundamental Research Funds for the Central
Universities/HUST (2016YXMS034, 2014TS015),
and the Director Fund of WNLO. The funders had
no role in study design, data collection and RESEARCH ARTICLE Abstract Understanding information coding is important for resolving the functions of visual neu-
ral circuits. The motion vision system is a classic model for studying information coding
as it contains a concise and complete information-processing circuit. In Drosophila, the
axon terminals of motion-detection neurons (T4 and T5) project to the lobula plate,
which comprises four regions that respond to the four cardinal directions of motion. The
lobula plate thus represents a topographic map on a transverse plane. This enables us
to study the coding of diagonal motion by investigating its response pattern. By using in
vivo two-photon calcium imaging, we found that the axon terminals of T4 and T5 cells in
the lobula plate were activated during diagonal motion. Further experiments showed
that the response to diagonal motion is distributed over the following two regions com-
pared to the cardinal directions of motion—a diagonal motion selective response region
and a non-selective response region—which overlap with the response regions of the
two vector-correlated cardinal directions of motion. Interestingly, the sizes of the non-
selective response regions are linearly correlated with the angle of the diagonal motion. These results revealed that the Drosophila visual system employs a composite coding
for diagonal motion that includes both independent coding and vector decomposition
coding. Understanding information coding is important for resolving the functions of visual neu-
ral circuits. The motion vision system is a classic model for studying information coding
as it contains a concise and complete information-processing circuit. In Drosophila, the
axon terminals of motion-detection neurons (T4 and T5) project to the lobula plate, OPEN ACCESS
Citation: Yue Y, Ke S, Zhou W, Chang J (2016) In
Vivo Imaging Reveals Composite Coding for
Diagonal Motion in the Drosophila Visual System. PLoS ONE 11(10): e0164020. doi:10.1371/journal. pone.0164020
Editor: Gregg Roman, University of Mississippi,
UNITED STATES
Received: March 4, 2016
Accepted: September 19, 2016
Published: October 3, 2016
Copyright: © 2016 Yue et al. This is an open access
article distributed under the terms of the Creative
Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in
any medium, provided the original author and
source are credited. Flies Flies were maintained on a 12:12-hour light:dark cycle at 25°C with 60% humidity on Bloom-
ington standard recipe food. Experiments were performed on 3–5-day-old female flies, with
the following genotype: Uas-GCamp5G/+; Gal4-R42F06/+. analysis, decision to publish, or preparation of the
manuscript. Competing Interests: The authors have declared
that no competing interests exist. Competing Interests: The authors have declared
that no competing interests exist. Previous studies found that there were some neurons that preferentially respond to diagonal
motions in the visual cortex of rats and cats [8–10]. Similar neurons were also observedin the
fly central brain [11]. Rodents have neurons that have robust direction selectivity to diagonal
motion, but have no discernible spatial patterns [10, 12, 13]. Currently, there are two possible
coding mechanisms for the detection of diagonal motion: (1) independent coding by segre-
gated neurons selectively responding to different directions [9, 10], or (2) vector coding of a
neuronal population, with the result a vector sum of all involved neurons [14, 15]. The Drosophila visual system is a classic model that provides excellent opportunities to
study the structures and organization of visual neural circuits and to explore neuronal signal-
processing [16–19]. The neuroanatomical architecture of the Drosophila motion vision circuit
has been well studied [7, 20]. The original motion signal is received by R1-R8 photoreceptor
cells in the retina and is then transmitted through interneurons L1 and L2 to the lamina, where
it results in distinct ON or OFF signals. Ultimately, the direction of the motion is perceived in
the T4 and T5 cells in the medulla [6, 7]. Both morphological and calcium imaging data indi-
cate that axon terminals of T4 and T5 cells project to the lobula plate and form four adjacent
layers corresponding to motion in the four cardinal directions [6, 7]. This provides an indepen-
dent topographic map that encodes cardinal directions and allows us to study the coding of
complex motion patterns, such as those seen in diagonal motion. In this study, we labeled motion-detectionneurons, T4 and T5, with the genetic calcium
probe GCamp5 and recorded the response of their axon terminals in the lobula plate to diago-
nal motions using in vivo two-photon calcium imaging in Drosophila [21]. Further, we investi-
gated the coding mode for diagonal motion in the visual system. Introduction In nature, the motion vision information is vital to living animals [1]. Previous studies have
shown that while flying, flies respond to approaching threats by executing rapid visually
directed banked turns, and generate the escape sequences that consisted of a series of diagonal
movements around yaw and roll axes [2, 3]. Thus, the visual information processing of diago-
nal motion is important for flying insects and other animals to successfully evade predators
and perform other essential movements [2, 4]. 1 / 12 PLOS ONE | DOI:10.1371/journal.pone.0164020
October 3, 2016 Composite Coding for Diagonal Motion in the Drosophila Visual System The presence of independent coding in directionally selective neurons in animals has been
previously reported. Previous studies on ganglion cells in the mammalian retina have shown
that there are only four subpopulations of neurons, each of which preferentially responds to
motion in one cardinal direction [5]. In addition, Maisak et al. and Takemura et al. have found
specific subpopulations of T4 and T5 cells in the optic lobes of flies, which are tuned to motion
in one of the four cardinal directions [6, 7]. However, how the visual system encodes diagonal
motion is still unclear. analysis, decision to publish, or preparation of the
manuscript. analysis, decision to publish, or preparation of the
manuscript. Visual stimulation The motion stimulation consisted of the movement of a grating pattern. The stimulator was
modifiedfrom a previous study [24]. The stimulator had a height of 65 mm and a diameter of
145 mm, and was constructed using 14 pieces of modular LED panels (SUNSCOB, China). Each panel contains 8 × 8 individual LEDs. The emission wavelength was 590 nm, the half-
width of the peak was 14 nm. The display spanned 180° in azimuth and 53° in elevation while
the fly was positioned at the center. The speed of the movement of the grating pattern was 1 Hz
(bars moving at 26.5° per second). For local field stimulation, we used an eighth of the left 100°
of the visual field in azimuth. We applied the following sequence of stimulation patterns: first,
bars staying still for 15 seconds; then bars moving for 4 seconds; at the end, bars stopping and
staying still for 20 seconds. Fly preparation A modifiedmethod was applied to prepare samples for analysis [22, 23]. Briefly, flies were
anesthetized on ice and then fixed on a custom holder with cyanoacrylateglue (Pattex, Ger-
many). The brain of the fly was bent anteriorly by approximately 85° to expose the posterior
surface. After fixing the fly on the holder, the legs and wings of the fly remained free moving. The posterior of the brain was exposed, making microscopic surgery convenient. To avoid
brain vibration, we removed the M16 muscle and fixed the extended proboscis with glue. In
HL-3 saline (70 mM NaCl, 5 mM KCl, 4 mM MgCl2, 10 mM NaHCO3, 115 mM sucrose, 5
mM Trehalose, 5 mM triethylsilane, 1.5 mM CaCl2), a small window was cut to expose the tar-
geted neurons using sharpened #5 forceps. This approach allowed us to image the T4 and T5
cells on the transverse plane of the lobula plate. 2 / 12 PLOS ONE | DOI:10.1371/journal.pone.0164020
October 3, 2016 Composite Coding for Diagonal Motion in the Drosophila Visual System Image acquisition and processing We obtained images using a two-photon microscope (Zeiss LSM 780NLS, Germany) with a
water immersion objective (20×, NA = 1.0). We used an excitation wavelength of 920 nm from
a femtosecond pulse laser (Maitai HP from Spectra-Physics, USA). Images were analyzed using
a custom-written program (Matlab, USA). Raw images were initially smoothed using a Gauss-
ian filter (HSIZE = [10 10], SIGMA = 5). The relative fluorescence change was calculated using
the formula ΔF/F = (Fstim−F0) / F0, in which Fstim was obtained by averaging all images during
the grating movement, and F0 was obtained by averaging all images taken during the two sec-
onds before stimulation. ΔF/F images were normalized to their maximum value. The ΔF/F values of each pixel were coded in pseudo-color images (0–255, blue to red) in
Figs 1C, 1D, 2A, 3D, 4A, and 5B. A threshold (25%) was then used to determine the contour of
the activated region on a topographic map, as previously reported [6]. The analysis below is all
based on this threshold. For the data shown in Figs 1E, 3A, 4B, 5D and 5F, pixels were assigned
to the activated regions providing ΔF/F values above the threshold. Otherwise,pixels were
assigned to background. The intensity of the response for each direction is defined as the maxi-
mum ΔF/F value during the movement of the grating pattern. The sizes of the activated regions
were analyzed using a two-sample t-test and the intensities of the activated regions were ana-
lyzed using the Mann-Whitney U-test. Results We investigated information coding for visual motion directions in Drosophila. The calcium
indicator GCaMP5 was specificallyexpressed in T4 and T5 cells using the Gal4/UAS system
[10, 18]. In vivo two-photon imaging technology was used to record calcium signals from the
axon terminals of T4 and T5 cells in the lobula plate (Fig 1A and 1B). During the motion of the
grating pattern in the four cardinal directions (upward, downward, frontward, and backward),
the axon terminals of the T4 and T5 cells were activated in specific sublayers of the lobula plate
(Fig 1C), forming a topographic map for the cardinal directions of motion, as previously
reported [10]. To determine whether diagonal directions also have a topographic map on the
lobula plate, four diagonal movements of the grating pattern (45° backward to upward, 45°
frontward to upward, 45° backward to downward, and 45° frontward to downward) were
applied as visual stimuli. We observeddistinct activated regions in the lobula plate for different
diagonal motions (Fig 1D). To clarify the relationship between the two response patterns resulting from diagonal and
cardinal directions of motion, we first compared the location of the resulting activated regions. For example, in response to the 45° frontward to upward motion (Fig 1D), the regions activated
during the diagonal motion do not completely overlap with those activated during the cardinal 3 / 12 PLOS ONE | DOI:10.1371/journal.pone.0164020
October 3, 2016 Composite Coding for Diagonal Motion in the Drosophila Visual System Fig 1. Diagonal motion activates selective response regions and non-selective response reg
the lobula plate. (A) The schematic drawing shows the projections of T4 and T5 cells and their axo
terminals in the lobula plate (LP). (B) Expression pattern of Gla4-R42F06 in the lobula plate. T4 and
are labeled with GFP and the neuropil is labeled by Mouse-anti-nc82 (red). Scale bar: 20 μm. (C)
Topographic maps of T4 and T5 axon terminals responding to motion in the four cardinal directions
indicate the directions). (D) Topographic maps of T4 and T5 axon terminals responding to motion in
45˚ diagonal directions. (E) Diagonal motion (45˚ frontward to upward) activates selective response
regions (red area in the right panel) and non-selective response (nSR) regions (yellow area in the rig
panel). These regions are compared to those responding to the four cardinal directions of motion (G
area in the middle panel). (F) The proportions of the two region types activated by diagonal motion. Results Statistical significance was assessed using the
Mann-Whitney U-test. Scale bar: 10 μm. significance was assessed using the two-sample t-test, p < 0.001. n = 15. (G) Intensities of responses for
diagonal motions and cardinal directions of motion (colors represent areas selective to the indicated
directions). n = 15. Data are presented as the mean ± SEM. Statistical significance was assessed using the
Mann-Whitney U-test. Scale bar: 10 μm. doi:10.1371/journal.pone.0164020.g001 directions of motion. These regions are divided into two groups: 1) a diagonal motion selective
response (SR) region (red area in the merged image of Fig 1E), and 2) a diagonal motion non-
selective response (nSR) region (yellow area in the merged image of Fig 1E) that overlaps with
the response regions of the cardinal directions of motion. The same pattern of activation is also
found in response to the other 45° diagonal motions. Our analysis reveals that the nSR region
occupies about two-thirds of the activated region for 45° diagonal motion (Fig 1F). We also
compared the intensities of the regions activated in response to diagonal and cardinal direc-
tions of motion. The results indicate that there were no significant differences in the intensities
of these regions (Fig 1G). Taken together, our data indicate that two types of response regions
are involved in the coding of diagonal motion in Drosophila. The distributions of the activated
regions also imply that there is a novel response pattern for diagonal motion in the lobula plate
of Drosophila. We investigated the characteristics of the diagonal motion SR regions. Our results indicate
that those regions activated by each diagonal motion distribute at the same layer with different
locations among individual flies (Fig 2A and 2B). We also found that diagonal motion SR
regions activated in response to the four 45° diagonal motions were separate from each other
(Fig 2C), only responded to one diagonal motion, and were silent during motion in other Fig 2. Locations and responses of the SR regions to diagonal motion. (A) Topographic maps of the response
to the 45˚ backward to downward diagonal motion obtained from three flies. (B) Compound representation of the
three SR regions in (A). The yellow, magenta, and cyan circles indicate the SR regions responding to the 45˚
backward to downward motion in three flies. (C) Compound representation of the SR regions responding to motion
in the four 45˚ diagonal directions. Arrows indicate directions. Results Fig 1. Diagonal motion activates selective response regions and non-selective response regions in
the lobula plate. (A) The schematic drawing shows the projections of T4 and T5 cells and their axon
terminals in the lobula plate (LP). (B) Expression pattern of Gla4-R42F06 in the lobula plate. T4 and T5 cells
are labeled with GFP and the neuropil is labeled by Mouse-anti-nc82 (red). Scale bar: 20 μm. (C)
Topographic maps of T4 and T5 axon terminals responding to motion in the four cardinal directions (arrows
indicate the directions). (D) Topographic maps of T4 and T5 axon terminals responding to motion in the four
45˚ diagonal directions. (E) Diagonal motion (45˚ frontward to upward) activates selective response (SR)
regions (red area in the right panel) and non-selective response (nSR) regions (yellow area in the right
panel). These regions are compared to those responding to the four cardinal directions of motion (Green
area in the middle panel). (F) The proportions of the two region types activated by diagonal motion. Statistica Fig 1. Diagonal motion activates selective response regions and non-selective response regions in
the lobula plate. (A) The schematic drawing shows the projections of T4 and T5 cells and their axon
terminals in the lobula plate (LP). (B) Expression pattern of Gla4-R42F06 in the lobula plate. T4 and T5 cells
are labeled with GFP and the neuropil is labeled by Mouse-anti-nc82 (red). Scale bar: 20 μm. (C)
Topographic maps of T4 and T5 axon terminals responding to motion in the four cardinal directions (arrows
indicate the directions). (D) Topographic maps of T4 and T5 axon terminals responding to motion in the four
45˚ diagonal directions. (E) Diagonal motion (45˚ frontward to upward) activates selective response (SR)
regions (red area in the right panel) and non-selective response (nSR) regions (yellow area in the right
panel). These regions are compared to those responding to the four cardinal directions of motion (Green
area in the middle panel). (F) The proportions of the two region types activated by diagonal motion. Statistical 4 / 12 PLOS ONE | DOI:10.1371/journal.pone.0164020
October 3, 2016 Composite Coding for Diagonal Motion in the Drosophila Visual System significance was assessed using the two-sample t-test, p < 0.001. n = 15. (G) Intensities of responses for
diagonal motions and cardinal directions of motion (colors represent areas selective to the indicated
directions). n = 15. Data are presented as the mean ± SEM. Results (D) Calcium transients from the four regions indicated
in (C). Arrows indicate the direction of grating motion. (E) Intensities of the responses to the diagonal motions in SR
regions. Data are presented as the mean ± SEM. Statistical significance was assessed using the Mann-Whitney U-
test. n = 15. Scale bar: 10 μm. doi:10.1371/journal.pone.0164020.g002 Fig 2. Locations and responses of the SR regions to diagonal motion. (A) Topographic maps of the response
to the 45˚ backward to downward diagonal motion obtained from three flies. (B) Compound representation of the
three SR regions in (A). The yellow, magenta, and cyan circles indicate the SR regions responding to the 45˚
backward to downward motion in three flies. (C) Compound representation of the SR regions responding to motion
in the four 45˚ diagonal directions. Arrows indicate directions. (D) Calcium transients from the four regions indicated
in (C). Arrows indicate the direction of grating motion. (E) Intensities of the responses to the diagonal motions in SR
regions. Data are presented as the mean ± SEM. Statistical significance was assessed using the Mann-Whitney U-
test. n = 15. Scale bar: 10 μm. doi:10 1371/journal pone 0164020 g002 doi:10.1371/journal.pone.0164020.g002 5 / 12 PLOS ONE | DOI:10.1371/journal.pone.0164020
October 3, 2016 Composite Coding for Diagonal Motion in the Drosophila Visual System Fig 3. Regions activated in response to motion in the two vector-correlated cardinal directions combined to code the
diagonal motion. (A) Compound display of the regions activated in response to diagonal motion (45˚ backward to downward) and
motion in the four cardinal directions. Red and green represent regions activated by motion in the diagonal and cardinal directions,
respectively; yellow represents the nSR regions that respond to both diagonal and cardinal. (B) The sizes of nSR regions activated by
diagonal motion and the four cardinal directions of motion. Data are presented as the mean ± SEM. Statistical significance was
assessed using the two-sample t-test. n = 15. (C) Intensities of responses in two nSR regions of 45˚ backward to downward diagonal
motion. The blue circle represents the nSR region activated by this diagonal and backward motions, the green circle represents the
nSR region activated by this diagonal and downward motions. Data are presented as the mean ± SEM. Statistical significance was
assessed using the Mann-Whitney U-test, p = 0.683. n = 15. Results (D) Topographic maps of responses to frontward, 45˚ frontward to
downward, and 45˚ frontward to upward motions. (E) The nSR regions activated by 45˚ frontward to downward and 45˚ frontward to
upward are separate in a region activated by frontward motion alone. Gray areas represent the regions activated by frontward
motion. Color circles represent the nSR regions responding to frontward and diagonal motions. Scale bar: 10 μm. Fig 3. Regions activated in response to motion in the two vector-correlated cardinal directions combined to code the
diagonal motion. (A) Compound display of the regions activated in response to diagonal motion (45˚ backward to downward) and
motion in the four cardinal directions. Red and green represent regions activated by motion in the diagonal and cardinal directions,
respectively; yellow represents the nSR regions that respond to both diagonal and cardinal. (B) The sizes of nSR regions activated by
diagonal motion and the four cardinal directions of motion. Data are presented as the mean ± SEM. Statistical significance was
assessed using the two-sample t-test. n = 15. (C) Intensities of responses in two nSR regions of 45˚ backward to downward diagonal
motion. The blue circle represents the nSR region activated by this diagonal and backward motions, the green circle represents the
nSR region activated by this diagonal and downward motions. Data are presented as the mean ± SEM. Statistical significance was
assessed using the Mann-Whitney U-test, p = 0.683. n = 15. (D) Topographic maps of responses to frontward, 45˚ frontward to
downward, and 45˚ frontward to upward motions. (E) The nSR regions activated by 45˚ frontward to downward and 45˚ frontward to
upward are separate in a region activated by frontward motion alone. Gray areas represent the regions activated by frontward
motion. Color circles represent the nSR regions responding to frontward and diagonal motions. Scale bar: 10 μm. directions (Fig 2D). In addition, the intensities of these regions were not significantly different
(Fig 2E). These results indicate that the diagonal motion SR regions could contribute to the
detection of the direction of diagonal motion. Our data also imply that diagonal motion has an
independent coding mode in motion-detectionneurons in flies. In order to investigate the characteristics of the diagonal motion nSR regions, we analyzed
their sizes, the intensities of their responses, and their composite features. PLOS ONE | DOI:10.1371/journal.pone.0164020
October 3, 2016 Results First, we compared
the sizes of nSR regions between each diagonal motion and the four cardinal directions of
motion. For example, the regions activated by 45° backward to downward motion overlapped
with those regions activated by backward (34.8 ± 5.7%) and downward (48.1 ± 5.2%) motions. However, these regions rarely overlapped with those activated by upward (5.6 ± 1.5%) and
frontward (3.0 ± 1.0%) motions (Fig 3A). Statistical analysis of responses to the other three
diagonal motions (45° backward to upward, 45° frontward to upward and 45° frontward to
downward) revealed that the nSR regions activated by one diagonal motion mainly overlap
with regions activated by the two vector-correlated cardinal directions of motion (Fig 3B). We
then analyzed the intensities of the responses in the nSR regions and found that there were no
significant differences between responses to diagonal and cardinal motions (Fig 3C). These PLOS ONE | DOI:10.1371/journal.pone.0164020
October 3, 2016 6 / 12 Composite Coding for Diagonal Motion in the Drosophila Visual System Fig 4. Diagonal motions are coded by the sizes of nSR regions. (A) Topographic maps of T4 and T5 axon terminals responding to
motion in the four cardinal directions, and 22.5˚ backward to upward and 67.5˚ backward to upward motions (arrows indicate directions). (B) Diagonal motions of 22.5˚ and 67.5˚ backward to upward activate SR regions (red area) and nSR regions (yellow area). Regions
responding to diagonal motion are compared to those responding to the four cardinal directions of motion (green area). (C) nSR regions
of diagonal motion along different angles. (D) The sizes of the nSR regions responding to backward motion and the three backward to
upward diagonal motions. (E) The sizes of the nSR regions activated by upward motion and the three backward to upward diagonal
motions. Data are presented as the mean ± SEM. Statistical significance was assessed using the two-sample t-test, * p < 0.05, **
p < 0.01, *** p < 0.001. n = 15. Scale bar: 10 μm. doi:10 1371/journal pone 0164020 g004 Fig 4. Diagonal motions are coded by the sizes of nSR regions. (A) Topographic maps of T4 and T5 axon terminals responding to
motion in the four cardinal directions, and 22.5˚ backward to upward and 67.5˚ backward to upward motions (arrows indicate directions). (B) Diagonal motions of 22.5˚ and 67.5˚ backward to upward activate SR regions (red area) and nSR regions (yellow area). PLOS ONE | DOI:10.1371/journal.pone.0164020
October 3, 2016 Results Regions
responding to diagonal motion are compared to those responding to the four cardinal directions of motion (green area). (C) nSR regions
of diagonal motion along different angles. (D) The sizes of the nSR regions responding to backward motion and the three backward to
upward diagonal motions. (E) The sizes of the nSR regions activated by upward motion and the three backward to upward diagonal
motions. Data are presented as the mean ± SEM. Statistical significance was assessed using the two-sample t-test, * p < 0.05, **
p < 0.01, *** p < 0.001. n = 15. Scale bar: 10 μm. doi:10.1371/journal.pone.0164020.g004 doi:10.1371/journal.pone.0164020.g004 doi:10.1371/journal.pone.0164020.g004 results suggest that the coding of diagonal motions in the nSR region was closely related to that
of the two vector-correlated cardinal directions. Next, to further study the regional coding of
motion, we compared the nSR regions activated by one cardinal direction of motion (front-
ward) and two orthogonal diagonal motions (45° frontward to upward and 45° frontward to
downward). The results indicate that the nSR regions responding to frontward motion and 45°
frontward to upward are completely separate from the regions responding to frontward motion
and 45° frontward to downward motion (Fig 3D and 3E). These data suggest that orthogonal
diagonal motions are coded by distinct parts of the regions activated by motion in the same
cardinal direction. To investigate whether the above rule also applies to other diagonal directions, we per-
formed the same analysis for other diagonal directions (by changing the angle between the
backward and upward movements to 22.5° and 67.5°, respectively) (Fig 4A). Our results indi-
cated that the coding patterns were the same for motion along those diagonal directions, as
they all included SR and nSR regions (Fig 4B). However, the sizes of the nSR regions were line-
arly correlated with the diagonal angle (22.5°: 0.69 ± 0.04, 45°: 0.47 ± 0.04, 67.5°: 0.37 ± 0.04; y
= -0.16x + 0.83, R2 = 0.34) (Fig 4C and 4D). We reached the same conclusion regarding the
nSR regions responding to upward motion and the three diagonal motions (Fig 4E). Therefore,
we suggest that the angles of the different diagonal motions are coded by the sizes of the nSR
regions. 7 / 12 PLOS ONE | DOI:10.1371/journal.pone.0164020
October 3, 2016 Composite Coding for Diagonal Motion in the Drosophila Visual System Fig 5. Response pattern of diagonal motion from local field stimulus. (A) Diagram of stimulation of local field (about 25˚ in
azimuth and elevation). (B) Topographic maps of T4 and T5 axon terminals responding to motion in the four cardinal directions and
45˚ frontward to upward diagonal direction. (C) Intensities of responses in regions activated by 45˚ frontward to upward diagonal
motion and cardinal directions of motion. (D) A comparison of regions responding to movement in 45˚ frontward to upward diagonal
motion to those responding to movement in the four cardinal directions. doi:10.1371/journal.pone.0164020.g004 (G) The
sizes of nSR regions between those activated by diagonal motion and motion in the four cardinal directions. (H), (J) Calcium
transients from the SR regions (H) of 45˚ frontward to upward diagonal motion and nSR regions (J) of 45˚ frontward to upward
diagonal motion and frontward motion. (I), (K) Responses in SR (I) and nSR (K) regions of 45˚ frontward to upward diagonal
motion. The blue circle in (K) represents the nSR region activated by diagonal and upward motion, the green circles in (K) are the
responses of the nSR region activated by diagonal and frontward motion. Relative fluorescence changes were normalized to the
maximum response before averaging. Data are presented as the mean ± SEM. n = 6. Scale bar: 10 μm. Fig 5. Response pattern of diagonal motion from local field stimulus. (A) Diagram of stimulation of local field (about 25˚ in
azimuth and elevation). (B) Topographic maps of T4 and T5 axon terminals responding to motion in the four cardinal directions and
45˚ frontward to upward diagonal direction. (C) Intensities of responses in regions activated by 45˚ frontward to upward diagonal
motion and cardinal directions of motion. (D) A comparison of regions responding to movement in 45˚ frontward to upward diagonal
motion to those responding to movement in the four cardinal directions. Diagonal motion of 45˚ frontward to upward activate SR
regions (red area in the merge image) and nSR regions (yellow area in the merge image). (E) The proportions of the SR and nSR
regions for diagonal motion. (F) Compound display of the regions activated by diagonal motion (45˚ frontward to upward) and
motion in the four cardinal directions of motion. Red and green regions represent regions activated by diagonal and cardinal
directions of motion, respectively; yellow represents the nSR regions between diagonal and cardinal directions of motions. (G) The
sizes of nSR regions between those activated by diagonal motion and motion in the four cardinal directions. (H), (J) Calcium
transients from the SR regions (H) of 45˚ frontward to upward diagonal motion and nSR regions (J) of 45˚ frontward to upward
diagonal motion and frontward motion. (I), (K) Responses in SR (I) and nSR (K) regions of 45˚ frontward to upward diagonal
motion. doi:10.1371/journal.pone.0164020.g004 Diagonal motion of 45˚ frontward to upward activate SR
regions (red area in the merge image) and nSR regions (yellow area in the merge image). (E) The proportions of the SR and nSR
regions for diagonal motion. (F) Compound display of the regions activated by diagonal motion (45˚ frontward to upward) and
motion in the four cardinal directions of motion. Red and green regions represent regions activated by diagonal and cardinal
directions of motion, respectively; yellow represents the nSR regions between diagonal and cardinal directions of motions. (G) The
sizes of nSR regions between those activated by diagonal motion and motion in the four cardinal directions. (H), (J) Calcium
transients from the SR regions (H) of 45˚ frontward to upward diagonal motion and nSR regions (J) of 45˚ frontward to upward
diagonal motion and frontward motion. (I), (K) Responses in SR (I) and nSR (K) regions of 45˚ frontward to upward diagonal
motion. The blue circle in (K) represents the nSR region activated by diagonal and upward motion, the green circles in (K) are the
responses of the nSR region activated by diagonal and frontward motion. Relative fluorescence changes were normalized to the
maximum response before averaging. Data are presented as the mean ± SEM. n = 6. Scale bar: 10 μm. doi:10 1371/journal pone 0164020 g005 Fig 5. Response pattern of diagonal motion from local field stimulus. (A) Diagram of stimulation of local field (about 25˚ in
azimuth and elevation). (B) Topographic maps of T4 and T5 axon terminals responding to motion in the four cardinal directions and
45˚ frontward to upward diagonal direction. (C) Intensities of responses in regions activated by 45˚ frontward to upward diagonal
motion and cardinal directions of motion. (D) A comparison of regions responding to movement in 45˚ frontward to upward diagonal
motion to those responding to movement in the four cardinal directions. Diagonal motion of 45˚ frontward to upward activate SR
regions (red area in the merge image) and nSR regions (yellow area in the merge image). (E) The proportions of the SR and nSR
regions for diagonal motion. (F) Compound display of the regions activated by diagonal motion (45˚ frontward to upward) and
motion in the four cardinal directions of motion. Red and green regions represent regions activated by diagonal and cardinal
directions of motion, respectively; yellow represents the nSR regions between diagonal and cardinal directions of motions. Discussion Investigating responses to diagonal motion in the visual system is a good model for the study
of information coding in neuronal circuits. In this study, we demonstrated the presence of a
novel topographic map for coding diagonal motion in the motion-detection neurons of Dro-
sophila. Specifically, we report that flies encode diagonal motions using regional composite
coding. Thus, an SR region responds to diagonal motion, while an nSR region is involved in
vector coding. In this work, the results from calcium imaging reveal the SR regions activated by diagonal
motion on the axon terminal of T4/T5 neurons. It suggests the function of T4/T5 neurons is
diverse, some of them can detect cardinal motions, and the others are preferred to sense diago-
nal motions. This property of diversity is consistent with the previous structural study on T4
and T5 motion detection circuit. 3D reconstruction of images from series-sectionelectron
microscopy indicated that single T4 or T5 neurons receive input from multiple upstream neu-
rons in multiple columns (T4 input from Mi1, Tm3, L5, C2 and C3; T5 input from spatially
segregated Tm1, Tm2, Tm4, and Tm9), and also showed that the receptive field of individual
T4 and T5 neurons covers multiple ommatidia, respectively [7, 25]. Furthermore, it is also sup-
ported by a recent functional study that shows single T4 neurons preferentially response to
diagonal motion [26]. Thus, it suggests that diagonal motion could be encoded by an indepen-
dent neural circuit. Although lobula plate tangential cells have been recognizedas the down-
stream neurons of T4 and T5 [27, 28], recent studies have indicated that there are other types
of neurons in the lobula plate. These neurons include lobula plate intrinsic interneurons and
Hx neurons, which can receive information regarding the direction of visual motion [29, 30]. These results imply that there may be several neuronal circuits for the detection of visual
motion in the lobula plate. The overlap of the region activated by diagonal motion with the regions activated by two
vector-correlated cardinal motions (Fig 3 and Fig 4) supports the population vector hypothesis
for motion information coding [14, 31]. This hypothesis considers each neuron to be a vector
and assumes that the direction is coded by the vector sum of the response of all neurons. Our
results suggest that vector coding is involved in the detection of diagonal motion in fly. doi:10.1371/journal.pone.0164020.g004 The blue circle in (K) represents the nSR region activated by diagonal and upward motion, the green circles in (K) are the
responses of the nSR region activated by diagonal and frontward motion. Relative fluorescence changes were normalized to the
maximum response before averaging. Data are presented as the mean ± SEM. n = 6. Scale bar: 10 μm. To verify the coding features of diagonal motion, we applied a local field stimulus of 25° in
azimuth and elevation (Fig 5A). We recorded the response to four cardinal and four diagonal PLOS ONE | DOI:10.1371/journal.pone.0164020
October 3, 2016 8 / 12 Composite Coding for Diagonal Motion in the Drosophila Visual System motions (45° backward to upward, 45° frontward to upward, 45° backward to downward, and
45° frontward to downward) (Fig 5B and 5C). Regions activated by diagonal motion in a local
field stimulus also demonstrated an SR region and an nSR region, similar to a wide field stimu-
lus (Fig 5D–5K). The response patterns from both wide field and local field stimuli suggest that
the Drosophila visual system employs regional composite coding to detect diagonal motion. PLOS ONE | DOI:10.1371/journal.pone.0164020
October 3, 2016 Supporting Information S1 File. Manuscript data. Data used in preparation of Figs 1F, 1G; 2E; 3B, 3C; 4D, 4E; 5C, 5E,
5G, 5I and 5K. S1 File. Manuscript data. Data used in preparation of Figs 1F, 1G; 2E; 3B, 3C; 4D, 4E; 5C, 5E,
5G, 5I and 5K. (XLSX) Writing – review& editing: JC WZ SK YY. Writing – review& editing: JC WZ SK YY. Acknowledgments Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were
used in this study. We thank the Optical Bioimaging Core Facility of WNLO-HUST for imag-
ing system assistance. We also thank the anonymous reviewers for their helpful and construc-
tive comments. Discussion Here, we reveal a novel pattern for the coding of diagonal motions by the axon terminals of
T4 and T5 neurons in the lobula plate. We also report that the different response regions (SR
and nSR regions) show differential preferences for motion in different diagonal directions. Direction and orientation are essential information for motion detection in animals. A recent
report also indicated that individual T4 and T5 axon terminals display prominent orientation
selectivity, and that this selectivity sharpens directional tuning via surround inhibition [29]. The co-extraction of orientation and direction information by T4 and T5 neurons may repre-
sent the first order of processing in the visual circuit. The preference and selectivity of the T4
and T5 axons may conform to a retinotopic organization, which is universal in the visual sys-
tems of insects, mammals, and primates [8, 11, 32]. Our study suggests that regional composite coding, which may combine independent cod-
ing with vector coding in neural circuits, processes diagonal directional information. It is PLOS ONE | DOI:10.1371/journal.pone.0164020
October 3, 2016 9 / 12 Composite Coding for Diagonal Motion in the Drosophila Visual System consistent with the efficiencyof the nervous system, where a small number of neurons code for
motion in a wide number of directions. consistent with the efficiencyof the nervous system, where a small number of neurons code for
motion in a wide number of directions. Writing – original draft: JC WZ SK YY. Writing – original draft: JC WZ SK YY. Methodology:WZ YY. Resources: WZ JC. Supervision:JC WZ. Visualization: JC WZ YY SK. Conceptualization: JC WZ. Formal analysis: YY SK. Funding acquisition: WZ JC. Investigation: YY. Methodology:WZ YY. Visualization: JC WZ YY SK. Writing – original draft: JC WZ SK YY. PLOS ONE | DOI:10.1371/journal.pone.0164020
October 3, 2016 References 1. Hupe JM, Rubin N. The oblique plaid effect. Vision Res. 2004; 44(5):489–500. doi: 10.1016/j.visres. 2003.07.013 PMID: 14680775 2. Muijres FT, Elzinga MJ, Melis JM, Dickinson MH. Flies evade looming targets by executing rapid visu-
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October 3, 2016 Composite Coding for Diagonal Motion in the Drosophila Visual System 32. Kremkow J, Jin J, Wang Y, Alonso JM. Principles underlying sensory map topography in primary visual
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cortex. Nature. 2016; 533(7601):52–57. doi: 10.1038/nature17936 PMID: 27120164 12 / 12
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O niepołączalności ról procesowych organów jednostek samorządu terytorialnego
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1 Tą intencją kierowano się, ustalając redakcję chociażby nieobowiązującego już art. 15 ust. 2
ustawy z 18 lipca 2001 r. – Prawo wodne (t.jedn.: Dz. U. 2017, poz. 1121, utraciła moc – Dz. U.
2017, poz. 1566). Przewidział on wszczęcie postępowania w przedmiocie ustalenia linii brzegu
dla cieków naturalnych, jezior i innych naturalnych zbiorników wodnych nie tylko na wniosek
podmiotu dysponującego w sprawie interesem prawnym, lecz również interesem faktycznym. Po
dobnie trzeba ocenić nakaz uwzględniania przez organy administracji interesów osób trzecich, na
które wynik postępowania ma bezpośredni wpływ, w trybie art. 7a § 1 in fine i art. 81a § 2 pkt 1
in fine ustawy z 14 czerwca 1960 r. – Kodeks postępowania administracyjnego, t.jedn.: – Dz. U.
2018, poz. 2096 ze zm. (dalej jako: k.p.a. lub kodeks). ZBIGNIEW KMIECIAK ZBIGNIEW KMIECIAK RUCH PRAWNICZY, EKONOMICZNY I SOCJOLOGICZNY
Rok LXXXII – zeszyt 4 – 2020 RUCH PRAWNICZY, EKONOMICZNY I SOCJOLOGICZNY
Rok LXXXII – zeszyt 4 – 2020 I. ZŁOŻONOŚĆ UKŁADU INTERESÓW W POSTĘPOWANIU
ADMINISTRACYJNYM W nauce prawa i postępowania administracyjnego tradycyjnie operuje się,
przynajmniej na poziomie ogólnych rozważań teoretycznych, prostym sche
matem odzwierciedlającym rozłączność ról procesowych podmiotów uczestni
czących w jakikolwiek sposób w czynnościach zmierzających do załatwienia
sprawy albo tylko umocowanych (legitymowanych) do udziału w nich. Wyróż
nia się zatem zawsze organ prowadzący postępowanie jako gospodarza proce
su oraz stronę lub strony postępowania. Poza nimi przepisy prawa stwarzają
podstawę do partycypacji w postępowaniu innych podmiotów. Są nimi w szcze
gólności organy zajmujące stanowisko w trybie współdziałania oraz podmioty
na prawach strony. Ten czytelny i przyjmowany do niedawna bez zastrzeżeń
podział skomplikował się nieco za sprawą rozwoju różnych koncepcji stakehol
ders, zakładających potrzebę dopuszczenia do postępowania w odpowiednich
formach podmiotów, które mogą być dotknięte skutkami rozstrzygnięcia, bądź
zainteresowanych wynikiem działań z innych powodów. Idea ta, korespon
dująca z kluczowym założeniem postępowań typu notice-and-comment, stała
się także źródłem inspiracji do poczynań prawodawcy związanych z regula
cją postępowań jurysdykcyjnych. Doskonałą ilustracją sformułowanej tezy są
pojawiające się w naszym ustawodawstwie przepisy mające chronić interesy
faktyczne jednostki1. Używając pojęcia stakeholders, uznaje się istnienie stosownej dywersy
fikacji preferencji, celów i interesów oznaczanych w ten sposób podmiotów:
poszczególnych jednostek, środowisk i społeczności lokalnych, organizacji https://doi.org/10.14746/rpeis.2020.82.4.7 84 Zbigniew Kmieciak pozarządowych, sektora prywatnego oraz władzy publicznej. W publikacjach
naukowych można nawet spotkać się z określeniem multi-stakeholders i kom
plementarnymi wobec niego terminami: multi-stakeholder-processing/multi
-stakeholder processes for governance, multiple viewpoints of different stake-
holders oraz multi-stakeholder dialogues and partnerships2. Identyfikacja
wskazanych pojęć i – co się z tym łączy – wyodrębnienie aktorów pierwszo-
i drugoplanowych oraz pozostałych, a z drugiej strony – tych, którzy wspierają
dane przedsięwzięcie lub są jemu przeciwni, pozwala wypracować adekwatny
do sytuacji sposób działania. W klasycznej teorii przedsiębiorstw, a w kon
sekwencji – teorii governance, w ramach kategorii stakeholders wyróżnia się
jeszcze grupę podmiotów mających bezpośredni wpływ na bieg spraw (głos
decydujący), traktowanych jako udziałowcy albo legitymowani do udziału
w ukształtowaniu pewnego rozwiązania, czyli shareholders. Nie oznacza to
oczywiście, że zapatrywania innych podmiotów są pomijane lub deprecjonowa
ne na etapie dochodzenia do pożądanego rezultatu3. Zaprezentowane spostrzeżenia prowadzą do wniosku, że realizowane
współcześnie zasady wypełniania zadań publicznych, uwarunkowane m.in. chęcią zapewnienia ekonomicznej efektywności oraz optymalnej alokacji za
sobów i środków pozostających w dyspozycji państwa, wyłamują się z tra
dycyjnych, stworzonych przed laty z myślą o ochronie praw podmiotowych,
konstrukcji prawa procesowego4. 4 Według O. Bähra całość prawa publicznego, w szczególności zaś prawa administracyjnego,
miała zostać zmodernizowana przez odwołanie się do pojęć i koncepcji prawa prywatnego oraz
procedury cywilnej. Autor postrzegał państwo jako część społeczeństwa, wyrażając przekonanie,
że tak jak jednostka powinno ono podlegać jurysdykcji sądów powszechnych. Stworzoną przez
niego teorię rozwinął O. Mayer, głosząc pogląd, że urzeczywistnienie idei państwa prawnego może
nastąpić tylko w razie przyjęcia za jego podstawę konstrukcji publicznych praw podmiotowych,
określającej stosunki w obrębie prawa publicznego – por. wywody Zoller (2008): 80–81, oraz Kün
necke (2007): 22 n. 5 Pojęcie „aktorzy postępowania” utrwalone w obcym piśmiennictwie zdaje się lepiej uka
zywać różnorodność pełnionych przez nie ról niż słowo „podmioty”, którym posługują się polscy
autorzy. Używane jest ono również w rozmaitych kontekstach, np. Blake (2011: 244) pisze o wnio
skodawcy (stronie) nadużywającym swoich praw procesowych jako „złym aktorze” (applicant is
a bad actor). 3 Kmieciak (2017a): 29–30. 2 Zob. Priddat (2008): 352 n.; Pollitt, Bouckaert (2011): 220, oraz Hemmati et al. (2002): 1 n.
Co do definicji słowa „stakeholders” zob. Supernat (2005): 515–516, oraz Garner (2009): 1534.
3 K
i
i k (
) I. ZŁOŻONOŚĆ UKŁADU INTERESÓW W POSTĘPOWANIU
ADMINISTRACYJNYM Pojęcie strony postępowania administra
cyjnego i podmiotów na prawach strony już nie wystarcza dla uformowania
proceduralnych mechanizmów wyważania wszystkich, uwikłanych w pewną
sprawę interesów. Samo zdefiniowanie relacji pomiędzy nimi i ustalenie ich
prawnej kwalifikacji oraz sprecyzowanie ról procesowych odgrywanych przez
poszczególnych „aktorów”, zwłaszcza w układzie zależności: organ prowadzący
postępowanie – strona, nastręcza w wielu przypadkach niemało trudności5. Równie poważne dylematy wyłaniają się na tle analizy innego rodzaju związ
ków, których istotę da się przedstawić, stawiając pytanie: czy organ upoważ
niony do wydania aktu współdziałania w trybie art. 106 k.p.a. (samorządu
zawodowego, taki jak izba aptekarska lub notarialna) może być jednocześnie 5 Pojęcie „aktorzy postępowania” utrwalone w obcym piśmiennictwie zdaje się lepiej uka
zywać różnorodność pełnionych przez nie ról niż słowo „podmioty”, którym posługują się polscy
autorzy. Używane jest ono również w rozmaitych kontekstach, np. Blake (2011: 244) pisze o wnio
skodawcy (stronie) nadużywającym swoich praw procesowych jako „złym aktorze” (applicant is
a bad actor). 85 O niepołączalności ról procesowych stroną postępowania?6 Tymczasem prowadzone w rodzimej literaturze docie
kania nadal zdominowane są dokonanymi niegdyś, wywodzącymi się po części
z innej epoki ustaleniami. Z tych względów oraz z racji dynamicznie zmienia
jącej się rzeczywistości społecznej nie zawsze łatwo uchwytna jest właściwa
dla sfery prawa administracyjnego dystynkcja: interes indywidualny – interes
społeczny (publiczny, ogólny). Wymownie potwierdzają to np. głosy tych auto
rów, którzy a priori sytuują organizację społeczną występującą jako podmiot
na prawach strony w postępowaniu administracyjnym w roli przedstawiciela
interesu społecznego. Okoliczność, że przepis art. 31 § 1 in fine k.p.a. uzależ
nia udział takiej organizacji w postępowaniu od uprzedniego zbadania przez
organ administracji, czy przemawia za tym interes społeczny, nie przesądza
bynajmniej o racjach, których może ona bronić. Nie sposób sprowadzać ich
wyłącznie do korzyści rozpatrywanych przez pryzmat interesu ogólnego lub
indywidualnego. Mogą one także mieścić się w obrębie kategorii pośredniej,
oznaczonej przez Giampaolo Rossiego zwrotem „interesy ponadjednostkowe”
(interessi sovraindividuali), będącym synonimem określenia: interesy zbio
rowości (interessi difussi)7. Jako pojęcie zbiorcze obejmuje ono wszakże pod
legające dalszej konkretyzacji interesy. Da się wśród nich wyróżnić interesy
rozmaitych grup społecznych i wspólnot terytorialnych (według Rossiego – in
teressi collettivi, di collettività territoriali) oraz właściwe interesy zbiorowości,
których znaczenie najlepiej zdają się oddawać wyrażenia: mieszane albo zło
żone. Ich istotą jest to, że są one wspólne dla całości grupy i jej poszczególnych
członków8. I. ZŁOŻONOŚĆ UKŁADU INTERESÓW W POSTĘPOWANIU
ADMINISTRACYJNYM Tak pojmowane interesy różnią się od interesów grupowych tym,
że dotyczą nieokreślonej bliżej liczby osób (un numero potenzialmente indefi
nito di sogetti sono interessi diffusi). Nie tworzą one homogenicznej struktury
ani tym bardziej zorganizowanego wedle jakichś przejrzystych reguł systemu. Przeciwnie, cechą takiej zbiorowości jest pluralizm poglądów, postaw i celów,
a jej sytuację zwięźle określają słowa – „stan płynny” (allo stato fluido)9. Nie
ulega jednak wątpliwości, że prawo musi poszukiwać form artykułowania
również tego typu oczekiwań, aspiracji i dążeń. 8 Lemetre, Miranda (2011): 205–206. 6 Zob. Kmieciak (1994): 242–243; (2016): 125 n.
7 Zob. Rossi (2011): 75
8 Lemetre, Miranda (2011): 205–206.
9 Lemetre, Miranda (2011): 205. Z uwagami tymi korespondują rozważania Iwańskiej (2013):
306–307, 326 n. oraz 342 n., dotyczące „odindywidualizowania” ochrony prawnej realizowanej
w formie „prawnośrodowiskowej skargi zbiorowej”; szerzej Kmieciak (2017b): 31–33. 7 Zob. Rossi (2011): 75 6 Zob. Kmieciak (1994): 242–243; (2016): 125 n. 9 Lemetre, Miranda (2011): 205. Z uwagami tymi korespondują rozważania Iwańskiej (2013):
306–307, 326 n. oraz 342 n., dotyczące „odindywidualizowania” ochrony prawnej realizowanej
w formie „prawnośrodowiskowej skargi zbiorowej”; szerzej Kmieciak (2017b): 31–33. II. PROCESOWE KONSEKWENCJE DUALNEGO STATUSU
ORGANÓW JEDNOSTEK SAMORZĄDU TERYTORIALNEGO Reaktywowanie w Polsce w 1990 r. samorządu terytorialnego zrodziło
konieczność dostosowania przepisów kodeksu o wyłączeniu pracownika oraz
organu (rozdział 5 działu I) do nowej formuły wypełniania zadań publicznych
w formach jurysdykcyjnych. Znaczną ich część przejęły wówczas organy gmin – 86 Zbigniew Kmieciak wójtowie, burmistrzowie i prezydenci miast. Są oni organami wykonawczy
mi podstawowych jednostek samorządu terytorialnego, mających osobowość
prawną i dysponujących prawem własności oraz innymi prawami majątko
wymi. Wypełniają zatem funkcje kojarzone ze sferą dominium – korzystania
z praw i wykonywania obowiązków, których korelatem jest pojęcie interesu
prawnego w rozumieniu art. 28 k.p.a. Organy te, niejako równolegle, realizują
zgoła odmienną funkcję, właściwą dla sfery imperium, co wynika z faktu usta
wowego określenia ich kompetencji. Jan Zimmermann nie wahał się napisać,
że organy te, „choć nie są »stronami«, uczestniczą merytorycznie w postępo
waniu”10. Jednak uczestniczą jako kto? Odpowiedź nie nastręcza większych
trudności: jako uosobienie władzy publicznej – podmiot usytuowany w jednym
z segmentów owej władzy, poza obszarem dominium. Rozłączność tych dwóch
ról nie podlega – jak sądzę – żadnej dyskusji11. Tym założeniem kierował się prawodawca, nowelizując w 1990 r. k.p.a. Ustawą z 24 maja 1990 r. o zmianie ustawy – Kodeks postępowania admini
stracyjnego12 dodano do kodeksu przepis oznaczony jako art. 27a. Stanowił
on o wyłączeniu organów gminy „od załatwiania spraw, w której stroną jest
gmina”. W takim przypadku legitymowany do załatwienia sprawy stawał się
organ innej gminy, wyznaczony odpowiednio przez kolegium odwoławcze przy
sejmiku (w sprawach należących do zadań własnych gmin) albo wojewodę
(w sprawach należących do zadań zleconych z zakresu administracji rządo
wej). Rozwiązanie, o którym mowa, mające chronić wartości w postaci bez
stronności orzekania w postępowaniu administracyjnym oraz samodzielności
gmin (odrębność ich prawnego bytu), nie przetrwało zbyt długo. Zamieszanie
wywołane podjęciem przez Trybunał Konstytucyjny (TK) uchwały z 23 czerw
ca 1993 r. (W 3/92)13 skłoniło ustawodawcę do uchylenia art. 27a kodeksu14. Wyciągnął on – ulegając perswazji niektórych przedstawicieli doktryny oraz
środowiska samorządowego – zbyt pochopne wnioski z wyrażonej przez Trybu
nał tezy, że „organy gminy podlegają na podstawie art. 27a k.p.a. wyłączeniu
od załatwienia sprawy rozstrzyganej w formie decyzji administracyjnej, jeżeli
przedmiotem sprawy jest stosunek cywilnoprawny, którego stroną jest gmina”. Krytyka, z którą spotkało się stanowisko TK, nie stwarzała podstaw do takiej
reakcji. 10 Zimmermann (2017): 111.
11 Por. też argumentację Kmieciaka (2017a): 75–78, odnoszącą się do sytuacji ujętej à re
bours, tj. procesowej pozycji organu administracji rządowej – dyrektora regionalnego zarządu
gospodarki wodnej, uznanego przez Sąd jednocześnie za organ umocowany do załatwienia sprawy
i reprezentanta strony postępowania administracyjnego.
12 Dz. U. Nr 34, poz. 201.
13 OTK 1993, nr 3, poz. 46.
14 Przepis ten, z mocy art. 25 pkt 3 ustawy z 12 października 1994 r. o samorządowych kole
giach odwoławczych (Dz. U. Nr 122, poz. 593), utracił moc z dniem 6 grudnia 1994 r.
15 Co do tej wartości – zob. Suwaj (2004): 13 n. 13 OTK 1993, nr 3, poz. 46. 12 Dz. U. Nr 34, poz. 201. 16 Szerzej Chróścielewski (2019): 211, 219–220.
17 Mimo przeprowadzanych ustawicznie, przekraczających rozsądną miarę, nowelizacji ko
deksu redakcja tego przepisu nie zmieniła się od czasu jego ustanowienia w całkowicie odmien
nych warunkach prawnoustrojowych.
18 T.jedn.: Dz. U. 2020, poz. 293.
19 T.jedn.: Dz. U. 2018, poz. 1474.
20 Zob. art. 92 ust. 1 pkt 2 ustawy z 5 czerwca 1998 r. o samorządzie powiatowym (t.jedn.:
Dz. U. 2020, poz. 920).
21 Szewczyk (2020): 85. II. PROCESOWE KONSEKWENCJE DUALNEGO STATUSU
ORGANÓW JEDNOSTEK SAMORZĄDU TERYTORIALNEGO Powinna ona raczej być traktowana jako dogodny punkt wyjścia dys
kusji na temat poszukiwania optymalnej formuły zespolenia zespołu standar
dów uchodzących za podstawowy atrybut rzetelnego procesu (bezstronności/
obiektywizmu orzekającego w sprawie organu)15 z instytucjami prawa ustro
jowego. Opisany zabieg można określić mianem zawoalowanej albo pośredniej 87 O niepołączalności ról procesowych (mocą przepisu ustawowego) derogacji wykładni dokonanej przez Trybunał16. Jego konsekwencje okazały się bardziej poważne i dalekosiężne, niż można
było przypuszczać. Brak gwarancji, którą na krótki czas ustanowił art. 27a, przy wąsko za
kreślonych przez art. 25 k.p.a. przesłankach wyłączenia organu administra
cji od załatwienia sprawy17, doprowadził do upowszechnienia się w obrębie
samorządu terytorialnego praktyki łączenia w jednej osobie ról procesowych
właściwych dla obszaru dominium i imperium. Jest ono widoczne np. w spra
wach lokalizacji inwestycji celu publicznego, podlegających załatwieniu we
dług reżimu przepisów art. 50–58 ustawy z 27 marca 2003 r. o planowaniu
i zagospodarowaniu przestrzennym18. Występując z wnioskami jako inwestor
(przedstawiciel strony postępowania), wójtowie, burmistrzowie albo prezy
denci zwykli rozpatrywać je w zakresie swojej właściwości, wchodząc w rolę
organu prowadzącego postępowanie. Granica pomiędzy tym, kto rozstrzyga
i kto o coś zabiega, zaciera się, wywołując mimowolnie pytania o logikę ta
kiego rozwiązania. Czy daje się ono pogodzić z paremią, że nie można być sę
dzią we własnej sprawie? O niebezpieczeństwie zespolenia dwóch ról proceso
wych w ręku jednego podmiotu nie zawsze możemy przekonać się, analizując
wyłącznie przepisy prawa materialnego, legitymujące do złożenia wniosku
i kreujące umocowanie do podjęcia rozstrzygnięcia. Niekiedy trzeba jeszcze
uwzględnić inne uregulowania, w szczególności prawa ustrojowego. Jest tak
chociażby w sprawach załatwianych decyzjami wydawanymi w trybie art. 11a
ustawy z 10 kwietnia 2003 r. o szczególnych zasadach przygotowania i realiza
cji inwestycji w zakresie dróg publicznych19. Zgodnie z jego ust. 1, w odniesie
niu do dróg powiatowych i gminnych, decyzję o zezwoleniu na realizację inwe
stycji drogowej na wniosek właściwego zarządcy drogi (którym może być organ
gminy) wydaje wykonujący zadania zlecone z zakresu administracji rządowej
starosta. Jednak w przypadku pewnej kategorii jednostek samorządowych –
miast na prawach powiatu to ich prezydenci pełnią funkcje starosty20. Według
utartej już praktyki rozpatrują oni składane przez siebie wnioski, jak można
mniemać – z pozytywnym wynikiem. Jak trafnie wywodzi Marek Szewczyk,
żaden inny podmiot nie może skorzystać z dobrodziejstw płynących z uregu
lowań tej ustawy, jak tylko organy administracji drogowej (w tym wójtowie,
burmistrzowie i prezydenci miast). Inne podmioty mogą natomiast zostać do
tknięte ich skutkami21. 20 Zob. art. 92 ust. 1 pkt 2 ustawy z 5 czerwca 1998 r. o samorządzie powiatowym (t.jedn.
U. 2020, poz. 920). II. PROCESOWE KONSEKWENCJE DUALNEGO STATUSU
ORGANÓW JEDNOSTEK SAMORZĄDU TERYTORIALNEGO Stanowisko doktryny w analizowanej materii razi niekonsekwencją. Nie
którzy jej przedstawiciele uznali za dopuszczalne prowadzenie przez organ 88 Zbigniew Kmieciak gminy postępowania w sprawie, w której jest ona stroną22. Prezentowany jest
również pogląd, że w takiej sytuacji gmina ma prawo uczestniczyć w postę
powaniu przed organem drugiej instancji, a nawet wnieść skargę do sądu
administracyjnego na wydaną w postępowaniu odwoławczym decyzję23. We
dług bardziej wyważonej opinii po uchyleniu art. 27a k.p.a. „upada stosowa
nie instytucji wyłączenia organu, ale mogą zachodzić podstawy do wyłącze
nia wszystkich pracowników urzędu gminy, łącznie z jego kierownikiem […]
z racji pozostawania w takim stosunku prawnym z gminą, który wpływa na
prawa i obowiązki pracowników, a to z tytułu treści obowiązków pracowni
czych spoczywających na pracownikach samorządowych”. Głoszący to zapa
trywanie Janusz Borkowski dodał, że problem ten może powstać także wtedy,
„gdy stroną będzie powiat lub województwo jako samodzielna wspólnota sa
morządowa”24. Autor wyraźnie nawiązał do wyrażonej w wielu publikacjach
koncepcji Wojciecha Chróścielewskiego, nieukrywającego, że z punktu widze
nia wymagań prawdy obiektywnej nie sposób wyobrazić sobie prowadzenia
postępowania administracyjnego „przez organy gminy wobec gminy będącej
stroną tego postępowania […] na takich samych zasadach jak postępowanie
prowadzone wobec innych podmiotów”. W jego ocenie trzeba przyjąć, że orga
ny gminy i pozostałych jednostek samorządu terytorialnego „podlegają, mimo
uchylenia art. 27a, wyłączeniu od załatwienia sprawy, której stroną jest dana
jednostka samorządu”, a jego podstawą jest art. 24 § 1 pkt 1 kodeksu25. Teza ta
spotkała się z aprobatą m.in. Romana Hausera i Marka Szewczyka26, Andrze
ja Wróbla27 i Piotra Przybysza28. Można również wskazać opracowania nauko
we, których autorzy unikają zajęcia stanowiska w tej kwestii, poprzestając na
zrelacjonowaniu osnowy trwającego już wiele lat sporu29. 22 Tak Klat-Wertelecka (2000): 138.
23 Kubalski (2008): 72.
24 Borkowski (2019): 198.
25 Chróścielewski (2019): 211–212.
26 Hauser, Szewczyk (1996): 45.
27 Wróbel (2018): 261.
28 Przybysz (2017): 133.
29 Tak m.in. Klonowski (2015): 177; Knysiak-Sudyka (2017): 177–179. 29 Tak m.in. Klonowski (2015): 177; Knysiak-Sudyka (2017): 177–179. III. PRÓBY ROZWIĄZANIA PROBLEMU
W RAMACH DZIAŁALNOŚCI ORZECZNICZEJ
SĄDÓW ADMINISTRACYJNYCH Zdecydowanie bardziej urozmaicone – choć nie zawsze klarowne i dogłęb
nie uzasadnione – stanowisko w przedmiocie dopuszczalności prowadzenia
przez organy jednostek samorządu terytorialnego postępowania w sprawach,
w których posiadają one przymiot strony (dysponują interesem prawnym),
prezentują sądy administracyjne. Od pewnego czasu można zaobserwować
wyraźną tendencję do negowania tradycyjnych zapatrywań na temat odno 26 Hauser, Szewczyk (1996): 45. 28 Przybysz (2017): 133. 89 O niepołączalności ról procesowych szącej się do takich przypadków gwarancji realizacji, ujętej obecnie wprost
w art. 8 § 1 kodeksu zasady bezstronności, wyrażonych m.in. w uchwale Na
czelnego Sądu Administracyjnego z 19 maja 2003 r. (OPS 1/03)30. Stwierdzono
w niej, że w sprawie dotyczącej zwrotu wywłaszczonej nieruchomości będącej
własnością miasta na prawach powiatu „prezydent tego miasta jako organ wy
konawczy miasta i reprezentujący je na zewnątrz oraz także jako pracownik
urzędu miasta, a jednocześnie sprawujący funkcję starosty, podlega wyłącze
niu na podstawie art. 24 § 1 pkt 1 i 4 k.p.a., co w konsekwencji wyłącza możli
wość upoważnienia przez niego do załatwienia sprawy jego zastępców i pozo
stałych pracowników urzędu miasta”. Zbieżna z tymi ustaleniami konstatacja
znalazła się w wyroku Sądu Najwyższego z 7 czerwca 2001 r. (III RN 104/00)31. ę
y
ą
j y
g
(
)
W odmiennym kierunku poszła argumentacja, którą posłużono się w wy
roku Wojewódzkiego Sądu Administracyjnego w Krakowie z 22 marca 2018 r. (II SA/Kr 185/18)32. Przyjęto w nim, że sfery władztwa administracyjnego i do
minium często w sposób zamierzony łączone są ze sobą, „co wynika ze specyfi
ki postępowania administracyjnego. Fakt, iż w stosunku do składnika mająt
kowego czy prawa należącego do jednostki, która jednocześnie jest organem
administracji, inne podmioty formują wnioski, żądania czy roszczenia inicju
jące wszczęcie postępowania administracyjnego, sam w sobie nie przesądza,
iż organ ten wyłączony jest z orzekania w sprawie tego wniosku czy roszcze
nia”. Wyłączenia takiego nie sposób również – zdaniem Sądu – zakładać z góry
wówczas, kiedy „organ orzekający jest jednocześnie inicjatorem postępowania. W szczególności w obowiązującym porządku prawnym nie ma ogólnej regula
cji uprawniającej do wyłączenia organu samorządu terytorialnego w sytuacji,
gdy sprawa będąca przedmiotem postępowania administracyjnego pozostaje
w związku z interesem prawnym danej wspólnoty samorządowej”. W wyroku
tego Sądu z 22 czerwca 2018 r. (II SA/Kr 374/18)33 zauważono natomiast, że
co do zasady „nie ma alternatywy w sytuacji, gdy inwestorem zamierzenia
inwestycyjnego jest gmina a podmiotem inicjującym postępowanie o ustalenie
warunków zabudowy jest wójt, burmistrz lub prezydent miasta. 34 Lex nr 562860. 33 Lex nr 2513878. 30 Orzecznictwo Naczelnego Sądu Administracyjnego 2003, nr 4, poz. 115.
31 Orzecznictwo Sądów Polskich 2002, nr 10, poz. 133. 32 Lex nr 2470522. 31 Orzecznictwo Sądów Polskich 2002, nr 10, poz. 133. 30 Orzecznictwo Naczelnego Sądu Administracyjnego 2003, nr 4, poz. 1
31 Orzecznictwo Sądów Polskich 2002, nr 10, poz. 133.
32 L Orzecznictwo Naczelnego Sądu Administracyjnego 2003, nr
31 Orzecznictwo Sądów Polskich 2002, nr 10, poz. 133.
32 III. PRÓBY ROZWIĄZANIA PROBLEMU
W RAMACH DZIAŁALNOŚCI ORZECZNICZEJ
SĄDÓW ADMINISTRACYJNYCH W takiej sytu
acji wójt, burmistrz lub prezydent miasta prowadzi to postępowanie i wydaje
decyzję w I instancji”. W wielu orzeczeniach nawiązuje się bezpośrednio do faktu utraty mocy
obowiązującej przez art. 27a k.p.a. Przykładowo w wyroku Naczelnego Sądu
Administracyjnego z 1 kwietnia 2009 r. (II OSK 460/08)34 zaznaczono, że nie
ma „wyraźnej normy, wzorem uchylonego art. 27a k.p.a., wyłączającej wójta,
burmistrza (prezydenta miasta) od orzekania w sprawie, w której jego gmina
jest stroną”. Obowiązku takiego nie można – w przekonaniu Sądu – dopatrzyć
się „w regulacjach art. 24”. 90 Zbigniew Kmieciak W opisanym nurcie wypowiedzi mieszczą się spostrzeżenia zawarte
w wyroku Wojewódzkiego Sądu Administracyjnego w Kielcach z 15 listopada
2017 r. (II SA/Ke 654/17)35. Nadano im postać następujących tez: 1. Przepisy u.p.z.p. wyraźnie wprowadzają kompetencję prezydenta miasta (wójta, burmi
strza) do wydawania decyzji o warunkach zabudowy (poza terenami zamkniętymi). Jed
nocześnie brak jest wyraźnego wyłączenia tej kompetencji w przepisach szczególnych. Kompetencja prezydenta miasta (wójta, burmistrza), co do zasady, nie ulega wyłączeniu
w sytuacji, gdy wydana decyzja może mieć wpływ na interes prawny gminy. Prezydent
miasta w takiej sytuacji reprezentuje przede wszystkim interes wspólnoty samorządowej
i działa dla dobra wspólnego. 1. Przepisy u.p.z.p. wyraźnie wprowadzają kompetencję prezydenta miasta (wójta, burmi
strza) do wydawania decyzji o warunkach zabudowy (poza terenami zamkniętymi). Jed
nocześnie brak jest wyraźnego wyłączenia tej kompetencji w przepisach szczególnych. Kompetencja prezydenta miasta (wójta, burmistrza), co do zasady, nie ulega wyłączeniu
w sytuacji, gdy wydana decyzja może mieć wpływ na interes prawny gminy. Prezydent
miasta w takiej sytuacji reprezentuje przede wszystkim interes wspólnoty samorządowej
i działa dla dobra wspólnego. 2. Przepisy regulujące kompetencje organów do wydania decyzji administracyjnej muszą być
rozumiane ściśle, zatem brak jest podstaw prawnych do przyjęcia w drodze wykładni są
dowej tych przepisów, że dany organ jest wyłączony od rozpatrywania określonej katego
rii spraw. W przeciwnym wypadku mogłoby dojść do naruszenia przepisów o właściwości
miejscowej. 3. Nie można skutecznie twierdzić, że dochodzi do naruszenia prawa w sprawie, gdy prezy
dent miasta wydaje decyzję, rozstrzygając przy tym o prawach i obowiązkach jednostki
samorządu terytorialnego (gminy), którą reprezentuje. Niestety, Sąd nie wyjaśnił, czy troska o „interes wspólnoty samorządowej”
i działanie „dla dobra wspólnego” zyskują prymat nad obiektywnym, zgodnym
z przepisami prawa rozstrzygnięciem sprawy. Nie zastanowił się on także nad
tym, czy pojęcia kompetencji i interesu prawnego nie są aby kategoriami roz
łącznymi. 35 Lex nr 2411782.
36 Lex nr 597931.
37 Orzecznictwo Naczelnego Sądu Administracyjnego i Wojewódzkich Sądów Administracyj
nych 2016, nr 4, poz. 54. 36 Lex nr 597931. 36 Lex nr 597931.
37 Orzecznictwo Naczelnego Sądu Administracyjnego i Wojewódzkich Sądów Administracyj
nych 2016, nr 4, poz. 54. 35 Lex nr 2411782. III. PRÓBY ROZWIĄZANIA PROBLEMU
W RAMACH DZIAŁALNOŚCI ORZECZNICZEJ
SĄDÓW ADMINISTRACYJNYCH Nie dostrzegł wreszcie tego, że przyznanie danemu organowi kom
petencji (wyznaczenie przez prawo jego właściwości w sprawie) nie oznacza
automatycznie posiadania zdolności do prowadzenia postępowania, gdyż gra
nice tej ostatniej wytyczają przepisy o wyłączeniu. Równie osobliwy pogląd sformułowano w wyroku Naczelnego Sądu Admi
nistracyjnego z 30 marca 2010 r. (II OSK 88/10)36. Zwrócono w nim uwagę, że
jeśli przepis prawa powierza organowi administracji kompetencję do podjęcia
decyzji i stwarza podstawę do wystąpienia z wnioskiem o wszczęcie postępo
wania, jest ono uruchamiane z urzędu. Połączenie ról procesowych organu
orzekającego w sprawie i wnioskodawcy zostało tym samym zinterpretowane
przez Sąd jako okoliczność kwalifikująca do zmiany formuły postępowania na
prowadzone ex officio. Trudno oprzeć się wrażeniu, że w niektórych orzeczeniach sądowych naj
wyraźniej mylona jest kolejność ustaleń co do konstrukcji prawa materialnego
legitymujących do udziału w postępowaniu w jednej z przewidzianych kodek
sem ról procesowych. Tego rodzaju błędu nie ustrzegł się Naczelny Sąd Ad
ministracyjny w uchwale składu 7 sędziów z 16 lutego 2016 r. (I OPS 2/15)37. Zgodnie z nią: „Powiat nie ma legitymacji procesowej strony w sprawie o usta
lenie odszkodowania od powiatu za nieruchomość przejętą pod drogę publicz
ną, która stała się własnością powiatu, jeżeli decyzję wydaje starosta na pod 91 O niepołączalności ról procesowych stawie art. 12 ust. 4a w związku z art. 11a ust. 1 ustawy z dnia 10 kwietnia
2003 r. o szczególnych zasadach przygotowania i realizacji inwestycji w za
kresie dróg publicznych (Dz.U. 2013 r. poz. 687 ze zm.) oraz art. 38 ust. 1
ustawy z dnia 5 czerwca 1998 r. o samorządzie powiatowym (Dz.U. z 2015 r. poz. 1445)”. Rzecz w tym, że w rozważanej sytuacji to starosta powinien zo
stać wyłączony od załatwienia sprawy, skoro dotyczyła ona interesu prawnego
powiatu, czyli podmiotu „legitymowanego pierwotnie” (mającego prawo do za
inicjowania postępowania przed właściwym organem administracji publicznej
i uczestnictwa w nim). Punkt widzenia Naczelnego Sądu Administracyjnego
podzielił Wojewódzki Sąd Administracyjny w Rzeszowie w wyroku z 11 kwiet
nia 2018 r. (II SA/Rz 1393/17)38. 38 Lex nr 2494627.
39 Maier (2001): 260.
40 Wade, Forsyth (2009): 402; podobnie Carroll (2002): 297.
41 Gellhorn, Levin (2006): 237. IV. USTROJOWY CHARAKTER ZASADY BEZSTRONNOŚCI
ORGANU PROWADZĄCEGO POSTĘPOWANIE
TYPU JURYSDYKCYJNEGO Podejmowane – w ramach drugiego z omówionych nurtów orzecznictwa –
próby rozwiązania problemu niemal całkowicie ignorują wnioski płynące
z faktu objęcia działalności jurysdykcyjnej organów jednostek samorządu te
rytorialnego ogólnym reżimem prawa procesowego – tym samym dla całej ad
ministracji publicznej. Bezstronność i obiektywizm orzekania są standardem
mocno zakotwiczonym w idei rządów prawa, wartością o wymiarze konstytu
cyjnym, której źródeł można też doszukać się w postanowieniach konwencyj
nych. Rozważając znaczenie tej zasady i jej związek z innymi standardami
postępowania administracyjnego, Tanja Maier nie wahała się przyznać, że
stanowi ona „dobro wspólne europejskiej tradycji prawnej” (gemeinsames Gut
europäischer Rechtstradition)39. W systemach common law wartość tę kojarzy się z jednym z wymagań na
tural justice, czyli reguł wynikających z naturalnego poczucia sprawiedliwo
ści. Przeciwdziałanie stronniczości (the rule against bias) ma tam umożliwić
realizację prawa strony do rzetelnego wysłuchania. Jak podkreśla się w lite
raturze, ze swojej istoty powinno ono być wysłuchaniem bezstronnym (a fair
hearing must be an unbiased hearing)40. W orzecznictwie sądów amerykań
skich bezstronność podmiotu podejmującego decyzję uznawana jest z kolei za
esencjonalny składnik konstytucyjnej klauzuli due proces41. Potrzeba docho
wania tego wymagania w trakcie postępowania przed organami administracji
publicznej nie jest także kwestionowana w systemach prawa kontynentalne
go. W niektórych z nich jego respektowanie łączy się wyraźnie z zakorzenioną
w tradycji prawa francuskiego koncepcją droits de la défense, będącą de facto 92 Zbigniew Kmieciak odmianą wyróżnionej doktryny „prawa do rzetelnego wysłuchania”42. Nie brak
także opinii, że bezstronność – jak process value – jest rdzeniem rozwijanej
w orzecznictwie sądowym, także poza Europą, doktryny sprawiedliwości pro
ceduralnej (procedural fairness)43. Co istotne, w rozważaniach na ten temat
nie różnicuje się specjalnie pozycji sądów i innych organów wypełniających
funkcje jurysdykcyjne, usytuowanych w obrębie władzy wykonawczej44. Te
dwie grupy podmiotów traktowane są nawet niekiedy jako jedność w sensie
instytucjonalnym, wobec których powinny obowiązywać te same gwarancje
ochrony bezstronności i obiektywizmu orzekania. W odniesieniu do organów
administracji publicznej akcentowany jest – co prawda – obowiązek troski
o poszanowanie interesu ogólnego, który jednak nie jest pojmowany jako prze
szkoda do realizacji utrwalonych w tradycji danego systemu zasad rzetelnego
postępowania. Przeciwnie, w ustawodawstwie wielu państw można doszukać
się przepisów formułujących powinność działania bez uszczerbku dla każdej
ze wskazanych wartości, tak aby zapewnić ich względną równowagę i respek
towanie w możliwie najszerszym zakresie. Nie najgorszą ilustracją przedstawionego twierdzenia jest art. 4f ustawy
boliwijskiej nr 2341 z 23 kwietnia 2002 r. 42 Schwarze (2010): 1243 n.
43 Co do tego pojęcia zob. Kmieciak (2014): 95–101.
44 Kmieciak (2011): 177 n.
45 Zob. Brewer-Carias (2009): 171.
46 Wyrzykowski (1986): 50.
47 Kmieciak (2014): 137–139. 47 Kmieciak (2014): 137–139. IV. USTROJOWY CHARAKTER ZASADY BEZSTRONNOŚCI
ORGANU PROWADZĄCEGO POSTĘPOWANIE
TYPU JURYSDYKCYJNEGO o postępowaniu administracyjnym,
który nakazując przestrzeganie zasady bezstronności (principio de imparcia
lidad), zobowiązał administrację do ochrony interesu ogólnego (defensa del in
terés general) oraz unikania zachowań, które prowadziłyby do dyskryminacyj
nego bądź zróżnicowanego traktowania administrowanych45. Trzeba zarazem
zaznaczyć, że ocena, czy pewne działanie leży w interesie publicznym (społecz
nym, państwowym, wspólnoty lokalnej albo nieokreślonej bliżej zbiorowości),
musi odbywać się zawsze w granicach obowiązującego prawa, w szczególności
w toku ustaleń dokonywanych dla celów stosowania przepisów zawierających
zwroty nieostre. Wraz z ogólnymi klauzulami kodeksowymi (takimi jak za
warte w art. 7, 7a, 8 § 1, 74 § 1, 81a, 137 albo 154 § 1 i 155) tworzą one pod
stawę do prawnej kategoryzacji i wyważania różnych interesów. Właściwością
tego rodzaju zwrotów normatywnych jest – co wyczerpująco uzasadnił swego
czasu Mirosław Wyrzykowski – uzyskiwanie treściowych konturów dopiero
w rezultacie zastosowania ich do konkretnych przypadków. Utożsamiane
z nimi pojęcia nieostre nie mają „przed zastosowaniem [...] konkretnej treści,
ich zawartość treściowa rzadko może zostać zdefiniowana, a na ogół może być
jedynie przedmiotem opisu ich cech zasadniczych”46. Wykładnia obowiązujące
go prawa jest zatem tym narzędziem, które pozwala na harmonizowanie ogól
nych, wiążących administrację zasad rangi konstytucyjnej, konwencyjnej lub
ustawowej, przy jednoczesnym zachowaniu ugruntowanej w poszczególnych
systemach prawnych hierarchii wyprowadzanych z nich dyrektyw47. 93 O niepołączalności ról procesowych Bez ryzyka popełnienia błędu można powiedzieć, że zasada bezstronno
ści organów prowadzących wszelkie postępowania typu jurysdykcyjnego, bez
względu na układ reprezentowanych w konkretnej sprawie interesów, ma wy
miar uniwersalny. Jest ona – w rozumieniu naszych pojęć – zasadą ustrojową,
wynikającą z konstytucyjnych wymagań demokratycznego państwa prawnego,
której stosowanie nie może być wyłączone mocą innych zasad, z powołaniem
się na wątpliwe metodologicznie założenia. Nie do przyjęcia jest w szczególno
ści teza, że odstępstwa od niej można uzasadnić przyczynami uchodzącymi za
pragmatyczne, takimi jak konieczność dochowania przepisów o właściwości
organów jednostek samorządu terytorialnego albo utratą mocy przez przepi
sy regulujące niegdyś wprost kwestię jednego z elementów wyłączenia tych
organów od załatwienia sprawy. Jak starałem się wykazać, operowanie tego
typu argumentacją stwarza tylko pozory odniesienia się do istoty problemu,
przesłaniając rzeczywiste, prawne tło wyłaniających się kontrowersji. 48 Szerzej Kmieciak (2013): 19 n. V. KONKLUZJE Zawarta w k.p.a. regulacja o wyłączeniu pracownika, organu i członków
organu kolegialnego od udziału w postępowaniu administracyjnym (załatwie
nia sprawy), zwłaszcza jeśli porównamy ją z unormowaniami obcymi, musi
być uznana za daleką od doskonałości48. Przetrwała ona w nieznacznie zmie
nionym kształcie od czasu uchwalenia kodeksu w 1960 r. Ustawodawca nie
wykorzystał kolejnych, skądinąd nazbyt często przeprowadzanych nowelizacji
tego aktu, do unowocześnienia tego zespołu przepisów i dostosowania go do
współczesnych potrzeb. Co więcej, dopuścił się on poważnego błędu, uchylając
w 1994 r. – po zaledwie kilku latach obowiązywania – art. 27a. Okoliczność ta
sprawiła, że aby dochować utrwalonego w naszej tradycji standardu bezstron
ności orzekania przez organy jednostek samorządu terytorialnego w spra
wach, w których jednostki te dysponują interesem prawnym, jako normatyw
ną podstawę wyłączenia zaczęto wskazywać (przyznając status pracownika
kierownikowi organu) art. 24 § 1 pkt 1 i 4 k.p.a., a jako organy umocowane
do wyznaczenia organu mającego prowadzić zamiast nich postępowanie – od
powiednio samorządowe kolegia odwoławcze albo wojewodów. Za poglądem
o dopuszczalności tego zabiegu opowiedziała się duża część doktryny, w mniej
szym stopniu zapatrywanie to uzyskało akceptację w orzecznictwie sądów
administracyjnych, co musi być traktowane jako swoiste i bardzo niepokoją
ce „rozdwojenie jaźni”. Utrzymywanie na dłuższą metę tego stanu rzeczy, tj. posługiwanie się swoistą „protezą prawną” dla zapewnienia stanu zgodności
praktyki z ogólnie uznanymi wymaganiami praworządności i sprawiedliwości
proceduralnej, stwarza nieznośną sytuację prawną, burzącą nasze wyobraże
nia o zasadach budowania ładu prawnego. Najprostszym rozwiązaniem rysu
jącego się dylematu byłoby uzupełnienie przepisów art. 25 kodeksu o uregu 94 Zbigniew Kmieciak lowanie stanowiące treść dawnego art. 27a, powiązane z niewielką korektą
art. 26. Czas pokaże, czy ustawodawcy wystarczy odwagi i determinacji, aby
zmienić te przepisy. Zbigniew Kmieciak
Uniwersytet Łódzki
zkmieciak@wpia.uni.lodz.pl
https://orcid.org/0000-0002-1297-4940 Zbigniew Kmieciak
Uniwersytet Łódzki
zkmieciak@wpia.uni.lodz.pl
https://orcid.org/0000-0002-1297-4940 Zbigniew Kmieciak
Uniwersytet Łódzki
zkmieciak@wpia.uni.lodz.pl
https://orcid.org/0000-0002-1297-4940 https://orcid.org/0000-0002-1297-4940 Blake, S. (2011). Administrative Law in Canada. Markham. Borkowski, J. (2019). Wyłączenie pracownika oraz organu, [w:] B. Adamiak, J. Borkowski, Kodeks
postępowania administracyjnego. Komentarz. Warszawa: 191–237. Borkowski, J. (2019). Wyłączenie pracownika oraz organu, [w:] B. Adamiak, J. Borkowski, Kodeks
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stracyjnego. Samorząd Terytorialny 10(1/2): 125–142. Klonowski, K. (2015). Wyłączenie pracownika oraz organu, [w:] H. Knysiak-Sudyka (red.), Kodeks
postępowania administracyjnego. Komentarz. Warszawa: 154–177. Kmieciak, M. (2011). Trybunały administracyjne w Zjednoczonym Królestwie (system jurysdykcji
po zmianach ustawodawstwa z 2007 r.). Zeszyty Naukowe Sądownictwa Administracyjnego
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go gminy. Samorząd Terytorialny 18(1/2): 72–85. Kubalski, G.P. (2008). Wójt jako organ orzekający w postępowaniu dotykającym interesu praw
go gminy. Samorząd Terytorialny 18(1/2): 72–85. Künnecke, M. (2007). Zbigniew Kmieciak
Uniwersytet Łódzki
zkmieciak@wpia.uni.lodz.pl
https://orcid.org/0000-0002-1297-4940 V. KONKLUZJE Tradition and Change in Administrative Law: An Anglo-American Compa
rison. Berlin–Heidelberg–New York. g
Lemetre, F., Miranda, R. (2011). Diritto amministrativo. Napoli. Lemetre, F., Miranda, R. (2011). Diritto amministrativo. Napoli. Lemetre, F., Miranda, R. (2011). Diritto am Maier, T. (2001). Befangenheit im Verwaltungsverfahren. Die Regelungen der EU-Mitgliedstaa
ten im Rechtsvergleich. Berlin. 95 O niepołączalności ról procesowych Pollitt, Ch., Bouckaert, G. (2011). Public Management Reform: A Comparative Analysis – New
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Public Management, Governance and the Neo-Weberian State. Oxford. g
Priddat, B.P. (2008). Zur Governancealisierung der Politik: Delegation, Führung, Governance,
Netzwerke, [w:] G. Schuppert, M. Zürn (Hrsg.), Governance in einer sich wandelnden Welt. Wiesbaden: 352–379. Priddat, B.P. (2008). Zur Governancealisierung der Politik: Delegation, Führung, Governance,
Netzwerke, [w:] G. Schuppert, M. Zürn (Hrsg.), Governance in einer sich wandelnden Welt. Wiesbaden: 352–379. Przybysz, P. (2017). Kodeks postępowania administracyjnego. Komentarz. Warszawa. Rossi, G. (2011). Diritto amministrativo. Principi. Padova. Schwarze, J. (2010). European Administrative Law. Sweet and Maxwell. Supernat, J. (2005). Zarządzanie. Wrocław. Suwaj, P. (2004). Gwarancje bezstronności organów administracji publicznej w postępowaniu ad
ministracyjnym. Wrocław. Szewczyk, M. (2020). O skali normatywnej i wadze teoretycznej specustaw inwestycyjno-budowal
nych oraz motywach ich stanowienia, [w:] T. Bąkowski (red.), Specustawy inwestycyjno-bu
dowlane. Gdańsk: 81–93. Wade, W., Forsyth, C. (2009). Administrative Law. Oxford. Wróbel, A. (2018). Wyłączenie pracownika oraz organu, [w:] A. Wróbel, M. Jaśkowska, Kodeks
postępowania administracyjnego. Komentarz. Warszawa: 230–261. Wróbel, A. (2018). Wyłączenie pracownika oraz organu, [w:] A. Wróbel, M. Jaśkowska, Kodeks
postępowania administracyjnego. Komentarz. Warszawa: 230–261. Wyrzykowski, M. (1986). Pojęcie interesu społecznego w prawie administracyjnym. Warszaw Zoller, E. (2008). Introduction to Public Law: A Comparative Study. Leiden. Zoller, E. (2008). Introduction to Public Law: A Comparative Study. Leiden. S u m m a r y The article presents an analysis of the permissibility of conducting administrative proceed-
ings by local self-government bodies (commune heads, mayors and city presidents) in cases in
which these bodies have a legal interest, that is, are treated as parties. In the author’s opinion, in
such cases local self-government bodies lose the ability to conduct the proceedings (are excluded
from settling a case). The author takes a critical stance towards the repeal of a provision in 1994
that explicitly referred to the exclusion of these bodies. Since then, both in legal science and in
judicial practice, there have been disputes as to whether such exclusion can be based on the pro
visions of Article 24 § 1 point 1 and 4 of the Code of Administrative Procedure. The author calls
for a rapid amendment of the Code, supplementing Article 25 with a regulation constituting the
content of Article 27a which was repealed in 1994, and making a minor revision of the existing
Article 26. The question of whether the legislature has enough courage to make these changes is
also addressed. Keywords: local self-government bodies; administrative proceedings; impartiality of adjudication;
exclusion of an authority from settling a case; principle of the rule of law
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Variations in Nutritional Requirements Across Bee Species
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Frontiers in sustainable food systems
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cc-by
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Variations in Nutritional Requirements Across Bee
Species Alexandre Barraud, Lena Barascou, Victor Lefebvre, Deborah Sene, Yves Le
Conte, Cedric Alaux, Francesca-Vittoria Grillenzoni, Francesca Corvucci,
Giorgia Serra, Cecilia Costa, et al. To cite this version: Alexandre Barraud, Lena Barascou, Victor Lefebvre, Deborah Sene, Yves Le Conte, et al.. Variations
in Nutritional Requirements Across Bee Species. Frontiers in Sustainable Food Systems, 2022, 6,
10.3389/fsufs.2022.824750. hal-03589111 Distributed under a Creative Commons Attribution 4.0 International License Variations in Nutritional
Requirements Across Bee Species We therefore developed original experiments in laboratory conditions to evaluate
the interspecific variations in bee nutritional requirements. We analyzed the chemical
content of eight pollen blends, different in terms of protein, lipid, amino acids, and sterols
total concentration and profiles. Each pollen blend was provided to four different bee
model species: honey bees (Apis mellifera), bumblebees (Bombus terrestris), mason
bees (Osmia bicornis and Osmia cornuta). For each species, specific protocols were
used to monitor their development (e.g., weight, timing, survival) and resource collection. Overall, we found that the nutritional requirements across those species are different,
and that a low-quality diet for one species is not necessarily low-quality for another
one. While honey bees are negatively impacted by diets with a high protein content
(∼40%), bumblebees and mason bees develop normally on these diets but struggle on
diets with a low total amino acid and sterol content, specifically with low concentrations
of 24-methylenecholesterol and β-sitosterol. Overall, our study supports the need of
conserving and/or introducing plant diversity into managed ecosystems to meet the
natural nutritional preferences of bees at species and community level. Keywords: bees, pollen, nutrition, bumble bee, honey bee, Osmia, nutrients Edited by: Edited by:
Kimberly Ann Stoner,
Connecticut Agricultural Experiment
Station, United States Reviewed by:
T’Ai Roulston,
University of Virginia, United States
Vanessa Corby-Harris,
Carl Hayden Bee Research Center,
United States Reviewed by:
T’Ai Roulston,
University of Virginia, United States
Vanessa Corby-Harris,
Carl Hayden Bee Research Center,
United States *Correspondence:
Alexandre Barraud
alexandre.barraud@umons.ac.be
Lena Barascou
lena.barascou@inrae.fr *Correspondence:
Alexandre Barraud
alexandre.barraud@umons.ac.be
Lena Barascou
lena.barascou@inrae.fr †These authors have contributed
equally to this work
‡These authors have equally
supervised this work Specialty section:
This article was submitted to
Agroecology and Ecosystem Services,
a section of the journal
Frontiers in Sustainable Food Systems Received: 29 November 2021
Accepted: 11 January 2022 Received: 29 November 2021
Accepted: 11 January 2022
Published: 04 February 2022 Variations in Nutritional
Requirements Across Bee Species 1 Laboratory of Zoology, Research Institute for Biosciences, University of Mons, Mons, Belgium, 2 INRAE, Abeilles et
Environnement, Avignon, France, 3 CREA Research Centre for Agriculture and Environment, Bologna, Italy, 4 Centre
d’Écologie Fonctionnelle et Évolutive (CEFE), Université de Montpellier, CNRS, EPHE, IRD, Montpellier, France With 2,000 species currently recorded in Europe, bees are a highly diversified and
efficient group of pollinating insects. They obtain their nutrients from nectar and pollen
of flowers. However, the chemical composition of these resources, especially of pollen
(e.g., protein, lipid, amino acids, fatty acids, or sterol content), is highly variable among
plant species. While it is well-known that bees show interspecific variation in their floral
choices, there is a lack of information on the nutritional requirements of different bee
species. We therefore developed original experiments in laboratory conditions to evaluate
the interspecific variations in bee nutritional requirements. We analyzed the chemical
content of eight pollen blends, different in terms of protein, lipid, amino acids, and sterols
total concentration and profiles. Each pollen blend was provided to four different bee
model species: honey bees (Apis mellifera), bumblebees (Bombus terrestris), mason
bees (Osmia bicornis and Osmia cornuta). For each species, specific protocols were
used to monitor their development (e.g., weight, timing, survival) and resource collection. Overall, we found that the nutritional requirements across those species are different,
and that a low-quality diet for one species is not necessarily low-quality for another
one. While honey bees are negatively impacted by diets with a high protein content
(∼40%), bumblebees and mason bees develop normally on these diets but struggle on
diets with a low total amino acid and sterol content, specifically with low concentrations
of 24-methylenecholesterol and β-sitosterol. Overall, our study supports the need of
conserving and/or introducing plant diversity into managed ecosystems to meet the
natural nutritional preferences of bees at species and community level. With 2,000 species currently recorded in Europe, bees are a highly diversified and
efficient group of pollinating insects. They obtain their nutrients from nectar and pollen
of flowers. However, the chemical composition of these resources, especially of pollen
(e.g., protein, lipid, amino acids, fatty acids, or sterol content), is highly variable among
plant species. While it is well-known that bees show interspecific variation in their floral
choices, there is a lack of information on the nutritional requirements of different bee
species. HAL Id: hal-03589111
https://hal.inrae.fr/hal-03589111v1
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abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License ORIGINAL RESEARCH
published: 04 February 2022
doi: 10.3389/fsufs.2022.824750 Citation: Experimental studies in controlled conditions have confirmed
that the nutritional quality of pollen (e.g., the concentration of
protein, and lipids, sterols, and amino acids) can have an impact
on the development and mortality of bumblebees (e.g., Taseï and
Aupinel, 2008a; Vanderplanck et al., 2014; Moerman et al., 2016,
2017; Barraud et al., 2020; Carnell et al., 2020) and mason bees
(Sedivy et al., 2011; Eckhardt et al., 2014). The floral diversity
of pollen diet does not seem to be the major factor of quality,
as bumblebees develop better on high-quality monofloral diets
compared to low-quality polyfloral diets (Moerman et al., 2017;
Carnell et al., 2020). The pattern for honey bees appears to be
similar at an individual level, with pollen quality (reflected by
protein content) having an impact on the physiology and survival
of adult honey bees (Brodschneider and Crailsheim, 2010; Di
Pasquale et al., 2013; Frias et al., 2016; Omar et al., 2017; Li et al.,
2019). Bees obtain their carbohydrate nutrient intake mainly from
nectar, and their protein and lipid from pollen (Roulston
and Cane, 2000; Nicolson, 2011). The chemical composition
of pollen is highly variable across floral species, between 2–
60 and 1–20% for protein and lipid contents, respectively
(Roulston and Cane, 2000; Vaudo et al., 2020). Field and semi-
field studies showed that this chemical composition can be
related to bee health (e.g., honey bee A. mellifera: Alaux et al.,
2010; Brodschneider and Crailsheim, 2010; Di Pasquale et al.,
2013, mason bee Osmia bicornis, Bukovinszky et al., 2017). Generalist bees seem able to assess pollen chemical quality and
balance multiple macronutrient resources when making foraging
decisions (Vaudo et al., 2016, 2018; Kraus et al., 2019; Ruedenauer
et al., 2020). Based on a large quantity and diversity of samples,
Vaudo et al. (2020) showed that honey bees collected pollens
between 1:1 and 2:1 protein to lipid (P:L) ratio. This species
appears to occupy a different nutritional space compared to
Bombus impatiens and Osmia cornifrons, which collect at P:L
ratios of 4:1 and 2:9, respectively. Furthermore, to satisfy the food
intake of colonies with numerous individuals, honey bees must
collect large amounts of pollen. Citation: However,
unmanaged species are still key pollinators as there are many
genus-specific plant-pollinator interactions, linking wild plant
diversity to wild bee diversity (Ollerton, 2017). Moreover, wild
bees have been shown to increase crop production by up to twice
as much as honey bees, underlining the importance of wild bees
even in agro-ecosystems (Garibaldi et al., 2013; Weekers et al.,
2022). honey bees and bumblebees, Osmia cornifrons, a solitary foraging
bee with a short flight period, has mixed preferences for Rosaceae
and Fabaceae pollen (Haider et al., 2014; Nagamitsu et al., 2018),
with average P:L ratios of 1.6 ± 0.3 and 3.8 ± 0.5, respectively. Regarding chemical profiles, particular lipids and proteins
seem more also important in bee nutritional requirement. For
example, sterols (e.g., β-sitosterol) are essential to synthetize
ecdysteroid, involved in the molting of the larvae and the
maturation of the ovaries of female imago. In case of sterol
deficiency, a delay in molting can be observed (Regali, 1996). Additionally, a good amino acid balance is also crucial for the
bee development (Moerman et al., 2016). They are involved
in growth, survival, flight ability or in immunity (Regali,
1996; Carter et al., 2006; Moerman et al., 2016). Some
amino-acids (methionine, lysine, threonine, histidine, leucine,
isoleucine, valine, phenylalanine, tryptophan) and sterols (24-
methylenecholestrol and β-sitosterol) cannot be synthetised by
the bee and are therefore considered as essential, meaning that
it is necessary to obtain them through pollen consumption (De
Groot, 1953; Svoboda et al., 1978; Behmer and Nes, 2003). Losses and declines in managed and wild bee populations
have been reported worldwide (Cameron et al., 2011; Goulson
et al., 2015; Duchenne et al., 2020). Habitat loss and agricultural
intensification, resulting in landscape simplification have been
identified as important drivers of pollinator decline (Winfree,
2010; Persson et al., 2015; Vray et al., 2019). These factors
can directly or indirectly affect the quality, the quantity and
the diversity of floral resources and thus the food sources of
bees (e.g., Roger et al., 2017b). This makes the abundance,
distribution/availability, quality and diversity of these resources
potentially a main proximal pressure explaining bee population
trends (Roulston and Goodell, 2011; Vaudo et al., 2015). Citation: Barraud A, Barascou L, Lefebvre V,
Sene D, Le Conte Y, Alaux C,
Grillenzoni F-V, Corvucci F, Serra G,
Costa C, Vanderplanck M and
Michez D (2022) Variations in
Nutritional Requirements Across Bee
Species. With more than 2,000 species recorded in Europe (Rasmont et al., 2017), bees represent a
highly diverse group of pollinators (Michener, 2007; Danforth et al., 2013). These species
show a wide variability in various traits such as body size (i.e., from 0.3 mm to 4.5 cm in
Europe), social behavior (e.g., cleptoparasitic, solitary, eusocial), nesting behavior (e.g., cavity-
or soil-nesting), foraging strategies (e.g., pollen generalist or specialist), or phenology (e.g.,
uni- or bivoltine) (Michener, 2007; Michez et al., 2019). This diversity is crucial for the
successful sexual reproduction of wild and domesticated plants, but it is also critical to With more than 2,000 species recorded in Europe (Rasmont et al., 2017), bees represent a
highly diverse group of pollinators (Michener, 2007; Danforth et al., 2013). These species
show a wide variability in various traits such as body size (i.e., from 0.3 mm to 4.5 cm in
Europe), social behavior (e.g., cleptoparasitic, solitary, eusocial), nesting behavior (e.g., cavity-
or soil-nesting), foraging strategies (e.g., pollen generalist or specialist), or phenology (e.g.,
uni- or bivoltine) (Michener, 2007; Michez et al., 2019). This diversity is crucial for the
successful sexual reproduction of wild and domesticated plants, but it is also critical to Front. Sustain. Food Syst. 6:824750. doi: 10.3389/fsufs.2022.824750 Front. Sustain. Food Syst. 6:824750. doi: 10.3389/fsufs.2022.824750 February 2022 | Volume 6 | Article 824750 Frontiers in Sustainable Food Systems | www.frontiersin.org Nutrition Across Bee Species Barraud et al. honey bees and bumblebees, Osmia cornifrons, a solitary foraging
bee with a short flight period, has mixed preferences for Rosaceae
and Fabaceae pollen (Haider et al., 2014; Nagamitsu et al., 2018),
with average P:L ratios of 1.6 ± 0.3 and 3.8 ± 0.5, respectively. understand this variability to implement efficient conservation
programs (Nieto et al., 2014). Indeed, bees are the dominant
pollinators of crop and wild plants in most ecosystems, visiting
more than 90% of crop varieties (Potts et al., 2016). Some
generalist bee species have been domesticated and are now used
for crop pollination like the western honey bee (Apis mellifera),
the buff-tailed bumblebee (Bombus terrestris) (Banda and Paxton,
1991; Velthuis and Doorn, 2006), and a few solitary species
(Gruber et al., 2011; Pitts-Singer and Cane, 2011). Frontiers in Sustainable Food Systems | www.frontiersin.org Citation: Therefore, honey bees collect
pollen from generalist, open floral morphologies such as mass
blooming trees (e.g., Quercus sp., Salix sp., Prunus sp.) and wild
herbs with high production of pollen (e.g., Asteraceae), which
may have a nutritional make up that falls in the lower P:L values
(i.e., 1–3:1 P:L) (Vaudo et al., 2020). Bumblebees look much
more picky in their choices, since many species mainly forage
on Fabaceae pollen showing a high P:L ratio value (3.8 ± 0.5)
(Leonhardt and Blüthgen, 2012; Wood et al., 2021). In contrast to Overall, these results suggest that a loss of a part of the plant
community, especially the families covering specific physiological
requirement (e.g., Fabaceae), is more likely to affect bumblebees
and solitary bees than honey bees (Leonhardt and Blüthgen,
2012). The more generalized the foraging behavior of a particular
bee species, the more likely it is to be able to switch to alternative
host plants and persist in an area, even if those host plants are of
a lower nutritional quality (Roger et al., 2017b). However, there
are multiple studies evaluating and comparing the development
of various generalist bee species in controlled conditions on
the same pollen diets (Moerman et al., 2016), and no study
considering a broad diversity of bee clades (e.g., different bee
tribes or bee families). To address these knowledge gaps, we evaluated the effect
of 8 pollen mixes of different qualities on key life-history
traits regulated by pollen consumption in four European bee
species (2 Apidae species: Apis mellifera (Apini) and Bombus
terrestris (Bombini); 2 Megachilidae species: Osmia bicornis and
O. cornuta). We first conducted palynological and chemical
analysis (total protein, total lipid, amino acid, and sterol
content) on these pollen blends to characterize their quality Frontiers in Sustainable Food Systems | www.frontiersin.org Frontiers in Sustainable Food Systems | www.frontiersin.org February 2022 | Volume 6 | Article 824750 2 Barraud et al. Nutrition Across Bee Species TABLE 1 | Frequency (%) of most represented pollen species in the 8 pollen blend. Pollen mixes
Dominant pollen species
Frequency (%)
C
Cistaceae: Cistus ladanifer
95
MS
Rosaceae: Malus/Pyrus f. 40
Salicaceae: Salix
27
TSo
Asteraceae: Taraxacum
21
Fabaceae: Sophora
17
QS
Fagaceae: Quercus robur gr. 51
Salicaceae: Salix
29
BQ
Brassicaceae
36
Fagaceae: Quercus robur gr. 35
SP
Salicaceae: Salix
43
Rosaceae: Prunus f. 34
S
Salicaceae: Salix
89
ST
Salicaceae: Salix
64
Asteraceae: Taraxacum
21 composition. Citation: We then developed experiments in controlled
conditions and monitored the key life-history traits in bees fed
with these pollen diets (e.g., survival for honey bees, brood
production for bumblebees, and larva development for mason
bees). We finally investigated which nutritional factors better
explain bee health and development across the four species. Our
hypothesis was that bee nutritional requirements are different
across species. Palynological Analyses One gram of pollen sample was inserted and centrifuged in a
50 ml centrifuge tube and then dissolved in 20 ml of distilled
water. Using a Pasteur pipette, a drop of sediment was placed
on a microscope slide and spread out over an area of about
18 × 18 mm. After drying, the sediment was included in
one drop of glycerine jelly and covered with the cover slip. Examination under the microscope were performed with 400X
magnification. After a first general check to identify all the
pollen types in the slide, a second read of the slide was
carried out until 500 pollen grains were counted. Abortive,
irregular, or broken pollen grains were still counted if they could
be identified. Bee Model Species p
This study was conducted on four common pollen generalist
bee species recorded in Europe which are foraging in the
same habitat for part of the year (Michez et al., 2019). We
selected the Western honey bee Apis mellifera (Hymenoptera,
Apidae, Apini), a domesticated eusocial species; the bufftailed
bumblebee Bombus terrestris (Hymenoptera, Apidae, Bombini),
a wild social species (Rasmont et al., 2008); and two mason bees
(Osmia bicornis and O. cornuta; Hymenoptera, Megachilidae,
Osmiini), wild solitary species. They are commonly used as
model species because of their easy management in laboratory
conditions. Bumblebee colonies were provided by Biobest
NV (Westerlo, Belgium); honey bees were obtained from
local apiaries at the “Institut National de la Recherche pour
l’Agriculture, l’Alimentation et l’Environnement” (INRAE) in
Avignon (France) and the mason bees were provided by
Wildbiene + Partner (Switzerland). and 45 µg/bee for tau-fluvalinate (oral exposure) (US EPA,
2021). Characterization of Pollen Diets Eight
organic
blends
of
honeybee-collected
pollen
were
purchased from the company “Abeille heureuse” (France). Each pollen blend was gamma irradiated to avoid parasite
infection, homogenized to reduce the risk of variation in
palynological composition in each pollen treatment, and
stored at −80◦C before the experiment. In addition, a
fraction of each pollen diet was lyophilized and stored at
−20◦C for palynological and chemical analyses (see below). Each pollen mix was named based on their palynological
analysis, using the first letter of dominant pollen species (see
Table 1). Recognition of pollen type was based on comparison between
the observed pollen forms and those present in the CREA-AA
collection of reference slides (built from anthers of identified
plants). For each pollen type, the percentage of each species
with respect to the total number of counted pollen grains
was calculated. Pesticide Analyses For each pollen diet, the presence of pesticide residues
was
determined
by
liquid
chromatography–tandem
mass
spectrometry (LC-MS/MS) with a limit of quantification
of 0.01 mg/kg and a limit of detection of 0.005 mg/kg
following the EN 15662:2018 procedure. Residues of 2,4
dimethylformamidine (DMF, degradation products of amitraz)
and tau-fluvalinate were detected in all pollen blends but
were below the limit of quantification. These compounds
used as chemical treatments against the honeybee parasite
Varroa destructor are consistently found in pollens (47.4 and
88.3% of trapped pollens for amitraz and tau-fluvalinate,
respectively; Mullin et al., 2010; Calatayud-Vernich et al.,
2019) and are considered as relatively safe for honeybees with
an oral LD50 of 75 µg/bee for amitraz (contact exposure) Honeybees (Apis mellifera) In honeybees, pollen is mostly consumed by young adult bees. Its consumption enables the development of mandibular glands
(Camilli et al., 2020) and especially hypopharyngeal glands
(Crailsheim et al., 1992), where jelly is produced to feed larvae,
the queen and drones (Crailsheim, 1992). We therefore tested
the influence of pollen quality at the individual level on the
fresh weight of individual heads, which is highly correlated to
the volume of acini from the hypopharyngeal glands (Hrassnigg
and Crailsheim, 1998). We also measured the survival rate of
bees. Experiments were performed in the spring. To obtain 1-
day-old bees, brood frames containing late-stage pupae were
taken from eight healthy colonies normally treated against the
parasitic mite Varroa destructor (Apis mellifera ligustica × Apis
mellifera mellifera), and were placed overnight into an incubator
under controlled conditions [34◦C, 50–70% of relative humidity
(RH)]. The next day, newly-emerged bees (<1 day old) were
collected, mixed and groups of 40 bees were placed in cages
(10.5 × 7.5 × 11.5 cm) (Pain, 1966). Caged bees, kept in an
incubator (30◦C and 50–70% RH), were provided ad libitum with
water, candy (Apifonda R⃝+ powdered sugar) and one of the
pollen diets (n = 10 cages per experimental group) (Figure 1A). Pollen diets were replaced every day for 10 days. To simulate
as much as possible colony rearing conditions, caged bees were
provided with a Beeboost R⃝(Ickowicz, France), releasing one
queen-equivalent of queen mandibular pheromone per day. Each
day, pollen diets were weighed to determine the amount of pollen
consumed per day and per bee. Pollen collection was corrected
for evaporation, which was estimated by placing two samples of
each pollen mixture in the same incubator for 24 h. Bee mortality
was recorded every day for 44 days by counting and removing
dead bees from cages. On day 7, 9 bees were sampled from each
cage and stored at −80◦C. The fresh weight of heads was then
measured on individual bees (n = 9 bees per cage giving a total of
90 bees per experimental group). Protein Analyses Pollen protein concentration was measured using the Bradford
assay according to Vaudo et al. (2020). We added 1.5 mL of
0.1 M NaOH to ∼1 mg of pollen sample (dry weight), and
conducted the Bradford assay with the Bio-Rad Protein Assay
Kit microassay 300 µL microplate protocol using bovine γ-
globulin as the protein standard (Bio-Rad Laboratories, Inc.,
Hercules, CA). We used three technical replications for each
biological replication and measured absorbance at 595 nm using
a SpectraMax 190 spectrophotometer (Molecular Devices, LLC,
Sunnyvale, CA). Protein concentrations were calculated using
polynomial 2nd analysis from the protein standards. February 2022 | Volume 6 | Article 824750 Frontiers in Sustainable Food Systems | www.frontiersin.org 3 Nutrition Across Bee Species Barraud et al. Lipid Analyses and 24-methylenecholesterol co-eluted. Therefore, the results are
pooled for these two compounds. Compounds were identified
according to their retention times by comparison with those of
sunflower oil as reference. The identifications were corroborated
by GC-FID (Vanderplanck et al., 2011). p
y
Pollen
lipid
concentrations
were
determined
using
a
modified protocol from Van Handel and Day (1988). In
2.0 mL microcentrifuge tubes, we added 200 µL 2% sodium
sulfate and 1.6 mL chloroform/methanol to ∼1 mg of each
pollen sample (dry weight) before a 5 min centrifugation. Supernatant was transferred to a clean glass tube with 600 µL
deionised water, and centrifuged for 5 min. We separated
the
top
carbohydrate/water/methanol
fraction
and
the
remaining chloroform fraction was used for lipid analysis. The lipid/chloroform fraction was left overnight in a fume hood
to completely evaporate the solvent. We added 200 µL sulfuric
acid to the sample and heated at 100◦C for 10 min. Then, 5 mL
vanillin/phosphoric acid reagent was added. We used three
300 µL technical replications for each biological replication
and measured absorbance at 525 nm. Lipid concentrations
were calculated using polynomial 2nd analysis from vegetable
oil standards. Pollen concentrations of protein and lipids are
reported as µg nutrient/mg pollen, and subsequent P:L ratios
were determined for each diet. Bee Nutrition Studies As the three genera (i.e., Apis, Bombus, and Osmia) show very
different life cycles and behavior, they could not be tested
following the same protocol in laboratory conditions. Thus, we
developed different experimental setup for each of the three
bee genera. Amino Acids Analyses For the analysis of total amino acids, 1 mL of hydrolysis solution
(6N HCl, 0.1% phenol and 500 µM norleucine) was added
to 3–5 mg (dry weight) of pollen (Vanderplanck et al., 2014)
and then incubated for 24 h at 110◦C. The hydrolysate was
evaporated until dryness under vacuum in a boiling bath at
100◦C. Afterwards, 1 mL of the sodium citrate buffer pH 2.2
was added into the tube. The sample solution was poured in an
HPLC vial after filtration (0.2 µm filter), and each amino acid
was measured separately with an ion-exchange chromatograph. A post-column ninhydrin reaction produced colored derivatives,
which was monitored via a UV detector. For amino acid
quantification, norleucine was used as internal standard. This
analysis includes essential amino acids that bee cannot synthesize,
as well as the non-essential ones. The essential amino acids were
established by De Groot (1953) for honey bees; namely arginine,
histidine, isoleucine, leucine, lysine, methionine, phenylalanine,
threonine, tryptophan and valine. Sterols Analyses Before each analysis, pollen samples were divided into a
minimum of three samples (i.e., 20 mg (dry weight) per analytical
replicate). Sterols were quantified by GC-FID after extraction and
purification according to the method described by Vanderplanck
et al. (2011). The multi-step procedure can be summarized
as follows: (i) saponification with 2M methanolic potassium
hydroxide, (ii) extraction of the unsaponifiable portion with
diethylether and several water washings, (iii) solvent evaporation,
(iv) fractionation of the unsaponifiable portion by TLC, (v)
trimethylsilylation of the sterols (scraped from the silicagel), and
(vi) separation by GC. The total sterol content was determined
considering all peaks above the limit of quantification [(LOQ);
LOQ = 9.6 ng/1.2 µl injected] whose retention time were
between cholesterol and betulin (internal standard). Individual
sterols were quantified on the basis of peak areas from analyses. Under the present analytical conditions applied, campesterol Bumblebees (Bombus terrestris) We tested the impact of pollen on bumblebees at micro-colony
level. Such a method to test the nutritive value of pollen diets
has been shown to be a good estimate of queenright colony
development at least under laboratory conditions with food ad
libitum (Taseï and Aupinel, 2008b). A total of five queen-right
colonies of 100 Bombus terrestris workers were used to build up
80 queen-less micro-colonies of five workers, placed in plastic February 2022 | Volume 6 | Article 824750 Frontiers in Sustainable Food Systems | www.frontiersin.org 4 Nutrition Across Bee Species Barraud et al. FIGURE 1 | Experimental set-ups for (A) honeybees, (B) bumblebees and (C) mason bees. Caged honeybees were provided with water, candy, and one of the pollen
diets. Five bumblebees (workers) were placed in plastic boxes provided with pollen and syrup. Eggs of mason bees developed in cell culture plates of 48 wells filled
with 400 mg of pollen. FIGURE 1 | Experimental set-ups for (A) honeybees, (B) bumblebees and (C) mason bees. Caged honeybees were provided with water, candy, and one of the pollen
diets. Five bumblebees (workers) were placed in plastic boxes provided with pollen and syrup. Eggs of mason bees developed in cell culture plates of 48 wells filled
with 400 mg of pollen. boxes (8 × 16 × 16 cm) (Figure 1B). This number of individuals
per micro-colony has been repeatedly used and is assumed to be
the most reliable for assessing diet effects (Gradish et al., 2013;
Moerman et al., 2016; Roger et al., 2017a; Vanderplanck et al.,
2018; Klinger et al., 2019). Micro-colonies were then distributed
in the different conditions (n = 10 micro-colonies for each
experimental treatment). All micro-colonies were maintained in
the same room in constant darkness at 26 ± 2◦C with a relative
humidity of 60–65%. They were manipulated under red light
to minimize disturbance (Sadd, 2011) for a period of 28 days. Pollen diets were provided ad libitum to the micro-colonies as
candies (mixed pollen with sugar syrup). New pollen candies
were provided every 2 days, while the previous ones were weighed
to assess the pollen collection. Pollen collection was corrected
for evaporation by monitoring the weight of two samples of
each diet placed in the rearing room for 48 h. Bumblebees (Bombus terrestris) To estimate the
performance and development of bumblebee micro-colonies, we
measured: (i) the total pollen and syrup collections, which can
impact brood production and development (e.g., Plowright et al.,
2008; Sutcliffe and Plowright, 2008); (ii) colony growth after 28
days of development [i.e., mass of individuals from all brood
stages (eggs, larvae, pupae, non-emerged, and emerged males)]
(Vanderplanck et al., 2014, 2018). For each micro-colony, all the
measured parameters were divided by the total mass of the five
workers to standardize the results and avoid potential effect of
worker activities related to their size (i.e., consumption and brood
care) (Cnaani and Hefetz, 1994). Additionally, we calculated the
pollen efficacy as the mass of total offspring divided by the total
pollen collection to estimate the colony performance. offspring were collected at the egg stage to avoid the consumption
of the original pollen supply by the freshly emerged larvae. In the
laboratory, cell culture plates of 48 wells were filled with 400 mg
of prepared pollen (mixed pollen with sugar syrup) (Figure 1C). A fine brush was used to pick the egg from its original brood
cell, and a single egg was placed cautiously onto each pollen
provision (n = 35–40 eggs per treatment group). Plates were
then placed into an incubator under controlled conditions (23◦C,
60% RH). Developmental stage of larvae was assessed every day
for 1 month and categorized into egg, larvae, feeding larvae,
feeding and defecating larvae, spinning larvae, light cocoon, and
cocoon. The time required to reach cocoon stage was used for the
analyses. On average 90 days after cocoon development, each of
them was taken out of the brood cells and weighed. Plates were
then kept at 12◦C for 4 days and at 4◦C for ∼120 days to mimic
hibernation. After 140 days, all cocoons were again kept at 12◦C
for 4 days before moving them into an incubator (25◦C, 60% RH)
to elicit emergence. After emergence, adults were weighed (fresh
weight) and determined as male or female. Statistical Analyses Statistical analyses were run using statistical software R version
3.3.3 (R Core Team, 2017). To analyse the influence of pollen
diets on honey bee survival, the number of dead bees per day
and cage throughout the experiments were transformed into a
survival table. A Cox proportional-hazards regression model was
then used to compare the different diets, with R functions (coxph)
and the package survival (Cox, 1970), considering the censored
data of the bees that were alive at the end of the study. Pollen
consumption and fresh weight of heads were analyzed using
Kruskal–Wallis followed by Dunn’s multiple comparison tests
since data were not normally distributed. Mason Bees (O. cornuta and O. bicornis)
We tested the impact of the pollen diet on the two species
of mason bees at the larval stage. Standard mason bee nesting
plates were installed close to the laboratory on the campus of the
University of Mons (Belgium). A total of 1,000 individuals were
released next to the nests. At regular time intervals, nests were
opened and investigated for brood cell production. After 3 weeks, For statistical analyses on bumblebees and Osmia data, two-
way crossed analyses of variance (Two-Way crossed ANOVA)
were conducted to evaluate the effect of diet. Since it is a
parametric test, homoscedasticity (Bartlett test) and normality of
the residuals (Shapiro test) were checked prior to the analyses. February 2022 | Volume 6 | Article 824750 Frontiers in Sustainable Food Systems | www.frontiersin.org 5 Nutrition Across Bee Species Barraud et al. When violation occurred, data were log- or z-transformed
to normality of residuals (“ztransform”function, R-package
“GenABEL,” Lenth, 2009) prior to the test. Multiple pairwise
comparisons were conducted using Tukey HSD post-hoc tests
when ANOVA detected significant difference between pollen
diets (p < 0.05). whereas TSo pollen mix had the highest protein content (397.66
± 11.5 mg/g, all p < 0.001). SP pollen mix had the lowest lipid
content (45.72 ± 2.42 mg/g, all p < 0.001) while ST and QS pollen
mixes had the significantly highest lipid content (89.93 and 82.87
mg/g, respectively, all p < 0.03). P:L ratio ranged from 2.93 to
3.44 for ST, C and S pollen mixes, to 6.01–6.19 for SP and TSo
diets (all p < 0.001). Statistical Analyses p
Differences in nutritional content were assessed using either
Kruskal–Wallis followed by Dunn’s multiple comparison tests
for proteins, lipids and P:L ratios, or perMANOVA for
sterols and amino acids (Euclidean distance, 999 permutations,
“adonis” command) after testing for multivariate homogeneity
(“betadisper” command) (R-package vegan, Oksanen et al.,
2020). Given the number of replicates, it was not possible to
run a multiple pairwise comparisons on amino acids and sterol
data. Differences were then visually assessed on UPGMA clusters
using Euclidean distance and multiscale bootstrap resampling
to calculate p-values for uncertainty in hierarchical cluster
(R- package pvclust, Suzuki et al., 2019). Indicator compound
analyses were performed to identify nutrients that were indicative
of the groups defined based on the hierarchical cluster (“indval”
command) (R-package labdsv, Roberts, 2019). All these analyses
were conducted using data expressed as mg/g. p
Total and essential amino acid content ranged from 109.73
mg/g (SP diet) and 233.28 mg/g (SP diet) to 52.57 mg/g
(C diet) and 125.04 mg/g (C diet), respectively. UPGMA
analyses identified two clusters: one composed of C diet only
(Cluster A, Supplementary Figure S1A) with a higher proline
concentration (p = 0.018), and a second cluster composed
of all other diets (Cluster B, Supplementary Figure S1A) with
a higher content of every amino acid except proline (all
p < 0.017). The different diets displayed concentrations of
total sterol from 4.54 mg/g (C diet) to 14.38 mg/g (ST
diet). UPGMA analyses identified two clusters: one composed
of ST and SP diets (Cluster B, Supplementary Figure S1B)
with a higher 24-Methylenecholesterol concentration compared
to the second cluster composed of all other diets (Cluster
A, Supplementary Figure S1B, p = 0.021). Other significant
differences out of these clusters have been identified: higher
concentrations of Cholesterol and δ7-Stigmasterol in TSo diet
(p = 0.024 and 0.01, respectively), a higher concentration of
Stigmasterol in BQ diet (p = 0.007) and higher concentration
of δ7-Avenasterol in ST diet (p = 0.007). Despite the low
concentrations of 24-Methylenecholesterol and δ5-Avenasterol
observed in C diet compared to other diets (Table 2), they were
not defined as significant by our analytical model, probably due to
the lack of replicates. Those differences were therefore considered
as tendencies. We analyzed the influence of each macro-nutrient in diets
(i.e., proteins and lipids) on species performance using Response
Surface Models (RSM). Statistical Analyses As it is standard for geometric analyses
of nutrition, the models included the linear and quadratic
components for protein and lipid intake as well as the interaction
term between proteins and lipids as explanatory variables. Regarding the response variable, we used the proportion of
individuals that survived for each diet treatment for Apis
mellifera, the pollen efficacy for Bombus terrestris and the adult
mass for Osmia bicornis and O. cornuta. As our response variables
were measured in different units, we standardized each response
variable to a mean of zero and a standard deviation of one using
a Z transformation prior to analysis (≪ztransform ≫function
from the GenABEL R-package; Ronnegard et al., 2016). We
performed these analyses using the ≪rsm ≫function from the
rsm R-package (Lenth, 2009), first considering lipid and protein
content (RSM 1), then sterol and amino acid content (RSM 2). Diet Effects on Bees
Honeybees (Apis mellifera) Significant differences in bee survival between pollen diets were
observed with the following order from the least to the most
beneficial pollen: TSo < MS – BQ – C < SP < ST - QS – S
(Figure 2A). Pollen diets were not consumed equally (p < 0.01,
Figure 2B). Bees consumed significantly more of the QS and
BQ than TSo and S pollen mixes. The head weight was also
affected by the type of pollen (p < 0.001, Figure 2C). Bees fed
with TSo pollen diet had a lighter head than bees fed with S, QS,
BQ, or ST pollen mixes. After normalization to the amount of
consumed pollen, head weights were the lowest for QS and BQ
pollens and the highest for TSo and S pollen mixes (p < 0.001;
Supplementary Figure S2). Palynological Analyses of Pollen Blends Palynological Analyses of Pollen Blends
The pollen mixtures were analyzed to verify the indication of the
dominant pollen (Table 1). Analyses confirmed the palynological
origin indicated by the seller only for two out of eight samples
(samples C, S). Only four out of eight samples showed the
presence of a dominant pollen (samples C, MS, BQ, SP); the other
samples were found to be more or less heterogeneous mixtures of
three or more pollen (see Supplementary Figure S1). Bumblebees (Bombus terrestris) The brood production was impacted by the diet (p < 0.01). Microcolonies fed with MS and S diets were more developed, in
term of total brood mass after 28 days, compared to those fed
with C diet (p = 0.026 and p = 0.022, respectively, Figure 3A). Pollen efficacy was also influenced by the different pollen diets. Microcolonies with bumblebees fed with MS, TSo, SP, S, and ST
pollen diets produced more brood per gram of pollen consumed
in 28 days compared to those fed with C pollen diet (all p < 0.04, S
QS
BQ
MS
SP
TSo
214.86 ± 10.5a
285.10 ± 12.4b
283.89 ± 7.23b
287.49 ± 9.00b
274.94 ± 18.1b
397.66 ± 11.5c
62.63 ± 4.3b
82.87 ± 2.75d
74.98 ± 2.53c
68.55 ± 2.74bc
45.72 ± 2.42a
64.24 ± 2.4b
3.44ac
3.44ac
3.79bc
4.19c
6.01d
6.19d
181.35 ± 25.04
194.18 ± 5.39
174.54 ± 24.48
218.37 ± 17.07
233.28 ± 16.01
198.57 ± 25.42
86.46 ± 10.45
90.45 ± 3.28
85.63 ± 8.05
103.54 ± 5.54
109.73 ± 7.48
91.54 ± 12.3
10.21 ± 1.28
10.87 ± 0.31
10.73 ± 1.27
12.79 ± 0.62
13.1 ± 0.87
11.28 ± 1.46
12.43 ± 1.28
12.39 ± 0.09
11.48 ± 0.75
12.33 ± 0.73
14.88 ± 0.76
10.4 ± 1.18
20.05 ± 2.96
20.62 ± 0.46
17.46 ± 3.51
22.52 ± 2.4
25.51 ± 1.58
19.68 ± 2.41
22.88 ± 4.55
25.32 ± 1.64
18.3 ± 5.51
24.65 ± 5.3
30.56 ± 3.8
24.06 ± 4.45
8.17 ± 1.03
8.43 ± 0.26
7.39 ± 1.43
9.14 ± 0.79
10.31 ± 0.86
8.73 ± 1.02
6.22 ± 1.15
7.17 ± 0.15
7.22 ± 0.58
6.1 ± 2.09
7.54 ± 0.68
7.95 ± 1.11
7.76 ± 0.96
8.16 ± 0.36
7.7 ± 0.86
10.16 ± 1.14
10.36 ± 0.82
8.6 ± 1.3
13.99 ± 1.79
14.82 ± 0.9
13.61 ± 1.48
18.4 ± 2.46
18.33 ± 1.84
15.6 ± 2.8
14.65 ± 1.25
14.71 ± 0.24
14.58 ± 1.59
16.29 ± 1.41
17.77 ± 1.06
14.55 ± 1.13
4.53 ± 0.37
4.25 ± 0.22
4.23 ± 0.47
5.38 ± 0.16
5.42 ± 0.35
4.57 ± 0.42
8.61 ± 1.14
9.7 ± 0.73
8.72 ± 1.1
12.42 ± 2.4
11.83 ± 1.55
10.37 ± 2.08
11.52 ± 0.63
14.55 ± 0.15
14.8 ± 1.34
20.59 ± 2.25
17.19 ± 0.96
18.87 ± 1.56
9.4 ± 1.76
10.14 ± 0.4
8.52 ± 2.24
11.17 ± 1.69
11.79 ± 1.14
10.74 ± 1.32
8.03 ± 1.07
8.38 ± 0.34
7.93 ± 1.18
9.39 ± 0.72
10.13 ± 0.84
8.48 ± 0.99
6.9 ± 1.23
7.59 ± 0.62
6.57 ± 1.18
9.08 ± 1.51
9.46 ± 0.96
8.05 ±1.55
10.23 ± 1.49
10.87 ± 0.45
10.17 ± 0.98
13.07 ± 1.12
13.47 ± 1.02
11 ± 1.76
8.22 ± 2.09
6.31 ± 1.6
6.86 ± 2.06
6.51 ± 0.16
12.68 ± 4.59
7.45 ± 0.44
0.1 ± 0.02
0.05 ± 0.03
0.06 ± 0.08
0.04 ± 0
0.06 ± 0.02
0.52 ± 0.14
2.48 ± 1.53
2.5 ± 0.76
3.21 ± 3.47
4.85 ± 0.15
10.26 ± 3.64
2.61 ± 1.71
0.1 ± 0.02
0.08 ± 0
0.79 ± 1.44
0.08 ± 0.01
0.02 ± 0.02
0.23 ± 0.1
2.42 ± 0.48
2.57 ± 0.66
1.5 ± 1.3
0.73 ± 0.04
1.2 ± 0.46
2.28 ± 1.16
1.41 ± 0.36
0.96 ± 0.17
1.27 ± 2.81
0.75 ± 0.04
1.05 ± 0.39
0.97 ± 0.5
0.02 ± 0.04
0.07 ± 0.05
0 ± 0
0.02 ± 0.01
0.03 ± 0.05
0.61 ± 0.15
1.69 ± 0.25
0.08 ± 0.02
0.04 ± 0.02
0.05 ± 0
± 0.02
0.22 ± 0.03
alues for each chemical are in bold as indicative. 21
6
18
86
1
1
2
2
8
6
7
1
1
4
8
1
8
1
8
2
2
1
0 Chemical Analyses of Pollen Blends Chemical Analyses of Pollen Blends
Pollen mixes had different chemical composition (Table 2,
perMANOVA, p < 0.001). Protein content varies from 209.61 to
397.66 mg/g of pollen (p < 0.001). C and S pollen blends had
the significantly lowest protein content compared to other mixes
(209.61 ± 9.13 and 214.86 ± 10.5 mg/g, respectively, all p < 0.03), February 2022 | Volume 6 | Article 824750 Frontiers in Sustainable Food Systems | www.frontiersin.org 6 Different letters indicate significant differences (Kruskal-Wallis tests followed by Dunn’s multiple Barraud et al. Nutrition Across Bee Species FIGURE 2 | Influence of pollen diets on Apis mellifera. (A) Survival probability (n = 30 bees per cage and 10 cages per pollen regime), (B) pollen collection and (C)
head weight. Boxes indicate the 1st and 3rd interquartile range with line denoting median (n = 30 pools of 3 bees per pollen). Whiskers include 90% of the individuals,
beyond which each outlier are represented by circles. Different letters indicate significant differences between pollen diets (Kruskal-Wallis tests followed by Dunn’s
multiple comparison tests: p < 0.01). FIGURE 2 | Influence of pollen diets on Apis mellifera. (A) Survival probability (n = 30 bees per cage and 10 cages per pollen regime), (B) pollen collection and (C)
head weight. Boxes indicate the 1st and 3rd interquartile range with line denoting median (n = 30 pools of 3 bees per pollen). Whiskers include 90% of the individuals,
beyond which each outlier are represented by circles. Different letters indicate significant differences between pollen diets (Kruskal-Wallis tests followed by Dunn’s
multiple comparison tests: p < 0.01). Figure 3B). The different pollen mixes had a limited impact on
resources collection. Bumblebees consume more of the TSo diet
than the SP diet (p = 0.024, Figure 3C). No significant differences
were observed between other diets nor regarding syrup collection
(p > 0.05, Figures 3C,D). range from 22 to 27 days. Cocoons of larvae fed with C diet were
significantly lighter compared to any other diets (all p < 0.002),
with a mean weight of 0.118 ± 0.011 and 0.095 ± 0.012 g for
females and males, respectively. Cocoons of larvae fed with MS
diet were ∼29, 12, and 11% heavier compared to those fed with
C, TSo or ST diet (all p < 0.02), respectively (Figure 4C). Adult
weight showed similar differences (Figure 4E). The time required
to reach cocoon stage for Osmia cornuta was significantly lower
when fed with BQ diet compared to any other diets (all p < 0.02,
Figure 4B). No significant differences were observed among the
other diets (all p > 0.05). Cocoons of larvae fed with C diet were
significantly lighter compared to any other diets (all p < 0.021),
with a mean weight of 0.102 ± 0.005 and 0.094 ± 0.011 g for
females and males, respectively. Cocoons of larvae fed with SP
diet were ∼27% heavier compared to those fed with C diet, with Geometric Analyses RSM analyses showed that A. mellifera survival variations
between diets were not explained by total amino acids (AAT)
or sterols (RSM 2, Supplementary Table S2), but rather by
protein and lipid content (RSM 1, Supplementary Table S2). On
the other hand, observed differences between diet-dependant
pollen efficacies in B. terrestris were not explained by protein or
lipid content (RSM 1, Supplementary Table S2), but rather by
AAT and sterols (RSM 2, Supplementary Table S2). O. bicornis
female mass variations were explained by the protein content Mason Bees (Osmia bicornis and O. cornuta) Osmia bicornis developed differently depending of their diet
(p < 0.001). Bees fed with C diet had a significantly higher
development time compared to any other diets (all p < 0.027),
with ∼25 and 31 days required to reach cocoon stage for females
and males, respectively (Figure 4A). In contrast, bees fed with
BQ diet had the lowest development time (all p < 0.004), with
20 and 23 days required to reach cocoon stage for females and
males, respectively (Figure 4A). Development time on other diets February 2022 | Volume 6 | Article 824750 Frontiers in Sustainable Food Systems | www.frontiersin.org 8 Barraud et al. Nutrition Across Bee Species FIGURE 3 | Influence of pollen diets on Bombus terrestris. (A) Brood mass production (g), (B) pollen efficacy (ratio between brood mass and pollen collection), (C)
pollen collection (g), and (D) syrup collection. Boxes indicate the 1st and 3rd interquartile range with line denoting median (n = 10 micro-colonies with 5 workers per
pollen diet). Different letters indicate significant differences between pollen diets (ANOVA tests followed by Tukey’s multiple comparison tests: p < 0.05). FIGURE 3 | Influence of pollen diets on Bombus terrestris. (A) Brood mass production (g), (B) pollen efficacy (ratio between brood mass and pollen collection), (C)
pollen collection (g), and (D) syrup collection. Boxes indicate the 1st and 3rd interquartile range with line denoting median (n = 10 micro-colonies with 5 workers per
pollen diet). Different letters indicate significant differences between pollen diets (ANOVA tests followed by Tukey’s multiple comparison tests: p < 0.05). a mean weight of 0.135 ± 0.012 and 0.115 ± 0.021 g for females
and males, respectively. They were also significantly heavier than
cocoons of larvae fed with QS, BQ, ST, and TSo diet (all p < 0.003,
Figure 4D). Adult weight showed similar differences, but with no
significant differences between C, Tso, and ST diets (all p > 0.07,
Figure 4F). only, while male mass was explained by proteins, AAT and
sterols (RSM 1, Supplementary Table S2). Finally, O. cornuta
adult mass variations between diets were explained in the
same way as for O. bicornis: proteins for females (RSM 1,
Supplementary Table S2); proteins, AAT and sterols for males
(RSM 2, Supplementary Table S2). Variation in Chemical Composition of
Pollen Diets Chemical composition of the tested diets was different, despite
some observed similarities. First, none of the pollen diets had
a protein content lower than 21%, which is already an average
amount: ∼25 plant genera contain <15% of protein in pollen
(Roulston and Cane, 2002). On the other hand, the protein-
richest diet of our experiments contained nearly 40% of proteins February 2022 | Volume 6 | Article 824750 Frontiers in Sustainable Food Systems | www.frontiersin.org 9 Barraud et al. Nutrition Across Bee Species FIGURE 4 | Influence of pollen diets on (left) Osmia bicornis and (right) Osmia cornuta. (A,B) Time required for larvae to reach the cocoon stage (days), (C,D) cocoon
weight (g) and (E,F) adult weight (g). Boxes indicate the 1st and 3rd interquartile range with line denoting median (n = 40 per pollen diet). Different letters indicate
significant differences between pollen diets (ANOVA tests followed by Tukey’s multiple comparison tests: p < 0.05). FIGURE 4 | Influence of pollen diets on (left) Osmia bicornis and (right) Osmia cornuta. (A,B) Time required for larvae to reach the cocoon stage (days), (C,D) cocoon
weight (g) and (E,F) adult weight (g). Boxes indicate the 1st and 3rd interquartile range with line denoting median (n = 40 per pollen diet). Different letters indicate
significant differences between pollen diets (ANOVA tests followed by Tukey’s multiple comparison tests: p < 0.05). in total content (Roulston and Cane, 2000; Roulston et al., 2000). With a variation from 4.6 to 9% of total lipid content, our diets are
again in range with no extreme values. Note that the lipid analysis
method may not completely lyse the pollen grains. There may
therefore be a slight difference between the results of the analysis
and what can be assimilated by the bee. (TSo diet). With more than 60 plant genera containing more
than 40% of protein in pollen (Roulston and Cane, 2002), our
diets are in the range of what can be find in natura, but no
pollen with a particularly low or high protein content was tested
here. As we used pollen blends and not monofloral diets, it was
expected that no extreme values would be observed (Roger et al.,
2017b). Lipids analyses also showed differences between the diets. Previous analyses showed that lipids can range from ∼1 to 20% Amino acids analyses showed similar amino acids profiles
were similar between pollen blends. Relationship Between Bee Performance
Traits and Pollen Chemical Composition B.terrestris, O.bicornis and O.cornuta worst performances were
observed when fed with C diet, whereas a different result was
recorded for A.mellifera, for which worst performance was
observed when fed with TSo diet. A.mellifera performances fed
with C diet were average, while TSo diet was good and average
for B.terrestris and Osmia species, respectively. Diet effects were
similar between O.bicornis and O.cornuta. As both species are
included in the same genus, it can be expected that nutritional
preferences are conserved. Finally, sterols analyses could explain observed results
regarding B. terrestris and O. cornuta measured performance
traits. Some sterolic compounds have already been reported
to be positively correlated with larval growth (Vanderplanck
et al., 2014). 24-methylenecholesterol is known to influence
molting and ovary development (Svoboda et al., 1978, 1983;
Human et al., 2007), while β-sitosterol and δ5-avenasterol are
supposedly involved in metabolic pathways of B. terrestris or have
a phagostimulant effect (Regali, 1996). Sterols in the C diet did
not affect A. mellifera performances. However, these results could
be explained by the performance traits tested in our experiment,
which did not take into account reproductive performances. In
this regard, it could be interesting to test the effect of diet on larval
development and not only on adults, to better evaluate the sterols
effect, known to have a positive effect on the larval development
on other bee species. Nevertheless, TSo diet, which caused the
lowest survival rate in A. mellifera, contained relatively high
concentrations of cholesterol and δ7-stigmasterol. If no negative
effect has been reported for cholesterol in the literature, maybe
this compound was in excess for A. mellifera. On the other hand,
δ7-stigmasterol have already been reported as not beneficial
for bumblebees (Vanderplanck et al., 2018). A. mellifera could
therefore be more sensitive to the presence of this compound
compared to other species. Finally, O. bicornis reached cocoon
stage quicker when fed with BQ pollen mix. This diet has
a relatively high concentration of stigmasterol, suggesting a
potential role in the larval development of this species. It has
recently been reported by Ruedenauer et al. (2021) that bees
can’t taste sterols, limiting the bee ability to forage on plants
with beneficial sterol composition or to avoid detrimental ones. Moreover, this work and previous studies showed that pollen
consumption is not adjusted to compensate low nutritional
quality of a diet (see Vanderplanck et al., 2014 for B. Variation in Chemical Composition of
Pollen Diets These results are in line Frontiers in Sustainable Food Systems | www.frontiersin.org February 2022 | Volume 6 | Article 824750 10 Barraud et al. Nutrition Across Bee Species (Standifer, 1967; Herbert et al., 1977), suggesting that high levels
of proteins can lead to deleterious effect. with previous studies, suggesting that amino acid profiles are
conserved among plants (Roulston and Cane, 2002; Weiner et al.,
2010; Vanderplanck et al., 2014). However, differences between
our diets have been observed in term of total and essential amino
acids, and the amount of specific amino acids. In this regard,
we have observed that C diet contains 54–72% less amino acids
and 48–61% less total and essential amino acids, compared to
other diets. Differences in amino acids content between the different
diets did not impact A. mellifera, but seems to explain results
observed with B. terrestis and Osmia species, with more than 3
times less pollen efficacy for B. terrestris, and up to −22 and
−26% adult weight for O.bicornis and O.cornuta, respectively,
when fed with C diet, which had the lowest amount of total
and essential amino acids. These results are similar to those of
Archer et al. (2021), who concluded that amino acid intake was
positively correlated with bumblebee body mass, and underlined
that the effects of total amino acids intake may depend on the
blend of individual amino acids. Interestingly, bumblebees can
perceive some amino acids via chemotactile antennal stimulation
and distinguish different concentrations without being able to
differentiate each amino acid (Ruedenauer et al., 2019). This way,
bumblebees could therefore assess the overall quantity of some
amino acids and adapt their foraging decisions. However, such
strategy could also lead wrong decisions, i.e., a rich pollen in
non-favorable amino acids proportions. Finally, among our different diets, total sterol concentration
and sterolic profiles were different with a lower total sterol
content in the C diet. Those results corroborate with previous
studies that showed a high variability in sterolic compounds
concentrations across plant species (Vanderplanck et al., 2014,
2018). Frontiers in Sustainable Food Systems | www.frontiersin.org ACKNOWLEDGMENTS We acknowledge all people that helped us to carry out this
experiment especially K. Przybyla, M. Folschweiller, E. Zambra,
M. Gerard, D. Evrard, and M. Bonneville for their help during
colony dissection. We thank also D. Evrard (University of Mons)
for the maintenance of bumblebee breeding room. This study does did consider every element that can
alter the quality of a pollen, such as pollenkit digestibility
or secondary metabolites (alkaloids, lactones, diterpenes, or
cyanogenic glycosides) (Peng et al., 1985; Detzel and Wink, 1993;
London-Shafir et al., 2003; Williams, 2003; Gunduz et al., 2008;
Kempf et al., 2010; Sedivy et al., 2012; Gosselin et al., 2013), and
did not evaluate directly the sterols effect on A. mellifera due to
measured parameters (only adults evaluated). Further research
is therefore needed to fully comprehend the importance of each
pollen-quality driver and interspecific differences. However, our FUNDING This research has received funding from the European Union’s
Horizon 2020 Research and Innovation Programme under grant
agreement N◦773921 for the POSHBEE project. DATA AVAILABILITY STATEMENT The original contributions presented in the study are included
in the article/Supplementary Material, further inquiries can be
directed to the corresponding author/s. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found
online
at:
https://www.frontiersin.org/articles/10.3389/fsufs. 2022.824750/full#supplementary-material Good Diets for Everyone study overall supports the need of conserving/introducing plant
diversity into agro-ecosystems to meet the nutritional preferences
of different bee species. y
Our results show that, if the 8 diet effects are different among
species, similarities can be noted. Because the best performances
of each species were shared across different diets, we could
not define only one specific favorable diet. However, a global
profile could be defined by comparing the nutritional factors
of these diets. Based on this and previous studies, a generally
good diet would require a high concentration of amino acids,
and of 24-methylenecholesterol, β-sitosterol and δ5-avenasterol
(Vanderplanck et al., 2014). P:L ratio did not impact measured
parameters of this study. While Vaudo et al. (2016) have shown
an impact of the P:L ratio on foraging behavior, the effects
of this ratio on colony development remain relatively unclear. More studies should be done to see if the foraging strategy in
favor of the P:L ratio results in effects on colony development,
by artificially modifying the protein and lipid content of the
same pollen without affecting the other compounds. Low protein
content did not affect bees in this study. However, as all diets
contained more than 20% of protein, we cannot conclude that
proteins are not playing a role in diet quality. Regarding the
literature, we hypothesize that protein content is important up
to a certain threshold that can be close to 20% depending
on the species (Regali and Rasmont, 1995; Génissel et al.,
2002; Taseï and Aupinel, 2008b; Alaux et al., 2010), and can
become detrimental if in excess (Standifer, 1967; Herbert et al.,
1977; Roulston and Cane, 2002; Human et al., 2007). Once the
minimum protein requirements are met, this work suggest that
amino acid and sterol compositions are playing a key role in
reproductive performances. Relationship Between Bee Performance
Traits and Pollen Chemical Composition terrestris
and Corby-Harris et al., 2018 for A. mellifera), suggesting that
a diet with a detrimental sterol composition could be regularly
consumed by the bees without them perceiving it and being able
to adapt their consumption. p
Several studies pointed out protein and/or protein:lipid ratio
as an indicator for pollen quality regarding developmental
performances (Roulston and Cane, 2002; Smeets and Duchateau,
2003; Alaux et al., 2010; Nicolson, 2011). However, observed
results and RSM analyses on Bombus terrestris showed that
performance cannot be explained by protein, lipid or P:L ratio,
while adult female mass of Osmia species is slightly affected by
protein content. Some good diets (see S pollen mix) and bad diets
(see C pollen mix) share the same protein, lipid, content and P:L
ratio. Those results indicate a difference in nutritional abilities
compared to A. mellifera for which RSM analyses highlighted an
importance of protein and lipids for their survivability. Despite
the common assumption that proteins are positively related to
performances (Regali and Rasmont, 1995; Génissel et al., 2002;
Roulston and Cane, 2002; Smeets and Duchateau, 2003; Alaux
et al., 2010; Nicolson, 2011; Stabler et al., 2015), in our case, the
only observed effects that can be related to protein content were
a negative one on Apis mellifera when fed with a protein-rich diet
(39.8%, TSo pollen mix) and a slight one on Osmia species that is
difficult to identify as positive or negative, as both pollen with the
highest (TSo) and lowest (C) protein content resulted in lighter
adults compared to other diets. Interestingly, Roulston and
Cane (2002) observed a higher mortality rate when Lasioglossum
zephyrum individuals were fed with a diet containing more
than 39% of total protein content. Moreover, it has already
been shown that in laboratory conditions, mortality and ovary
development in A.mellifera workers were negatively impacted
when fed with pollen containing 32% of proteins compared
to bees fed with 15% of proteins (Human et al., 2007). Other
authors observed similar mortality results with protein-rich diet February 2022 | Volume 6 | Article 824750 11 Barraud et al. Nutrition Across Bee Species AUTHOR CONTRIBUTIONS AB and LB wrote the manuscript and conducted experiments
on the bees. VL did all nutrient analyses. DS was involved in
Apis experiments. YL and CA supervised Apis experiments
and
redaction. F-VG,
FC,
GS,
and
CC
perfomed
the
palynological/pesticide residues analyses and wrote results
about it. MV and DM supervised the whole work. All authors
contributed to the article and approved the submitted version. REFERENCES Protein and amino acid requirements of the honey bee
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nectar and pollen - facts and possible roles. Plant Syst. Evol. 238, 87–95. doi: 10.1007/s00606-003-0272-y Michener, C. D. (2007). The Bees of the World, second edition. Baltimore, MD:
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the publisher, the editors and the reviewers. Any product that may be evaluated in
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Corvucci, Serra, Costa, Vanderplanck and Michez. This is an open-access article
distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the
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https://journal.unisa-bandung.ac.id/index.php/jka/article/download/164/110
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Indonesian
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PENGARUH SELF MANAGEMENT DIETARY COUNSELLING (SMDC) TERHADAP KUALITAS HIDUP PADA PASIEN HEMODIALISIS
|
Jurnal Keperawatan 'Aisyiyah
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ABSTRAK Pasien hemodialisis 40% mengalami malnutrisi. Salah satu upaya untuk menghindarinya
melalui Self Management Dietary Counselling (SMDC). SMDC bertujuan meningkatkan
kualitas hidup pasien hemodialisis. Penelitian ini bertujuan untuk mengetahui pengaruh
SMDC terhadap kualitas hidup pada pasien hemodialisis. Metode penelitian yaitu pretest
and posttest with control group design (quasi experiment with control). Sampel yaitu pasien
hemodialisis sebanyak 40 orang, dengan rerata usia 43 tahun dan jenis kelamin laki-laki. Teknik sampling menggunakan non pobablity sampling dengan pendekatan consecutive
sampling. Penelitian ini terdapat 2 kelompok yaitu kelompok intervensi dan kelompok kontrol. Intervensi dilakukan berupa SMDC. Sebelumnya dilakukan pengukuran kualitas hidup. Observasi dilakukan sebelum diberi perlakuan dan setelah diberi perlakuan. Pengambilan
data menggunakan Kidney Disease and Quality of Life Short Form Questionnaire-36 version
(KDQoL SF-36) dan SMDC. Hasil penelitian diperoleh peningkatan skor KDQoL-SF36 dari
rata-rata skor sebelum intervensi 60 menjadi 80. Pada uji beda terdapat perbedaan kualitas
hidup pada kelompok intervensi (78,30 ±,4,23) memiliki kualitas hidup lebih baik dibanding
kelompok kontrol (68,00±,4,23). Hal ini menggambarkan semakin baiknya kualitas hidup
pasien. SMDC dapat meningkatkan kualitas hidup pada pasien hemodialisis, rumah sakit
hendaknya dapat melakukan konseling secara terus menerus, bagi peneliti selanjutnya
dapat menambahkan waktu pelaksanaan SMDC. Kata kunci : hemodialisa, kualitas hidup, self management dietary counselling PENGARUH SELF MANAGEMENT DIETARY COUNSELLING (SMDC) TERHADAP
KUALITAS HIDUP PADA PASIEN HEMODIALISIS Hermawati1, Ika Silvitasari2
1,2) Universitas ‘Aisyiyah Surakarta
hermawatifarid@rocketmail.com ARTIKEL PENELITIAN
JKA.2020;7(1):39-47
PENGARUH SELF MANAGEMENT DIETARY COUNSELLING (SMDC) TERHADAP
KUALITAS HIDUP PADA PASIEN HEMODIALISIS JKA.2020;7(1):39-47 ARTIKEL PENELITIAN Keywords: hemodialysis, quality of life, self management dietary counselling METODOLOGI Dilaporkan dari USRDS (The United States
Renal Data System) tahun 2013 menunjukkan
angka prevalensi pasien gagal ginjal kronik yang
menjalani dialisis pada tahun 2013 di Amerika
Serikat sebesar 1.924 per 1 juta penduduk, di
Singapura sebesar 1.661 per 1 juta penduduk, dan
di Jepang sebesar 2.309 per 1 juta penduduk per
tahun (USRDS dalam Kusuma, 2013). Penderita
gagal ginjal kronik yang menjalani hemodialisa
di Indonesia pada tahun 2009 prevalensinya
12,5% atau 18 juta orang dewasa. Indonesia
termasuk negara dengan tingkat penderita gagal
ginjal kronik yang cukup tinggi (Dinkes Pemprop
Jateng, 2014). Penelitian
ini
menggunakan
desain
penelitian pretest and posttest with control group
design (quasi experiment with control) dengan
teknik
pengambilan
sampel
menggunakan
nonpobablity sampling. Pendekatan menggunakan
consecutive sampling setelah melalui tahap
persetujuan etika yang dikeluarkan oleh rumah
sakit tempat penelitian dilakukan. Alat dan bahan
yang digunkan adalah lembar pengumpulan data,
kuesioner KDQoL-SF36, catatan medik, serta
SMDC. Penilaian kuesioner dilakukan sesuai
panduan pada A Manual for Use and scoring
KDQoL-SF36 yang dikembangkan oleh Research
and Development (RAND) universitas Arizona. Prosedur penilaian kuesioner diadopsi dari RAND
dengan rentang 0 hingga 100. Semakin besar
skor akhir kuesioner, menandakan semakin
baik kualitas hidup pasien. Populasi dalam
penelitian ini adalah semua pasien gagal ginjal
kronik yang menjalani hemodialisis di RSUD Dr. Moewardi. Teknik pengambilan sampel dengan
menggunakan nonpobablity sampling dengan
pendekatan consecutive sampling didapatkan 40
sampel berdasarkan kriteria inklusi dan eksklusi. Kriteria inklusi terdiri dari pasien yang menjalani
terapi hemodialisis 2 kali seminggu, IMT/BMI ≤
21 kg/m2,
periode hemodialisa ≥ 3 bulan. Kriteria
eksklusi yaitu pasien yang mengidap penyakit
kanker, sepsis dan AIDS, gangguan fungsi kognitif
(dengan skore MMSE< 24). Pasien yang menjalani hemodialisis 50%-
70% mengalami malnutrisi (Moattari et.al., 2012),
sehingga memiliki morbiditas dan mortalitas yang
lebih tinggi dibanding populasi normal (Syamsiah,
2011). Keadaan ini akan mempengaruhi kualitas
hidup
pasien. Kecenderungan
terjadinya
malnutrisi pada pasien hemodialisis, maka
penilaian status nutrisi menjadi bagian penting. Penelitian ini dilakukan menggunakan
kuesioner KDQoL-SF36 yag merupakan kuesioner
spesifik untuk menilai kualitas hidup pasien
hemodialisis. Penelitian
lain
yang
melihat
hubungan Hb terhadap kualitas hidup pasien
gagal ginjal kronik berdasarkan usia serta
pemberian terapi anemia Namun demikian, belum
ada penelitian yang menggunakan intervensi Self
Management Dietary Counselling (SMDC). SMDC
merupakan intervensi yang digunakan sebagai
terapi pendekatan behaviour cognitive. Tindakan
ini lebih menitikberatkan pada komunikasi
yang efisien kepada pasien sehingga membantu
pasien
mengevaluasi
setiap
keputusannya
dalam membuat keputusan baru yang lebih
tepat (Valsaraj, 2014) serta untuk meningkatkan
kepatuhan dan outcome klinik. Abstract Hemodialysis patients 40% experience malnutrition. One effort to avoid this is through
Self Management Dietary Counselling (SMDC). SMDC aims to increase the quality of life of
hemodialysis patients. This study aims to determine the effect of SMDC on quality of life
in hemodialysis patients. The research method is pre-test and post-test with control group
design (quasi experiment with control). Samples were 40 hemodialysis patients, with an
average age of 43 years and male sex. The sampling technique uses non-probability sampling
with consecutive sampling approach. This study has 2 groups: an intervention group and a
control group. The intervention was carried out in the form of SMDC. Previously measured
quality of life. Observations were made before being treated and after being treated. Retrieval
of data using Kidney Disease and Quality of Life Short Form Questionnaire-36 version (KDQoL
SF-36) and SMDC. The results obtained increase KDQoL-SF36 scores from the average score
before intervention 60 to 80. In the different tests there were differences in quality of life
in the intervention group (78.30 ± 4.23) had a better quality of life than the control group
(68, 00 ±4.23). SMDC can improve the quality of life in hemodialysis patients, the hospital
should be able to conduct counseling continuously, for researchers can then add the time of
implementing SMDC. Keywords: hemodialysis, quality of life, self management dietary counselling Keywords: hemodialysis, quality of life, self management dietary counselling 39 Jurnal Keperawatan ‘Aisyiyah 40 METODOLOGI Hal ini akan
meningkatkan kualitas hidup pasien. Kriteria inklusi terdiri dari pasien yang menjalani
terapi hemodialisis 2 kali seminggu, IMT/BMI ≤
21 kg/m2,
periode hemodialisa ≥ 3 bulan. Kriteria
eksklusi yaitu pasien yang mengidap penyakit
kanker, sepsis dan AIDS, gangguan fungsi kognitif
(dengan skore MMSE< 24). Penelitian
ini
dilaksanakan
selama
5 minggu, dimulai pada bulan Juli 2019 dan
penelitian ini diawali terlebih dahulu dengan
pengambilan data pre intervensi pada minggu
ke 0 dan terdapat 2 kelompok yaitu kelompok
yang mendapat perlakuan dan kelompok kontrol. Kelompok perlakuan diberikan Self Management
Dietary Counseling (SMDC). Pre test dilakukan
pada minggu ke 1 dan minggu ke 3 sedangkan
evaluasi dilakukan pada minggu ke 2 dan minggu
ke 4 pada masing-masing kelompok. JKA | Volume 7 | Nomor 1 | Juni 2020 Pengaruh Self Management Dietary Counselling (SMDC) terhadap Kualitas Hidup
pada Pasien Hemodialisis Pengaruh Self Management Dietary Counselling (SMDC) terhadap Kualitas Hidup
pada Pasien Hemodialisis 41 41 perbedaan hasil pengukuran kualitas hidup
menggunakan
uji
Mann-Whitney. Sebelum
dianalisis, data dilakukan uji normalitas dengan
uji Kolmogorov-Smirnov. Peneliti menggunakan etika penelitian
yang terdiri dari 3 prinsip yaitu Prinsip manfaat
(beneficience), The principle of respect for human
dignity, Prinsip keadilan (right to justice). Selain itu
peneliti telah mendapatkan surat keterangan lolos
uji etik dari rumah sakit tempat dilaksanakannya
penelitian. JKA | Volume 7 | Nomor 1 | Juni 2020 HASIL Hasil penelitian didapatkan data distribusi
frekuensi, pengaruh SMDC terhadap kualitas
hidup, dan perbedaan hasil pengukuran pengaruh
SMDC terhadap kualitas hidup yang dapat dilihat
pada tabel 1. Analisis data penelitian ini menggunakan
uji statistik yang digunakan adalah uji non
parametrik,
untuk
mengetahui
pengaruh
SMDC terhadap kualitas hidup menggunakan
uji Wilcoxon, sedangkan untuk mengetahui . Distribusi Frekuensi& Uji Homogenitas Karakteristik Demografi Responden Table 1. Distribusi Frekuensi& Uji Homogenitas Karakteristik Demografi Resp Table 1. Distribusi Frekuensi& Uji Homogenitas Karakteristik Demografi R Table 1. Distribusi Frekuensi& Uji Homogenitas Karakteristik Demografi Responden
yang Menjalani Hemodialisis di RSUD Dr. Moewardi Surakarta Bulan Juli-Agustus 2019 (n=40)
Karakteristik
Kelompok
Pvalue*
I
II
n=40
%
n=40
%
Jenis Kelamin
Laki-laki
10
50,0
15
75,0
0,26
Perempuan
10
50,0
5
25,0
Tingkat pendidikan
SMP
5
25,0
8
40,0
0,93
SMA
11
55,0
9
45,0
Diploma
2
10,0
1
10,0
Sarjana (S1)
2
10,0
1
5,0
Umur (tahun)
21 - 30
1
100
0
0
0,41
31 - 40
5
22,7
10
45,5
41 - 50
8
47,1
3
17,6
51 - 60
6
30,0
7
35,0
Penghasilan Keluarga
> 1 juta
8
40,0
7
35,0
0,93
< 1 juta
12
60,0
13
65,0
Dukungan Sosial
Baik
0
0
0
0
-
Tidak baik
20
100
20
100
Riwayat Penyakit
Ya
19
95,0
18
90,0
0,80
Tidak
1
5,0
2
10,0
Medikasi
Ya
17
85,0
17
85,0
0,87
Tidak
3
15,0
3
15,0 JKA | Volume 7 | Nomor 1 | Juni 2020 Jurnal Keperawatan ‘Aisyiyah 42 Karakteristik
Kelompok
Pvalue*
I
II
n=40
%
n=40
%
Kebiasaan Merokok
Ya
3
15,0
6
30,0
0,23
Tidak
17
85,0
14
70,0
Tingkat Aktivitas
Ringan
2
10,0
2
10,0
0,73
Sedang
17
85,0
18
90,0
Berat
1
5,0
0
0
Depresi
Ringan
16
80,0
16
80,0
1,00
Sedang
4
20,0
4
20,0
Berat
0
0
0
0
I: (Intervensi), II: (kontrol)
*≥ 0.05 distribusi merata le 2. Distribusi Frekuensi Kualitas Hidup Responden pada Pasien Hemodialisis Table 2. Distribusi Frekuensi Kualitas Hidup Responden pada Pasien Hemodialisis
di RSUD Dr. Moewardi Surakarta Bulan Juli-Agustus 2019 (n=40)
Variabel
Kelompok
Total
I
II
n=40
%
n=40
%
n=40
%
Kualitas hidup(Pre intervensi)
Kurang berkualitas
18
90,0
16
80,0
34
85,0
Kulaitas baik
2
10,0
4
20,0
6
15,0
Post intervensi
Kurang berkualitas
8
40,0
16
60,0
24
60,0
Kualitas baik
12
60,0
4
40,0
16
40,0
I: (Intervensi), II: (kontrol) Table 3. HASIL Analisis Pengaruh SMDC terhadap Kualitas Hidup pada Kelompok Intervensi dan Kelompok
Kontrol pada Pasien yang Menjalani Hemodialisis di RSUD Dr. Moewardi Surakarta
Bulan Juli-Agustus 2019 (n=40)
Perlakuan
Mean (SD)
Z
pValue*
(95% CI)
Kelompok II
Post intervensi
68,00(±,10,75)
-2,14
0,00 (62,97-73,03)
Kelompok I
Post intervensi
78,30 (±,4,23)
-3,84
0,00 (76,32-80,28)
I (Intervensi), II (kontrol)
*< 0.05 berarti signifikan JKA | Volume 7 | Nomor 1 | Juni 2020 Pengaruh Self Management Dietary Counselling (SMDC) terhadap Kualitas Hidup
pada Pasien Hemodialisis 43 Table 4. Analisis Perbedaan Hasil Pengukuran Pengaruh SMDC terhadap Kualitas Hidup antar
Kelompok Pre Intervensi & Intervensi pada Pasien yang Menjalani Hemodialisis di RSUD Dr. Moewardi Surakarta Bulan Juli-Agustus 2019 (n=40)
Perlakuan
Mean (SD)
Z
pValue*
(95% CI)
Kualitas Hidup
Kelompok I& II
Pre intervensi
54.35 (±9.89)
-,76
0,06 (48,95-59,94)
Post intervensi
67.30(±12,83)
-3,58
0,00 (62,97-73,03)
I (Intervensi, II (kontrol),
*< 0.05 berarti signifikan Table 4. Analisis Perbedaan Hasil Pengukuran Pengaruh SMDC terhadap Kualitas Hidup antar
Kelompok Pre Intervensi & Intervensi pada Pasien yang Menjalani Hemodialisis di RSUD Dr. Moewardi Surakarta Bulan Juli-Agustus 2019 (n=40) erbedaan Hasil Pengukuran Pengaruh SMDC terhadap Kualitas Hidup antar
tervensi & Intervensi pada Pasien yang Menjalani Hemodialisis di RSUD Dr. kualitas hidup. kualitas hidup. Pemberian konseling hendaknya diberikan
kepada pasien yang menjalani hemodialisis
karena konsep SMDC perlu diterapkan dalam
keperawatan untuk menangani pasien-pasien
dengan
penyakit
kronis,
seperti
diabetes
mellitus dan gagal hinjal kronis yang menjalani
hemodialisis (Hidayati, 2012). Salah satu tugas
seorang perawat nefrologi adalah memberikan
konseling kepada pasien untuk membantu pasien
dalam
meningkatkan
kemandirian
sehingga
pasien dapat mempertahankan kualitas hidup
ke arah yang lebih baik atau normal. Konseling
yang diberikan dapat membantu perawat dalam
mendeteksi tingkat pemahaman dan kepatuhan
pasien dalam menjalankan diet yang harus
dijalankan
sehingga
dapat
mengoptimalkan
asuhan
keperawatan
yang
akan
diberikan
(Ulfah, et.al., 2018). Studi lain menunjukkan
bahwa pasien gagal ginjal kronik yang menjalani
hemodialisis mengalami keputusasaan sehingga
mereka berpotensi tidak mematuhi terapi, salah
satunya pembatasan nutrisi sehingga hal ini
dapat menurunkan kualitas hidup pasien menjadi
kualitas hidup yang kurang baik (Kusuma, 2013). Pemberian SMDC memberikan perubahan
kualitas hidup yang lebih baik. Self Management
Dietary Counseling (SMDC) merupakan suatu
proses dengan tujuan untuk meningkatkan self
care dalam pengelolaan diet. Konseling gizi yang
diberikan merupakan suatu proses yang yang
sistematis dalam upaya mengidentifikasi masalah
gizi serta penyebabnya. Menurut Anita (2012)
pemberian konseling meningkatkan pengetahuan,
sikap dan tindakan atau kemampuan individu
atau keluarga tentang pengelolaan nutrisi. Setelah
melakukan konseling, individu dan keluarga
mampu
mengambil
langkah-langkah
untuk
mengatasi masalah gizinya termasuk perubahan
pola makan serta memecahkan masalah terkait
gizi ke arah kebiasaan hidup sehat. Kemampuan pengelolaan nutrisi yang
tepat dapat meningkatkan outcome klinik yang
baik. Salah satunya yang dapat diukur yaitu anemia. Anemia berdampak negatif terhadap kadar energi
dan akivitas, perilaku makan dan tidur, status
kesehatan umum, kehidupan seksual, dan dapat
menyebabkan kelelahan otot, kesemutan, serta
nafas pendek. Tabel
perbedaan
hasil
pengukuran
SMDC dalam kelompok pada penelitian ini
menunjukkan bahwa pemberian intervensi Self
Management Dietary Counselling (SMDC) pada Nilai rata-rata pada kelompok intervensi,
menunjukkan rata-rata kualitas hidup yang
baik (Mean 78,3) menunjukkan bahwa terdapat
pengaruh yang signifikan terhadap peningkatan JKA | Volume 7 | Nomor 1 | Juni 2020 Jurnal Keperawatan ‘Aisyiyah 44 kelompok I menunjukkan secara signifikan dapat
meningkatkan kualitas hidup.Tabel frekuensi dan
persentase responden berdasarkan kualitas hidup
pada follow up minggu ke 4 menunjukkan bahwa
responden yang mengalami kualitas hidup yang
kurang baik semakin sedikit. tetapi bisa juga melakukan kajian data ulang
serta menganalisis intervensi gizi yang telah
diberikan sebelumnya sehingga dapat mendeteksi
perubahan status nutrisi yang dapat terjadi pada
pasien hemodialisis. Kemampuan
self
care
pasien
yang
menjalani hemodialisis dalam penelitian ini
mengacu pada teori self care Orem. kualitas hidup. Self care
menurut Dorothea Orem adalah tindakan yang
mengupayakan orang lain memiliki kemampuan
untuk dikembangkan ataupun mengembangkan
kemampuan yang dimiliki agar dapat digunakan
secara tepat untuk mempertahankan fungsi
optimal
(Alligood,
2014). Ketidakmampuan
melaksanakan self care ini mengakibatkan gejala
yang dirasakan semakin berat dan menjadi
penyebab pasien menjalani hospitalisasi. Hal
ini dapat menurunkan nilai kualitas hidup pada
pasien (Britz & Dunn, 2010). Diketahui bahwa konseling yang dilakukan
pada kelompok I lebih efektif pada minggu ke
4. Kuantitas konseling merupakan salah satu
faktor yang memberikan perbedaan output
pada kemampuan peningkatan kualitas hidup. Kita ketahui bahwa pada kelompok II diberikan
konseling 2x seminggu dibandingkan kelompok I
yang hanya diberikan konseling 1x seminggu. Perubahan perilaku dapat terjadi karena
konseling merupakan siklus dari serangkaian
langkah-langkah
yang
saling
berkaitan,
berlangsung
terus
menerus,
dan
berulang. Pengulangan yang dilakukan terus menerus akan
melatih daya pikir dan keterampilan-keterampilan
sehingga akan membantu responden dalam
melakukan pemecahan masalah atau mengatur
dirinya sendiri (self management). Menurut teori
asosiasi menyatakan bahwa perubahan perilaku
terjadi melalui proses penyampaian informasi
yang diulang-ulang sehingga terjadi asosiasi
yang erat antara tanggapan-tanggapan yang
lama dan tanggapan-tanggapan baru yang akan
dimunculkan ke alam sadar (Kusuma, 2013). Hal ini sejalan dengan penelitian yang
dilakukan (Tovazzi & Mazzoni, 2012) yang
menyatakan bahwa sangat penting bagi pasien
untuk memberikan perhatian khusus dalam hal
kemampuan self care (perawatan diri) secara
mandiri karena kondisi pasien yang semakin
memburuk sering dikaitkan dengan kurangnya
perhatian pada self care. Kurangnya perhatian
pada self care terutama pada pengelolaan diet
nutrisi dapat menurunkan kualitas hidup. Faktor
yang dominan berhubungan dengan kualitas
hidup pada penelitian ini adalah hipertensi karena
sebagian besar pasien yang menjalani hemodialisis
di RSUD Dr.Moewardi mengalami hipertensi. Hal
ini diakibatkan oleh adanya riwayat hipertensi
yang tidak terkontrol, riwayat penyakit lain
misalnya Diabetes Mellitus (DM) kronis maupun
hipertensi yang ditimbulkan akibat adanya
penyakit gagal ginjal kronik. Hasil penelitian dan didukung oleh
penjelasan
di
atas
menunjukkan
bahwa
pemberian SMDC selama satu bulan lebih
bermakna dalam perubahan kualitas hidup yang
lebih baik. Self Management Dietary Counselling
(SMDC) merupakan suatu proses dengan tujuan
untuk meningkatkan self care dalam pengelolaan
diet. Konseling gizi yang diberikan merupakan
suatu proses yang didalamnya terdapat kegiatan
pengumpulan, verifikasi, dan interpretasi data
yang sistematis dalam upaya mengidentifikasi
masalah
gizi
serta
penyebabnya. kualitas hidup. Kegiatan
inibukan
hanya
mengumpulkan
data
awal, Sebagian besar pasien yang menjalani
hemodialisis diberikan obat anti hipertensi karena
hipertensinya tidak terkontrol, namun dari hasil
wawancara didapatkan umumnya pasien tidak JKA | Volume 7 | Nomor 1 | Juni 2020 Pengaruh Self Management Dietary Counselling (SMDC) terhadap Kualitas Hidup
pada Pasien Hemodialisis 45 kenaikan interdialytic weight gain pada pasien
hemodialisis, menunjukkan bahwa dari 110
pasien yang mendapatkan konseling tentang
pembatasan asupan cairan, 72% dari pasien
tersebut
menunjukkan
adanya
penurunan
inter dialytic weight gain. Hasil penelitian yang
dilakukan oleh Molfino et.al (2012), tidak sesuai
dengan hasil pada penelitian ini. Molfino et.al
(2012) yang melakukan penelitian yang berjudul
“Effect Of Intensive Nutritional Counselling And
Support On Clinical Outcomes Of Hemodialysis
Patients” menyatakan bahwa konseling nutrisi
yang diberikan tidak terikat waktu. Misalkan 1
(satu) kali seminggu atau 2 (dua) kali seminggu
karena
seorang
konselor
tetap
membuka
kesempatan kepada klien untuk dilakukan follow-
up atau seorang pasien dapat kembali ke konselor
bila diperlukan atau sesuai kebutuhan pasien. teratur meminum obat antihipertensi karena
beralasan tidak merasakan efek yang berbeda
antara sebelum maupun sesudah meminum obat
hipertensi. Menurut pasien, untuk menghilangkan
gejala penyakitnya yang terutama adalah dengan
dilakukan hemodialisis, sehingga segala gejala-
gejala ketidaknyamanan dapat hilang, misalnya
sesak nafas, oedema, mual, muntah, pusing. Hasil ini mendukung penelitian Ghaddar
(2012) di Lebanon pada pasien yang menjalani
hemodialisis, intervensi yang diberikan yaitu SMDC
yang diberikan sebanyak 2x dalam seminggu. Penelitian ini menunjukkan perubahan yang
signifikan pada peningkatan pengetahuan pasien,
kepatuhan, serta nilai biochemical clinic. Ghaddar
(2012) dalam penelitiannya menjelaskan bahwa
Self Management Dietary Counselling (SMDC)
merupakan intervensi yang digunakan sebagai
terapi pendekatan behavior cognitive. Terapi ini
bertujuan untuk meningkatkan pengetahuan
pasien dan partisipasi pasien dalam pengambilan
keputusan kesehatan serta untuk meningkatkan
kepatuhan dan outcome klinik. Berdasarkan hasil penelitian di atas
menununjukkan adanya pengaruh yang signifikan
terhadap perubahan kualitas hidup yang baik
dibandingkan pre intervensi. Self
Management
Dietary Counselling (SMDC) merupakan salah
satu konseling yang berisikan proses konseling
gizi dengan pendekatan behaviour cognitive. Peningkatan
pengetahuan
dan
kemampuan
individu
ini
akan
berpengaruh
terhadap
peningkatan kemampuan self care dan perubahan
status nutrisi. SMDC berisikan langkah-langkah
yang saling berkaitan, berlangsung terus menerus
dan berulang (Ghaddar, 2012). Diawali dari
langkah pertama yaitu membangun dasar-dasar
konseling, langkah kedua pengkajian gizi, langkah
ke tiga yaitu diagnosis gizi, langkah keempat
yaitu intervensi gizi, serta langkah ke lima yaitu
monitoring dan evaluasi. Penelitian lainnya yang dilakukan oleh
Hidayati (2012), pemberian konseling dapat
merubah
prilaku
seseorang
menjadi
lebih
baik. JKA | Volume 7 | Nomor 1 | Juni 2020 SIMPULAN DAN SARAN Konseling yang diberikan pada pasien
berisikan intervensi gizi dengan tujuan untuk
meningkatkan
pengetahuan
pasien
tentang
pemilihan bahan makanan sumber protein dan
pemahaman tentang pola makan tinggi protein
serta
meningkatkan
pengetahuan
tentang
penggunaan suplemen makanan. Selanjutnya
dalam konseling juga melakukan perhitungan
kebutuhan energi dan zat gizi serta menetapkan
preskripsi
diet
dengan
menekankan
pada
perubahan pola makan protein tinggi. Asupan
protein energi yang menurun, asidosis metabolik
dan
retriksi
protein
yang
dilakukan
juga
mempercepat terjadinya malnutrisi (Tovazzi,
2012). Nilai rata-rata menunjukkan peningkatan
kualitas hidup ke arah yang baik pada kelompok
yang
mendapatkan
SMDC. Nilai
rata-rata
menunjukkan 76,30 (kelompok I) dan 76,05
(kelompok II) yang berarti responden memiliki
kemampuan
untuk
meningkatkan
kualitas
hidup. Terdapat pengaruh dan perbedaan pada
kualitas hidup yang positif pada kelompok yang
mendapatkan SMDC, terutama pada follow-up
minggu ke 4, kualitas hidup meningkat dari kualitas
hidup kurang baik menjadi kualitas hidup yang
baik. Peneliti selanjutnya dapat mengembangkan
penelitian keperawatan dengan menambah waktu
pelaksanaan SMDC. Hasil penelitian dapat digunakan oleh
pusat pelayanan kesehatan terutama di rumah
sakit, institusi pendidikan serta bagi para
peneliti selanjutnya sehingga bisa berperan aktif
dalam meningkatkan kualitas hidup pasien yang
menjalani
hemodialisis
melalui
peningktana
self care dengan cara self management dietary
counselling. Langkah terakhir SMDC yaitu melakukan
monitoring dan evaluasi yaitu melalui perubahan
asupan protein sesuai preskripsi diet, perubahan
asupan energi, natrium, kalium, kalsium dan
fosfor sesuai dengan yang dianjurkan. Perubahan
Index Massa Tubuh (IMT) jika sebelumnya
kurang, perubahan nilai laboratorium (ureum,
kreatinin, hemoglobin, GFR dan elektrolit) ke arah
normal. Berkurangnya keadaan asites dan edema,
perubahan keluhan klinis yaitu berkurangnya
keluhan mual, muntah, mudah lelah dan gatal-
gatal serta perubahan pemilihan jenis makanan
(Ghaddar, 2012). kualitas hidup. Intensitas konseling dengan pendekatan
behaviour cognitive dapat diterapkan pada
pasien hemodialisis yang kurang patuh terhadap
diet yang telah dianjurkan. Konseling dengan
pendekatan behaviour cognitive yang berasumsi
setiap pribadi memiliki potensi untuk memilih
dan mengarahkan ulang atau membentuk ulang
nasibnya sendiri. Teori ini lebih menitikberatkan
pada komunikasi yang efisien kepada klien
sehingga membantu klien mengevaluasi setiap
keputusannya dalam membuat keputusan baru
yang lebih tepat sehingga dengan keputusan yang
tepat ini dapat membantu dalam meningkatkan
self care (Valsaraj, 2014). Langkah-langkah
konseling
di
atas
menggunakan pendekatan behaviour cognitive,
sehingga
diharapkan
terjadinya
perubahan
perilaku. Perubahan perilaku ke arah yang positif
dapat mempengaruhi terjadinya peningkatan
self care dalam mengelola diet nutrisi pasien Hal senada juga dikemukakan
oleh
Hidayati
(2012)
dalam
penelitian
tentang JKA | Volume 7 | Nomor 1 | Juni 2020 Jurnal Keperawatan ‘Aisyiyah 46 hemodialisis. Langkah ke tiga konseling yaitu
bersama-sama pasien memilih rencana alternatif
sebagai upaya perubahan perilaku diet yang
dapat diimplementasikan serta komitmen untuk
melaksanakan perlakuan diet serta membuat
rencana yang realistis dan dapat diterapkan. Komitmen yang dibuat tersebut merupakan
gambaran terhadap adanya upaya dari konseling
untuk meningkatkan kemampuan self care dalam
mengelola nutrisi. Peningkatan self care akan
berpengaruh pada perubahan status nutrisi ke
arah yang lebih baik (Moattari, 2012). hal ini berarti bahwa tetap ada proses belajar di
dalamnya. Proses belajar merupakan kegiatan
yang menghasilkan perubahan diri individu yang
sedang belajar, baik aktual maupun potensial. Hasil yang diberikan memang berbeda dengan
kelompok yang diberikan SMDC, karena proses
belajar pada SMDC terdapat stimulus sedangkan
pada kelompok kontrol sebaliknya. Makin banyak
dan sering diberikan stimulus, maka makin
memperkaya tanggapan pada subjek belajar. JKA | Volume 7 | Nomor 1 | Juni 2020 DAFTAR PUSTAKA Alligood, M.R., (Eds). (2014). Nursing Theory and
Their Work. St.Louis, Missouri : Elsevier
Mosby. Anita, A.Y. (2012). Hubungan Self Care dan Depresi
dengan Kualitas Hidup Pasien Heart Failure
di RSUP Prof. DR.R.D. Kandou Manado. Responden
pada
kelompok
kontrol
dapat membaca melalui leaflet yang diberikan, JKA | Volume 7 | Nomor 1 | Juni 2020 Pengaruh Self Management Dietary Counselling (SMDC) terhadap Kualitas Hidup
pada Pasien Hemodialisis 47 Controll Trial.Health and quality of life
outcomes journal. 10:115 Tesis.Universitas Indonesia. Britz, J.A., & Dunn, K.S. (2010).Self Care and Quality
of Life among Patients with Heart Failure. Journal of the american academy of nurse
practicioners, 22, 480-487. Molfino, A., Chiappini M. G., Laviano, A., Ammann,
T., Bollea, M.R., Alegiani, F., Fanelli, F. R.,
Muscaritoli, M. (2012). Effect of intensive
Nutritional
Counseling
and
Support
on Clinical Outcomes of Hemodialysis
Patients.Elsevier Journal. Dinkes Pemprov Jateng. (2014). Profil Kesehatan
Jawa Tengah tahun 2014.6 Januari, 2015. http://www.dinkesjatengprov.go.id/ Syamsiah,
N. (2011). Faktor-Faktor
yang
Berhubungan dengan Kepatuhan Pasien
CKD yang Menjalani Hemodialisa di
RSPAU Dr. Esnawan Antariksa Halim
Perdana Kusuma Jakarta.Tesis.Universitas
Indonesia. Ghaddar, S. (2012).Nutritional Education for
The
Management
of
Osteodystrophy
(NEMO) in Patients on Hemodialysis : A
Randomized Control Trial. Journal of renal
care, 39 (1), 19-30. Hidayati, S. (2012). Efektifitas Konseling Analisis
Transaksional tentang Diet Cairan terhadap
Penurunan
Interdialytic
Weight
Gain
(IDWG) Pasien Gagal Ginjal Kronis yang
Menjalani Hemodialisa Di RSUD Kardinah
Tegal.Tesis.Universtitas Indonesia. Tovazzi, M.E., & Mazzoni, V. (2012). Personal
Paths Of Fluid Retriction In Patient On
Hemodialysis, nephrology nursing journal. 39 (3), 207-215. Ulfah, Yuniarti, Rahayuni. (2018). Efek Konseling
Gizi
Terhadap
Pengetahuan
Dan
Kepatuhan Pembatasan Intake Cairan
Pada Pasien Chronic Kidney Disease
(CKD) Di Rsud Abdul Wahab Sjahranie
Samarinda. Jurnal Nutrisia. Vol. 20 No. 1,
Maret 2018. ISSN 1693-945X (Print), ISSN
26147165 (Online). DOI 10.29238/jnutri. v20i1.253. Kusuma, H. (2013). Efektifitas Program Edukasi
Predialisis Berbasis Empowerment pada
Pasien Penyakit Ginjal Kronik untuk
Meningkatkan Efikasi Diri Pasien dalam
Melakukan Self Care, In : Aplikasi evidence
based
nursing
dalam
meningkatkan
patients safety. FK Universitas Diponegoro,
Semarang, PP 21 Juni 2014. Valsaraj, B.P., Valsaraj, P., Bhat, M.S., Prabhu, R.,
George, A. (2014). Development of Dietary
Guidelines
for
Persons
Undergoing
Haemodialysis.IOSR Journal of Nursing and
Health Science. Volume 3 issue 1 ver. II. PP
31-35. Moattari, M., Ebrahimi, M., Sharifi, N., & Rouzbeh,
J. (2012).The Effect Of Empowerment
On The Self-Efficacy, Quality Of Life And
Laboratory Indicators Of Patients Treated
With Hemodialysis : A Randomized JKA | Volume 7 | Nomor 1 | Juni 2020
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https://openalex.org/W4210652684
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https://hal.inrae.fr/hal-03719480/document
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English
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On an association between fear-induced aggression and striatal-enriched protein tyrosine phosphatase (STEP) in the brain of Norway rats
|
Biomedicine & pharmacotherapy
| 2,022
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cc-by
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On an association between fear-induced aggression and striatal-enriched
protein tyrosine phosphatase (STEP) in the brain of Norway rats On an association between fear-induced aggression and striatal-enriched
protein tyrosine phosphatase (STEP) in the brain of Norway rats Vitalii S. Moskaliuk a, Rimma V. Kozhemyakina a, Darya V. Bazovkina a, Elena Terenina b,
Tatyana M. Khomenko c, Konstantin P. Volcho c, Nariman F. Salakhutdinov c, Alexander
V. Kulikov a, Vladimir S. Naumenko a, Elizabeth Kulikova a,* a Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences (SB RAS), Pr. Lavrentyeva 10, 630090 Novosibirsk, Russia
b GenPhySE, Universit´e de Toulouse, INRA, INPT, ENVT, F-31326 Castanet-Tolosan, France a Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences (SB RAS), Pr. Lavrentyeva 10, 630090 Novosibirsk,
b GenPhySE, Universit´e de Toulouse, INRA, INPT, ENVT, F-31326 Castanet-Tolosan, France
c N.N. Vorozhtsov Institute of Organic Chemistry, SB RAS, 9 Lavrentieva Avenue, 630090 Novosibirsk, Russia Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences (SB RAS), Pr. Lavrentyeva
b GenPhySE, Universit´e de Toulouse, INRA, INPT, ENVT, F-31326 Castanet-Tolosan, France
c N.N. Vorozhtsov Institute of Organic Chemistry, SB RAS, 9 Lavrentieva Avenue, 630090 Novosibirsk, Russia GenPhySE, Universite de Toulouse, INRA, INPT, ENVT, F-31326 Castanet-Tolosan, France
c N.N. Vorozhtsov Institute of Organic Chemistry, SB RAS, 9 Lavrentieva Avenue, 630090 Novosibirsk, Russia A R T I C L E I N F O Keywords:
Genetically determined aggression
Rats
Ptpn5
Striatal-enriched protein tyrosine phosphatase
Fluoxetine
TC-2153
Chemical compounds:
TC-2153 (PubChem CID: 92042875)
Fluoxetine (PubChem CID: 62857) Striatal-enriched protein tyrosine phosphatase (STEP) is a signal transduction protein involved in the patho
genesis of neuropathologies. A STEP inhibitor (TC-2153) has antipsychotic and antidepressant effects. Here, we
evaluated the role of STEP in fear-induced aggression using Norway rats selectively bred for 90 generations for
either high aggression toward humans (aggressive rats) or its absence (tame rats). We studied the effects of acute
administration of TC-2153 on behavior and STEP expression in the brain of these animals and the influence of
chronic treatment with TC-2153 on the behavior and STEP expression in aggressive rats in comparison with
classic antidepressant fluoxetine, which is known to exert antiaggressive action. Acute TC-2153 administration
decreased the aggressive reaction to humans in aggressive rats, while having no impact on the friendly behavior
of tame rats. Moreover, in the elevated plus-maze test, the drug had an anxiolytic effect on both aggressive and
tame rats. Aggressive rats demonstrated elevated levels of a STEP isoform (STEP46) as compared to tame ani
mals, whereas acute TC-2153 administration significantly reduced STEP46 protein concentration in the brain of
aggressive rats. Chronic treatment of aggressive rats with either TC-2153 or fluoxetine attenuated fear-induced
aggression. Chronic administration of fluoxetine enhanced the exploratory activity in the elevated plus-maze test
and decreased the STEP46 protein level in aggressive rats’ hippocampus, whereas chronic TC-2153 adminis
tration did not affect these parameters. Thus, STEP46 can play an important role in the mechanisms of aggression
and may mediate antiaggressive effects of TC-2153 and fluoxetine. Chemical compounds:
TC-2153 (PubChem CID: 92042875)
Fluoxetine (PubChem CID: 62857) Russia) for enhancement (aggressive rats) or absence (tame rats) of
aggression toward humans. In addition to pronounced fear-induced
aggression, the aggressive rats manifest high anxiety [1] and demon
strate deficits of learning and memory in the Morris water maze test [59]
as compared to the tame rats. It is worth noting that aggression toward
humans is a distinct type of aggression and differs from other types in
ethological expression and molecular mechanisms [47,70]. The tame
rats show friendly behavior and trust and express interest in humans. Furthermore, the aggressive and tame rats differ in brain 5-HT and Contents lists available at ScienceDirect Contents lists available at ScienceDirect Available online 29 January 2022
0753-3322/©
2022
The
Authors.
Published
by
Elsevier
Masson
SAS.
This
is
an
open
access
article
under
the
CC
BY
license
(http://creativecommons.org/licenses/by/4.0/).
https://doi.org/10.1016/j.biopha.2022.112667
Received 11 November 2021; Received in revised form 14 January 2022; Accepted 24 January 2022 E-mail addresses: moskaliukvs@bionet.nsc.ru (V.S. Moskaliuk), korimma@bionet.nsc.ru (R.V. Kozhemyakina), daryabazovkina@gmail.com (D.V. Bazovkina),
elena.mormede@inra.fr (E. Terenina), chomenko@nioch.nsc.ru (T.M. Khomenko), volcho@nioch.nsc.ru (K.P. Volcho), anvar@nioch.nsc.ru (N.F. Salakhutdinov),
akulikov@ngs.ru (A.V. Kulikov), naumenko2002@mail.ru (V.S. Naumenko), lisa_kulikova@ngs.ru, kulikova.elisa@gmail.com (E. Kulikova). Biomedicine & Pharmacotherapy 147 (2022) 112667 Biomedicine & Pharmacotherapy 147 (2022) 112667 y
0753-3322/©
2022
The
Authors.
Published
by
Elsevier
Masson
SAS.
This
is
an
open
access
article
under
the
CC
BY
license
(http://creativecommons.org/licenses/by/4.0/). Available online 29 January 2022
0753-3322/©
2022
The
Authors.
Published
by
Elsevier
Masson
SAS.
This
is
an
open
access
article
under
the
CC
BY
license
(http://creativecommons.org/licenses/by/4.0/).
Corresponding author.
E-mail addresses: moskaliukvs@bionet.nsc.ru (V.S. Moskaliuk), korimma@bionet.nsc.ru (R.V. Kozhemyakina), daryabazovkina@gmail.com (D.V. Bazovkina),
elena.mormede@inra.fr (E. Terenina), chomenko@nioch.nsc.ru (T.M. Khomenko), volcho@nioch.nsc.ru (K.P. Volcho), anvar@nioch.nsc.ru (N.F. Salakhutdinov),
akulikov@ngs.ru (A.V. Kulikov), naumenko2002@mail.ru (V.S. Naumenko), lisa_kulikova@ngs.ru, kulikova.elisa@gmail.com (E. Kulikova).
https://doi.org/10.1016/j.biopha.2022.112667
Received 11 November 2021; Received in revised form 14 January 2022; Accepted 24 January 2022 2.2. Experimental procedures and drugs Experiment 1. First of all, we evaluated the impact of acute admin
istration of the STEP inhibitor (TC-2153) on the behavior and STEP
expression in the brain of the aggressive and tame rats. To this end, 60
rats (30 aggressive and 30 tame) were used. Two days before the drug
administration, the animals were evaluated in the glove test to deter
mine the basic scores of their aggressive or tame behavior. The STEP
selective inhibitor TC-2153 [8-(trifluoromethyl)−1,2,3,4,5-benzo
pentathiepin-6-amine hydrochloride, NIOCh, Novosibirsk, Russia] was
diluted in a solution of 0.05% Tween 20 and 0.05% DMSO as described
earlier [40]. Three groups of 10 rats of both strains were treated intra
peritoneally (100 µL/100 g of body weight) with 10 or 20 mg/kg
TC-2153 or vehicle (a 0.05% solution of Tween 20 and DMSO) as a
control. Three hours after single TC-2153 or vehicle injection, the glove
test and, immediately after, the elevated plus-maze (EPM) test were
performed. Two hours after the behavioral tests, the rats were decapi
tated; their brains were rapidly removed on ice; the whole hypothala
mus, hippocampus, and midbrain were dissected, frozen in liquid
nitrogen, and stored at −80 ◦C. l STEP selective inhibitor TC-2153 [8-(trifluoromethyl)−1,2,3,4,5-
benzopentathiepin-6-amine hydrochloride], first synthesized at Novo
sibirsk Institute of Organic Chemistry (Novosibirsk, Russia) [29], has
strong psychotropic effects. TC-2153 possesses anxiolytic [29], anti
cataleptic [37] and antidepressant properties [36,38–40]. Furthermore,
administration of TC-2153 increases the level of brain-derived neuro
trophic factor (BDNF) [37] and significantly affects the serotonergic
(5-HT) system [35,39,40]. Accordingly, TC-2153 has been proposed as a
promising new-generation antidepressant [38,41]. Classic antidepres
sant fluoxetine raises the BDNF level and—just as other anti
depressants—diminishes aggression [2,4,9,18,24,64,80]. Taking into
account the close interactions among STEP, neurodegenerative disor
ders, BDNF, and 5-HT, we hypothesized STEP involvement in the
mechanism of fear-induced aggression and the potential antiaggressive
activity of a promising new-generation antidepressant, TC-2153. g
Experiment 2. We studied the influence of chronic TC-2153 or
fluoxetine administration on behavior and STEP expression in the brain
of aggressive rats. In this experiment, 29 aggressive male rats were
subdivided into three groups and were treated with a drug dissolved in
drinking water for 30 days. The administration of drugs via drinking
water was chosen to avoid handling effects and minimize the stress of
animals. 1. Introduction Aggressive behavior (including overt fighting) plays an important
role in the life of animal populations and their evolution [45]. In the
wild, aggression is important for protection, reproduction, and survival. The molecular mechanisms underlying this complicated type of
behavior are still unknown. Suitable models for studying the mechanism
of aggression are two strains of wild Norway rats selectively bred for 90
generations at the Institute of Cytology and Genetics (Novosibirsk, Abbreviations: 5-HT, serotonergic system; ANOVA, analysis of variance; BDNF, brain-derived neurotrophic factor; DMSO, dimethyl sulfoxide; EPM, elevated plus-
maze; PTPN5, protein tyrosine phosphatase nonreceptor type 5; SD, standard deviation; SDS, sodium dodecyl sulfate; STEP, striatal-enriched tyrosine protein
phosphatase; TC-2153, 8-(trifluoromethyl)-1,2,3,4,5-benzopentathiepin-6-amine hydrochloride. Biomedicine & Pharmacotherapy 147 (2022) 112667 V.S. Moskaliuk et al. BDNF systems [25,30,51,66,68,69,86], indicating a potential alteration
of neuroplasticity in the brain of these two strains. BDNF systems [25,30,51,66,68,69,86], indicating a potential alteration
of neuroplasticity in the brain of these two strains. and Use of Laboratory Animals approved by the Ministry of Public
Health of Russia (Supplement to decree No. 267, June 19, 2003) and
with National Institutes of Health “Guide for the Care and Use of Lab
oratory Animals” (NIH Publication No. 8023, revised in 1978) and were
approved by the ethical committee of the Institute of Cytology and
Genetics. Moreover, it has been shown that aggression is often associated with
neurodegenerative disorders [16,43,46,55,87]. Therefore, the proteins
involved in the regulation of neuronal plasticity may be implicated in
the mechanism of aggression. Every effort was made to minimize the number of animals used and
their suffering. The study was conducted at the Centre for Genetic Re
sources of Laboratory Animals at the federal research center Institute of
Cytology and Genetics, the Siberian Branch of the Russian Academy of
Sciences (RFMEFI62119×0023). The animals’ maintenance was funded
by the Basic Research Project for a Young Researcher, grant number
FWNR-2022-0010. Striatal-enriched protein tyrosine phosphatase (STEP, also known as
protein tyrosine phosphatase nonreceptor type 5 [PTPN5]) is encoded
by the Ptpn5 gene, and its transcript undergoes alternative splicing
resulting in two active isoforms: STEP61 and STEP46 [44]. Both STEP61
and STEP46 contain a kinase-interacting motif and a protein tyrosine
phosphatase domain [44]. In contrast to the cytosolic isoform (STEP46),
membrane-associated STEP61 is well studied and has additional 172
amino acid residues at the N terminus including two transmembrane
domains and two polyproline domains for specific substrate binding [5]. 2.1. Animals 2.1. Animals The experiments were performed on adult outbred male rats (Rattus
norvegicus) (4–5 months old, weighing 300–350 g) selectively bred from
wild rats for 90 generations at the Institute of Cytology and Genetics
(Novosibirsk) for the absence (tame rats, n = 30) or enhancement
(aggressive rats, n = 59) of an aggressive reaction to humans [49,61]. In
this study, we used male rats to avoid potential effects of the estrous
cycle of female rats on behavior and on gene expression in the brain. 1. Introduction STEP61 is highly expressed throughout the central nervous system [26],
whereas STEP46 has so far been found only in the striatum, amygdala,
optic nerve, and retina [19,78]. STEP regulates synaptic plasticity by
dephosphorylating and inactivating neuronal substrates, including ki
nases ERK1/2 and p38 [48], FYN [57], PYK2 [89], and subunits of
NMDA receptor [42] and AMPA receptor [90]. STEP dysregulation is
associated with several neurodegenerative and psychiatric disorders, in
particular Alzheimer’s disease, Huntington’s disease, schizophrenia,
Martin–Bell syndrome, ischemia, and stress-related disorders [17,27]. l 2.2. Experimental procedures and drugs Nine animals received an aqueous solution of 0.04% DMSO and
0.08% Tween 20 (control group); 12 rats were treated with 80 mg/L TC-
2153 in an aqueous solution of 0.04% DMSO and 0.08% Tween 20; and
eight rats received 80 mg/L fluoxetine hydrochloride, i.e., ( ± )-N-
methyl-γ-[4-(trifluoromethyl)phenoxy]benzenepropanamine
hydro
chloride (Merck, USA), in the same solution of vehicle. The approximate
amount of drinking solutions was 35–45 mL per rat per day. The drugs
and vehicle were refreshed every 2 days. The concentration of 80 mg/L
in water corresponds to a daily dose of approximately 10 mg/kg [20,28,
71,82]. Before the drug administration, the animals were assessed by the
glove test to determine their basic aggression scores. Subsequently,
aggression scores were obtained on the 4th, 11th, 18th, 25th, and 29th
days of treatment. On the 25th day of drug administration, the animals
were isolated in individual cages to remove the group effect. On the 29th
day of treatment, the EPM test was performed, and on the 30th day, the
rats were decapitated. The brains were rapidly excised on ice; the whole
hypothalamus, hippocampus, and midbrain were dissected, frozen in
liquid nitrogen, and stored at −80 ◦C. Thus, the aim of this study was to evaluate the role of STEP isoforms
in fear-induced aggression and a potential antiaggressive activity of TС-
2153. For this purpose, we investigated the association between the
expression of two isoforms of STEP in the brain and fear-induced
aggression in the rats selectively bred for aggressive and tame
behavior toward humans as well as the effects of acute TC-2153
administration on the behavior and expression of STEP in the brain of
the aggressive and tame rats. Furthermore, we tested whether these
effects would persist during chronic TC-2153 administration to aggres
sive rats and compared its action with the classic antidepressant
fluoxetine. 2.6. Western blot analysis For assessment of the STEP protein level, 100 µL of the homogenate
in Tris-HCl buffer (50 mM, pH 7.6) was added to 100 µL of homogeni
zation buffer (300 mM NaCl, 100 mM Tris-HCl pH 8, 4 mM EDTA, 0.2%
of Triton X-100) with protease inhibitors (Pierce Protease Inhibitor
tablets [Thermo Scientific], 1 mM sodium orthovanadate, and 2 mM
PMSF) and rested for 1 h on ice. Then, the homogenate was centrifuged
for 15 min at 12000 × g (4 ◦C), and the supernatant was transferred to a
fresh 1.5 mL tube and kept at −80 ◦C. p
The concentration of total protein was evaluated by a bicinchoninic
acid assay (Pierce BCA Protein Assay Kit) according to the manufac
turer’s instructions with the help of an Eppendorf spectrophotometer
(BioPhotometer plus, Eppendorf, USA) followed by adjustment of the
samples to equal total-protein concentrations with 2 × Laemmli sample
buffer (4% of SDS, 20% of glycerol, 120 mM Tris-HCl pH 6.8, 10% of
mercaptoethanol, 0.02% of bromophenol blue) and denaturing by
boiling for 5 min at 95 ◦C. The extracts (20 µg per lane) were resolved on
an SDS 10% polyacrylamide gel and blotted onto a nitrocellulose
membrane (Thermo Fisher Scientific Inc., USA) with a TE77 PWR
Semidry Blotter (GE Healthcare Bio-Sciences AB, Sweden) at a 50 mA
current and room temperature overnight. The membranes were blocked
in Tris-buffered saline supplemented with 0.1% of Tween 20 (TBST) and
5% of BSA for 1 h, rinsed, and next incubated with a polyclonal mouse
anti-STEP antibody (dilution 1:1000 in 5% BSA, cat. # sc-23892, lot #
KO314, Santa Cruz Biotechnology, USA) at 4 ◦C overnight and then with
a goat anti-mouse IgG antibody conjugated with horseradish peroxidase
(secondary antibody; dilution 1:10000, cat. # 31430, lot # TL272497,
Invitrogen, USA) for 1 h at room temperature. Between the incubation
steps, the membranes were washed in TBST five times for 5 min each
time. The blots were treated with the SuperSignal West Femto Maximum
Sensitivity Substrate (Thermo Fisher Scientific Inc., USA) according to
the manufacturer’s instructions. Protein bands were detected using a
Fusion FX7–820 system (Vilber Lourmat, France). Quantification of
protein bands was performed by volume densitometry in the ImageJ
software (NIH, USA). As a reference, we probed the blots with an anti-
GAPDH antibody (dilution 1:2000 in 5% BSA, cat. Table 1 Table 1
The primer sequences, annealing temperatures, and amplicon lengths. Gene
Sequence
Annealing
temperature,
◦C
Product
length,
bp
Polr2a
F: 5′-
TTGTCGGGCAGCAGAACGTG-3′
R: 5′-
CAATGAGACCTTCTCGTCCTCCC-
3′
63
186
Ptpn5_ex8
(exon 8)
F: 5′-
GACAGACGACAATCAGTGAGC-3′
R: 5′-
AGTCAACGAGGTGGGATCAG-3′
63
162
Ptpn5_ex16
(exon 16)
F: 5′-
CGTGTTTTTATCGCTGACTTTAA-
3′
R: 5′-
GACACCAAATGTCTCTGTATG-3′
61
59 The primer sequences, annealing temperatures, and amplicon lengths. 2.6. Western blot analysis # ab9485, lot #
GR192141–5, Abcam, UK) at 4 ◦C overnight and a goat anti-rabbit IgG
antibody conjugated with horseradish peroxidase (secondary antibody;
dilution 1:10000, cat. # 31460, lot # SH253595, Invitrogen, USA) for 1
h at room temperature to detect the signal. STEP61 and STEP46 protein
levels were evaluated as a percentage ratio of fluorescence intensity of
STEP to the fluorescence intensity of the GAPDH band. STEP61 and
STEP46 were detected at 61 and 46 kDa, respectively. GAPDH migrated
at the 37 kDa level. The whole structures of the brain (hippocampus, hypothalamus, and
midbrain) were homogenized in 300 µL of Tris-HCl buffer (50 mM, pH
7.6) at 4 ◦C by means of a motor-driven grinder (Z359971, Sigma-
Aldrich, USA), and aliquots of the homogenate were used for total-
RNA and protein extraction. 2.4. The glove (handling) test The behavior of rats was assessed judging by the expression of a
positive or negative emotional response to a gloved hand, depending on
the distance between the hand and the rat in the glove test. The gloved
hand was placed into a cage in which a rat was kept, and behavior to
ward the human being was evaluated on a scale ranging from −4 (as
aggressive as possible) to −1 (slightly aggressive) in aggressive rats and
from + 4 (maximally tame) to + 1 (somewhat tame) in tame rats [22,51,
62]. Negative behavior or aggressiveness was scored as follows: −4: as
the hand approached a closed cage door, the rat emits threatening
vocalization and attacks the hand; −3.5: as the cage door opens, the rat
attacks the hand as soon as it is placed on the open door; −3: as the cage
door opens, the rat retreats to the back wall, emits threatening vocali
zation, and attacks the approaching hand; −2.5: the rat stays at the back
wall, actively resists handling, and attempts to bite the hand; −2: the rat
stays at the back wall, resists handling, and when handled, tries to
escape and vocalizes; −1.5: the rat stays at the back wall, turns away
from the approaching hand, hides in the corner, resists handling to some
extent, and vocalizes; −1: the rat flees to the back wall, avoids human
touch and handling, and does not vocalize. Tameness was scored as
follows: 4: the rat freely explores the hand, as the door opens, is
completely relaxed when handled, displays interest in the researcher;
3.5: the rat freely explores the hand, as the door opens, is completely
relaxed when handled; 3: the rat freely explores the hand, as the door
opens, does not attempt to escape when handled but is somewhat tense;
2.5: the rat approaches the hand as the door opens and makes a weak
attempt to escape when handled; 2: the rat does not approach the hand
as the door opens, and when handled, makes a weak attempt to escape;
1.5: the rat stays at the back wall, approaches the extended hand, and
avoids handling; 1: the rat stays at the back wall, does not explore the
hand, and avoids the human touch and handling. 2.3. The EPM test The EPM device consisted of two crosswise arms (closed and open,
50 × 10 cm each) with a central area (10 × 10 cm). The closed arms
bordered 40-cm-high walls. This device was elevated by 50 cm above the
floor. A Panasonic camera on a tripod was placed at 50 cm from the end
of the open arm. A rat was placed in the center of the maze facing a The animals were housed in metal cages (50 × 33 × 20 cm) under
standard laboratory conditions on a natural light–dark cycle with free
access to water and food in groups of four individuals. All experimental
procedures were carried out in compliance with the Guide for the Care 2 Biomedicine & Pharmacotherapy 147 (2022) 112667 Biomedicine & Pharmacotherapy 147 (2022) 112667 V.S. Moskaliuk et al. closed arm, and the experimenter was able to watch the behavior of the
rat on a computer from an adjacent room [32,60]. The duration of the
test was 5 min. The behavior of rats was videotaped, and the time spent
(%) in the center and closed and open arms as well as time points and
numbers of peeks and head dips were analyzed by an experienced rater
blinded to the group assignment of the rats. The arena was cleaned with
wet and dry napkins after each test. 2.7. Statistical analysis 2.7. Statistical analysis The results of the glove test after acute administration of a drug are
presented as block plots with whiskers, where blocks indicate mean ±
SD, and whiskers min and max values. The data were analyzed by a
nonparametric test for two dependent samples (the Sign test) followed
by the Bonferroni correction. The data of the glove test in the chronic-
treatment experiment are presented as means of scores ± SD received
on each testing day. To evaluate the effect of timing (experiment day) on
the aggression scores in each group, we carried out a nonparametric test
for multiple dependent samples: the Friedman test. The Mann–Whitney
test is performed to assess the difference between the control and TC-
2153 as well as between the control and fluoxetine groups on each
day. The data from the Friedman and Mann–Whitney tests were sub
jected to the Bonferroni correction. The data from the EPM test and
assays of Ptpn5 mRNA and STEP protein expression were checked for
normality and equality of dispersion by Lilliefors and Barlett’s test,
respectively. These results are presented as mean ± standard error of the
mean and were analyzed in the first experiment by two-way ANOVA
with “Genotype” (strain) and “Treatment” as two independent factors
that can interact, and in the second experiment, by one-way ANOVA. If
any effect was significant, the difference between groups was assessed
by Fischer’s LSD post hoc multiple pair-wise comparisons. Statistical
significance was set to p < 0.05. The Dixon criteria were applied to
identify outliers. We noticed a strong influence of the genotype on all parameters
evaluated in the EPM test. Tame rats demonstrated longer time spent in
the center (F1,42 = 44.04, p < 0.001) (Fig. 2b), open arms (F1,42 = 11.14,
p < 0.01) (Fig. 2a), and longer rearings (F1,42 = 41.94, p < 0.001)
(Fig. 2e) and head dips (F1,42 = 18.75, p < 0.001) (Fig. 2d), whereas
time spent in the closed arms (F1,42 = 72.02, p < 0.001) (Fig. 2c) was
significantly shorter for tame rats than for aggressive ones. These results
revealed a higher exploratory activity and lower anxiety in tame rats
than in the aggressive animals. 2.7. Statistical analysis Moreover, we registered a significant
impact of TC-2153 on the time spent in the open (F2,42 = 4.65, p < 0.05)
and closed (F2,42 = 6.42, p < 0.01) arms, but there were no effects on the
time spent in the center (F2,42 < 1), and duration of rearings (F2,42 < 1)
and head dips (F2,42 = 1.56, p > 0.05). TC-2153 in the dose of 20 mg/kg
significantly prolonged the time spent in open arms for tame rats
(p < 0.05), shortened the time spent in the closed arms for both tame
(p < 0.01) and aggressive rats (p < 0.05), and increased the duration of
head dips in aggressive rats (p < 0.05). At the same time, there were no
effects of genotype × drug interaction on any investigated EPM test
parameters [time spent in the center (F2,42 < 1), open arms (F2,42 = 1.26,
p > 0.05), and closed arms (F2,42 < 1) and duration of rearings (F2,42 <
1) and head dips (F2,42 < 1)]. 3.2. Effects of acute TC-2153 treatment on the behavior of aggressive and
tame rats in the EPM test 3.2. Effects of acute TC-2153 treatment on the behavior of aggressive and
tame rats in the EPM test 2.5. Real-time quantitative PCR Total RNA was extracted from 60 µL of a homogenate in Tris-HCl
buffer (50 mM, pH 7.6) mixed with 300 µL of the TRIzol Reagent
(Ambion, USA) according to the manufacturer’s instructions. The
extracted RNA was treated with RNA-free DNase (Promega, USA) and
diluted to 0.125 µg/µL with diethyl pyrocarbonate–treated water. The
obtained total RNA was subjected to cDNA synthesis with a random
hexanucleotide mixture (BioLab Mix, Novosibirsk, Russia). To estimate
the number of Ptpn5 cDNA copies, we employed two pairs of primers:
the first one was selected in the 8th exon encoding a part of the STEP61
isoform, and the second pair in the 16th exon encoding a part of both the
STEP46 and STEP61 isoform (Table 1). SYBR Green I fluorescence
detection (R-402 Master mix, Syntol, Moscow, Russia) was performed. As external standards, we used genomic DNA (0.06, 0.125, 0.25, 0.5, 1,
2, 4, 8, 16, 32, and 64 ng/µL) extracted from the rat liver [34,52,53]. Gene expression was evaluated as the number of cDNA copies per 100
copies of a housekeeping gene: DNA-dependent RNA polymerase II
(Polr2a). To control amplification specificity, we performed melting
curve analysis at the end of each run for each pair of primers. 3 3 Biomedicine & Pharmacotherapy 147 (2022) 112667 V.S. Moskaliuk et al. V.S. Moskaliuk et al. 3.1. Effects of acute TC-2153 treatment on the aggressive and tame
behavior in the glove test We detected no effects of a drug (midbrain F2,35 < 1; hippocampus
F2,32 < 1; hypothalamus F2,33 < 1), genotype (midbrain F1,35 = 1.71,
p > 0.05; hippocampus F1,32 < 1; hypothalamus F1,33 = 1.30, p > 0.05),
or genotype × drug interaction (midbrain F2,35 < 1; hippocampus F2,32
< 1, hypothalamus F2,33 < 1) on the mRNA level of the STEP61 isoform
as measured by the Ptpn5_ex8 pair of primers (binding to exon 8 specific
to the STEP61 isoform) (Fig. 3a–c) in the assayed brain structures. Moreover, with the Ptpn5_ex16 pair of primers (binding to exon 16 and
detecting both STEP61 and STEP46 expression), no influence of a drug
(midbrain F2,35 = 1.49, p > 0.05; hippocampus F2,32 < 1; hypothalamus
F2,34 < 1), genotype (midbrain F1,35 < 1; hippocampus F1,32 < 1; hy
pothalamus F1,34 < 1), or genotype × drug interaction (midbrain F2,35 <
1; hippocampus F2,32 < 1, hypothalamus F2,34 = 2.11, p > 0.05) in the
studied brain structures was detected either (Fig. 3d−f). We found that acute TC-2153 treatment had an antiaggressive effect
on aggressive rats (Fig. 1a), without affecting tame animals (Fig. 1b). Vehicle administration to the aggressive control group (Z = 0.48, p >
0.05) did not alter the behavior of aggressive animals in the handling
test. TC-2153 at 10 mg/kg (Z = 2.27, p > 0.05) exerted a slight anti
aggressive action, whereas 20 mg/kg TC-2153 significantly reduced
aggression (Z = 2.67, p < 0.05) in these rats. TC-2153 in the dose of 10
mg/kg (Z = 1.22, p > 0.05) and 20 mg/kg (Z = 1.15, p > 0.05) as well as
vehicle administration had no effect on the glove reaction of tame rats
(Z = 1.15, p > 0.05) (Fig. 1b). Fig. 1. Effects of acute treatment with vehicle (control) or TC-2153 at 10 or 20 mg/kg on the behavior of aggressive (a) and tame (b) rats in the glove test. Data are
presented as block plots with whiskers, where blocks indicate mean ± SD, and whiskers min and max values. $p < 0.05 in comparison with the data point before
treatment; 10 animals per group. Fig. 1. Effects of acute treatment with vehicle (control) or TC-2153 at 10 or 20 mg/kg on the behavior of aggressive (a) and tame (b) rats in the glove test. 3.4. The impact of acute TC-2153 treatment on STEP61 and STEP46
protein levels in aggressive and tame rats in the aggression scores between the groups were detectable [H(2)
= 0.62, p > 0.05]. p
We found a sequential decrease of aggression in the TC-2153 group
[X2(5) = 38.22, p < 0.000001] and fluoxetine group [X2(5) = 29.37,
p < 0.0002] but not in the control group [X2(5) = 9.88, p = 0.079] in all
these experiments. Fluoxetine reduced the aggression on the 4th day
(p < 0.05) in comparison to the control group, whereas on the 11th day
(p > 0.05), this difference disappeared and returned only on the 18th
day (p < 0.05) and lasted until the end of the experiment. Meanwhile,
TC-2153 lowered aggression on the 11th day (p < 0.05) until the 25th
day (p < 0.05). On the 29th day, 4 days after the separation of the an
imals into individual cages, all the rats demonstrated elevation of
aggression
scores
[TC-2153
(−2.75 ± 0.26);
fluoxetine
(−2.375 ± 0.35); control (−3.11 ± 0.22)]. There aggression scores
diminished in the fluoxetine group (p < 0.05) but not in the TC-2153
group (p > 0.05) in comparison to the control animals. This phenome
non can be explained by the separation of the animals 4 days before the
test. We documented a significant effect of the genotype on the STEP61
level in the hippocampus (F1,37 = 5.59, p < 0.05) (Fig. 4b), while there
was no difference between the rat strains in this protein level in the
midbrain (F1,41 = 2.45, p > 0.05) (Fig. 4a) and hypothalamus (F1,37 < 1)
(Fig. 4c). No influence of a drug (midbrain F2,41 < 1, hippocampus F2,37
< 1; hypothalamus F2,37 < 1) or genotype × drug interaction (midbrain
F2,41 < 1, hippocampus F2,37 = 1.26, p > 0.05, hypothalamus F2,37 =
1.06, p > 0.05) was found in all the analyzed brain structures in terms of
the STEP61 level. i A significant effect of the genotype on the STEP46 protein level was
observed. Aggressive animals had an elevated concentration of the
STEP46 protein in the midbrain (F1,41 = 53.04, p < 0.001) (Fig. 5a),
hippocampus (F1,40 = 36.67, p < 0.001) (Fig. 5b), and hypothalamus
(F1,40 = 31.86, p < 0.001) (Fig. 5c) in comparison with tame rats. 3.6. Effects of chronic TC-2153 or fluoxetine treatment on the behavior of
aggressive rats in the EPM test Here we registered an effect of the drugs on the number of head
peeks from the closed arms (F2,26 = 4.32, p < 0.05). The group that
received fluoxetine (p < 0.01) demonstrated an increased number of
head peeks in comparison to the control group, while TC-2153
(p > 0.05) did not affect this parameter. Additionally, we failed to
detect an influence of the drugs on the time spent in the closed arms
(F2,24 < 1), in the center (F2,25 < 1), and in the open arms (F2,24 < 1) and
on the numbers of head dips (F2,26 < 1) and rearings (F2,26 = 1.13,
p > 0.05) (Table 2). 3.4. The impact of acute TC-2153 treatment on STEP61 and STEP46
protein levels in aggressive and tame rats An
influence of TC-2153 administration was also detectable in all the
investigated brain structures (midbrain F2,41 = 4.89, p < 0.05; hippo
campus F2,40 = 4.16, p < 0.05; hypothalamus F2,40 = 5.24, p < 0.01). TC-2153 in the dose of 20 mg/kg reduced the STEP46 protein level in
the midbrain (p < 0.01), hippocampus (p < 0.01), and hypothalamus
(p < 0.001) of aggressive rats, without affecting the STEP46 protein
level in tame rats. The genotype × drug interaction was not significant
in the studied brain regions (midbrain F2,41 = 2.09, p > 0.05; hippo
campus F2,40 = 2.44, p > 0.05, hypothalamus F2,40 = 2.73, p > 0.05). 3.1. Effects of acute TC-2153 treatment on the aggressive and tame
behavior in the glove test Dat
presented as block plots with whiskers, where blocks indicate mean ± SD, and whiskers min and max values. $p < 0.05 in comparison with the data point b
treatment; 10 animals per group. Fig. 1. Effects of acute treatment with vehicle (control) or TC-2153 at 10 or 20 mg/kg on the behavior of aggressive (a) and tame (b) rats in the glove test. Data are
presented as block plots with whiskers, where blocks indicate mean ± SD, and whiskers min and max values. $p < 0.05 in comparison with the data point before
treatment; 10 animals per group. Biomedicine & Pharmacotherapy 147 (2022) 112667 V.S. Moskaliuk et al. py
(
)
Fig. 2. Effects of acute treatment with either vehicle (control) or TC-2153 at 10 or 20 mg/kg on anxious behavior of aggressive and tame rats. * *p < 0.01,
* **p < 0.001 for the genotype effect; @p < 0.05, @@p < 0.01 in comparison with the control group. #p < 0.05, ##p < 0.01 in comparison with the TC-2153 10 mg/
kg group (8 animals per group). Fig. 2. Effects of acute treatment with either vehicle (control) or TC-2153 at 10 or 20 mg/kg on anxious behavior of aggressive and tame rats. * *p < 0.01,
* **p < 0.001 for the genotype effect; @p < 0.05, @@p < 0.01 in comparison with the control group. #p < 0.05, ##p < 0.01 in comparison with the TC-2153 10 mg/
kg group (8 animals per group). 3.4. The impact of acute TC-2153 treatment on STEP61 and STEP46
protein levels in aggressive and tame rats 3.5. The influence of chronic TC-2153 or fluoxetine treatment on fear-
induced aggression in the glove test We noticed a significant antiaggressive effect of chronic adminis
tration of TC-2153 or fluoxetine (Fig. 6). Before the treatment was
started, all animals were subjected to the glove test, and no differences 5 Biomedicine & Pharmacotherapy 147 (2022) 112667
Fig. 3. Effects of acute administration of vehicle (control) or TC-2153 at 10 or 20 mg/kg on Ptpn5 mRNA expression in the midbrain, hippocampus, and hypo
thalamus of aggressive and tame rats. The expression levels were measured using primers Ptpn5_ex8 (binding to the beginning of the 8th exon and detecting only
STEP61 expression) and Ptpn5_ex16 (binding to the 16th exon and detecting the expression of both isoforms, STEP61 and STEP46). The expression levels were
evaluated as the number of transcript copies per 100 copies of Polr2a mRNA (5–7 animals per group). V.S. Moskaliuk et al. Biomedicine & Pharmacotherapy 147 (2022) 112667 V.S. Moskaliuk et al. Biomedicine & Pharmacotherapy 147 (2022) 112667
V.S. Moskaliuk et al. Fig. 3. Effects of acute administration of vehicle (control) or TC-2153 at 10 or 20 mg/kg on Ptpn5 mRNA expression in the midbrain, hippocampus, and hypo
thalamus of aggressive and tame rats. The expression levels were measured using primers Ptpn5_ex8 (binding to the beginning of the 8th exon and detecting only
STEP61 expression) and Ptpn5_ex16 (binding to the 16th exon and detecting the expression of both isoforms, STEP61 and STEP46). The expression levels were
evaluated as the number of transcript copies per 100 copies of Polr2a mRNA (5–7 animals per group). Fig. 3. Effects of acute administration of vehicle (control) or TC-2153 at 10 or 20 mg/kg on Ptpn5 mRNA expression in the midbrain, hippocampus, and hypo
thalamus of aggressive and tame rats. The expression levels were measured using primers Ptpn5_ex8 (binding to the beginning of the 8th exon and detecting only
STEP61 expression) and Ptpn5_ex16 (binding to the 16th exon and detecting the expression of both isoforms, STEP61 and STEP46). The expression levels were
evaluated as the number of transcript copies per 100 copies of Polr2a mRNA (5–7 animals per group). Fig. 4. Effects of acute administration of either vehicle (control) or TC-2153 at 10 or 20 mg/kg on the STEP61 protein level in the midbrain, hippocampus, and
hypothalamus of aggressive and tame rats. The protein levels are presented as a percentage of the GAPDH protein level (6–8 animals per group). An example of a
western blot for the quantification of the STEP61 protein. 3.5. The influence of chronic TC-2153 or fluoxetine treatment on fear-
induced aggression in the glove test *p < 0.05: for the genotype effect. Groups: A0, aggressive rats, control; A10, aggressive rats TC-2153 at
10 mg/kg; A20, aggressive rats TC-2153 at 20 mg/kg; T0, tame rats, control; T10, tame rats TC-2153 at 10 mg/kg; and T20, tame rats TC-2153 at 20 mg/kg. Fig. 4. Effects of acute administration of either vehicle (control) or TC-2153 at 10 or 20 mg/kg on the STEP61 protein level in the midbrain, hippocampus, and
hypothalamus of aggressive and tame rats. The protein levels are presented as a percentage of the GAPDH protein level (6–8 animals per group). An example of a
western blot for the quantification of the STEP61 protein. *p < 0.05: for the genotype effect. Groups: A0, aggressive rats, control; A10, aggressive rats TC-2153 at
10 mg/kg; A20, aggressive rats TC-2153 at 20 mg/kg; T0, tame rats, control; T10, tame rats TC-2153 at 10 mg/kg; and T20, tame rats TC-2153 at 20 mg/kg. 3.7. Effects of chronic treatment with TC-2153 or fluoxetine on Ptpn5
mRNA expression in aggressive rats 3.7. Effects of chronic treatment with TC-2153 or fluoxetine on Ptpn5
mRNA expression in aggressive rats Ptpn5_ex16 pair of primers (binding to exon 16 and detecting the
expression of both STEP61 and STEP46) in the midbrain (F2,25 < 1),
hippocampus (F2,24 = 1.20, p > 0.05), and hypothalamus (F2,25 = 2.94,
p > 0.05; Fig. 7d–f). With the Ptpn5_ex8 pair of primers (binding to exon 8 and specific to
the STEP61 isoform), no effect of drugs on the STEP61 mRNA level were
found in the midbrain (F2,26 < 1), hippocampus (F2,26 < 1), and hypo
thalamus (F2,25 = 1.14, p > 0.05; Fig. 7a–c). Furthermore, chronic
administration of TC-2153 or fluoxetine did not change the signals of 6 Biomedicine & Pharmacotherapy 147 (2022) 112667 V.S. Moskaliuk et al. Fig. 5. Effects of acute administration of either vehicle (control) or TC-2153 at 10 or 20 mg/kg on STEP46 protein concentration in the midbrain, hippocampus, and
hypothalamus of aggressive and tame rats. The protein levels are presented as a percentage of the GAPDH protein level. * **p < 0.001: for the genotype effect,
@@p < 0.01, @@@p < 0.001 in comparison with the control group (7–8 animals per group). An example of a western blot for the measurement of STEP46 protein
concentration. Table 2 3.8. Effects of chronic TC-2153 or fluoxetine treatment on STEP61 and
STEP46 protein levels in aggressive rats
content of the hippocampus (F2,21 = 4.01, p < 0.05). The protein con
centration of STEP46 was lowered by the chronic treatment with
fluoxetine (p < 0.05) (Fig. 9b), whereas TC-2153 failed to alter this
Table 2
Effects of chronic treatment with vehicle (control), TC-2153, or fluoxetine on anxious behavior of aggressive rats. Behavioral parameter
Control
TC-2153
Fluoxetine
F
p
Number of head peeks
4.00 ± 1.12
9.50 ± 1.79
11.38 ± 2.19
F2,26 = 4.32
p < 0.05
Time spent in open arms, %
5.25 ± 2.67
3.90 ± 1.89
7.64 ± 2.77
F2,24 < 1
Time spent in closed arms, %
87.04 ± 6.71
82.73 ± 5.78
80.17 ± 4.61
F2,24 < 1
Time spent in center, %
10.68 ± 4.68
15.42 ± 4.26
9.45 ± 2.63
F2,25 < 1
Duration of head dips, s
3.36 ± 1.48
3.11 ± 1.14
4.38 ± 1.80
F2,26 < 1
Duration of rearings, s
1.76 ± 0.90
1.83 ± 0.82
0.31 ± 0.23
F2,26 = 1.13
p > 0.05 3.8. Effects of chronic TC-2153 or fluoxetine treatment on STEP61 and
STEP46 protein levels in aggressive rats
Western blot assays of the STEP61 protein in the midbrain, hippo
campus, and hypothalamus of aggressive rats did not show any impact of
either TC-2153 or fluoxetine (midbrain: F2,23 < 1, hippocampus F2,22 =
2.23, p > 0.05; hypothalamus F2,23 = 1.71, p > 0.05) (Fig. 8a–c). We found a significant effect of drug administration on the STEP46
content of the hippocampus (F2,21 = 4.01, p < 0.05). The protein con
centration of STEP46 was lowered by the chronic treatment with
fluoxetine (p < 0.05) (Fig. 9b), whereas TC-2153 failed to alter this
protein’s expression. In the midbrain and hypothalamus, no drug effect
was detectable (midbrain: F2,22 < 1, hypothalamus F2,23 < 1; Fig. 9a,c). Table 2
Effects of chronic treatment with vehicle (control), TC-2153, or fluoxetine on anxious behavior of aggressive rats. 3.5. The influence of chronic TC-2153 or fluoxetine treatment on fear-
induced aggression in the glove test Groups: A0, aggressive rats, control; A10, aggressive rats TC-2153 10 mg/kg; A20, aggressive rats TC-2153 20 mg/kg; T0, tame rats, control; T10,
tame rats TC-2153 10 mg/kg; and T20, tame rats TC-2153 20 mg/kg. Fig. 5. Effects of acute administration of either vehicle (control) or TC-2153 at 10 or 20 mg/kg on STEP46 protein concentration in the midbrain, hippocampus, and
hypothalamus of aggressive and tame rats. The protein levels are presented as a percentage of the GAPDH protein level. * **p < 0.001: for the genotype effect,
@@p < 0.01, @@@p < 0.001 in comparison with the control group (7–8 animals per group). An example of a western blot for the measurement of STEP46 protein
concentration. Groups: A0, aggressive rats, control; A10, aggressive rats TC-2153 10 mg/kg; A20, aggressive rats TC-2153 20 mg/kg; T0, tame rats, control; T10,
tame rats TC-2153 10 mg/kg; and T20, tame rats TC-2153 20 mg/kg. Fig. 6. Effects of chronic treatment with vehicle (control), TC-2153, or fluoxetine on fear-induced aggressive behavior of aggressive rats in the glove test. * **p < 0.001: for the effect of the chronic treatment, @p < 0.05, @@p < 0.01 in comparison to the control group on the same day (8–12 animals per group). Fig. 6. Effects of chronic treatment with vehicle (control), TC-2153, or fluoxetine on fear-induced aggressive behavior of aggressive rats in the glove test. * **p < 0.001: for the effect of the chronic treatment, @p < 0.05, @@p < 0.01 in comparison to the control group on the same day (8–12 animals per group). Table 2
Effects of chronic treatment with vehicle (control), TC-2153, or fluoxetine on anxious behavior of aggressive rats
Behavioral parameter
Control
TC-2153
Fluoxetine Table 2 Behavioral parameter
Control
TC-2153
Fluoxetine
F
p
Number of head peeks
4.00 ± 1.12
9.50 ± 1.79
11.38 ± 2.19
F2,26 = 4.32
p < 0.05
Time spent in open arms, %
5.25 ± 2.67
3.90 ± 1.89
7.64 ± 2.77
F2,24 < 1
Time spent in closed arms, %
87.04 ± 6.71
82.73 ± 5.78
80.17 ± 4.61
F2,24 < 1
Time spent in center, %
10.68 ± 4.68
15.42 ± 4.26
9.45 ± 2.63
F2,25 < 1
Duration of head dips, s
3.36 ± 1.48
3.11 ± 1.14
4.38 ± 1.80
F2,26 < 1
Duration of rearings, s
1.76 ± 0.90
1.83 ± 0.82
0.31 ± 0.23
F2,26 = 1.13
p > 0.05 3.8. Effects of chronic TC-2153 or fluoxetine treatment on STEP61 and
STEP46 protein levels in aggressive rats content of the hippocampus (F2,21 = 4.01, p < 0.05). The protein con
centration of STEP46 was lowered by the chronic treatment with
fluoxetine (p < 0.05) (Fig. 9b), whereas TC-2153 failed to alter this
protein’s expression. In the midbrain and hypothalamus, no drug effect
was detectable (midbrain: F2,22 < 1, hypothalamus F2,23 < 1; Fig. 9a,c). content of the hippocampus (F2,21 = 4.01, p < 0.05). The protein con
centration of STEP46 was lowered by the chronic treatment with
fluoxetine (p < 0.05) (Fig. 9b), whereas TC-2153 failed to alter this
protein’s expression. In the midbrain and hypothalamus, no drug effect
was detectable (midbrain: F2,22 < 1, hypothalamus F2,23 < 1; Fig. 9a,c). Western blot assays of the STEP61 protein in the midbrain, hippo
campus, and hypothalamus of aggressive rats did not show any impact of
either TC-2153 or fluoxetine (midbrain: F2,23 < 1, hippocampus F2,22 =
2.23, p > 0.05; hypothalamus F2,23 = 1.71, p > 0.05) (Fig. 8a–c). i We found a significant effect of drug administration on the STEP46 7 Biomedicine & Pharmacotherapy 147 (2022) 112667 V.S. Moskaliuk et al. Fig. 7. Effects of chronic treatment with vehicle (control), TC-2153, or fluoxetine on Ptpn5 mRNA expression in the midbrain, hippocampus, and hypothalamus of
aggressive rats. The expression levels were measured with primers Ptpn5_ex8 located in the beginning of the 8th exon (detecting only STEP61 expression) and
primers Ptpn5_ex16 located in the 16th exon (detecting the expression of both isoforms, STEP61 and STEP46). The expression level was evaluated as the number of
transcript copies per 100 copies of Polr2a mRNA (8–12 animals per group). Fig. Table 2 7. Effects of chronic treatment with vehicle (control), TC-2153, or fluoxetine on Ptpn5 mRNA expression in the midbrain, hippocampus, and hypothalamus of
aggressive rats. The expression levels were measured with primers Ptpn5_ex8 located in the beginning of the 8th exon (detecting only STEP61 expression) and
primers Ptpn5_ex16 located in the 16th exon (detecting the expression of both isoforms, STEP61 and STEP46). The expression level was evaluated as the number of
transcript copies per 100 copies of Polr2a mRNA (8–12 animals per group). Fig. 8. Effects of chronic treatment with vehicle (control), TC-2153, or fluoxetine on the STEP61 protein content of the midbrain, hippocampus, and hypothalamus in
aggressive rats. The protein amounts are presented as a percentage of the GAPDH protein level (7–10 animals per group). An example of a western blot for the
quantification of the STEP61 protein in aggressive rats. Groups: C, Control; TC, TC-2153; and F, fluoxetine. Fig. 8. Effects of chronic treatment with vehicle (control), TC-2153, or fluoxetine on the STEP61 protein content of the midbrain, hippocampus, and hypothalamus in
aggressive rats. The protein amounts are presented as a percentage of the GAPDH protein level (7–10 animals per group). An example of a western blot for the
quantification of the STEP61 protein in aggressive rats. Groups: C, Control; TC, TC-2153; and F, fluoxetine. 4. Discussion Notably, STEP knockout mice do not demon
strate changes in the aggressive behavior in comparison to the wild-type
mice in the resident-intruder test [27]. Nevertheless, this finding can be
explained by differences in the mechanisms of fear-induced and inter
male aggression types [70] and/or species-specific factors. ruling out the handling effect in this behavioral test. Here for the first
time, we revealed that chronic treatment with TC-2153 or fluoxetine
significantly reduces fear-induced aggression. At the same time, the
timelines of effects of these drugs differed: fluoxetine reduced aggressive
behavior on the 4th day of treatment, whereas TC-2153 only on the 11th
day of administration, and there was an increase in the aggressive score
on the 29th day of TC-2153 treatment but not fluoxetine treatment. These results may be explained by possible stress induced by the sepa
ration of the animals on the 29th day. The animals that received
TC-2153 were more vulnerable to this stress than those in the fluoxetine
group. Besides, in the present study, we unexpectedly did not see any
anxiolytic effect of chronic TC-2153 or fluoxetine administration in the
EPM test, which was also performed on the 29th day of treatment. Earlier, anxiolytic effects of TC-2153 [29,33,75] and fluoxetine [72]
have been reported. Moreover, it has been shown that prolonged
administration (more than 30 days) of TC-2153 has adverse effects on
the behavior of rats [73]. Therefore, TC-2153 is more effective during
acute administration. In addition, during chronic treatment, TC-2153 is
less effective than fluoxetine. This difference in the timeline of effects of
TC-2153 and fluoxetine may result from dissimilarity of the pharma
cokinetics of these drugs. l i
Numerous studies have proven the association between aggression
and anxiety. Nevertheless, this link is inconsistent and depends on ani
mal models and types of aggressive behavior [3,11,31,54,55,83,84]. Earlier, it was demonstrated that the aggression toward humans of the
rat strains analyzed here is associated with increased fear [50]. Accordingly, in the present study, we decided to evaluate the effects of
TC-2153 on anxiety-like behavior and exploratory activity by the EPM
test. Earlier, an anxiolytic effect of TC-2153 has been reported for mice
[29] in a hole-board test and for the fish Danio rerio in a novel tank test
[33,75]. Here, for the first time, we demonstrated that acute TC-2153
administration produces a strong anxiolytic effect on both strains of
rats in the EPM test. 4. Discussion For example, the aggressive and tame rats treated
with TC-2153 spent more time in the open arms and less time in the
closed arms. It is known that classic anxiolytic drugs prolong the time
spent in the open arms [15]. Moreover, TC-2153 increased the duration
of head dips and rearings, indicating elevated exploratory activity. Furthermore, in this test, we demonstrated an effect of the genotype on
anxiety-related behavior of these strains of rats. Consequently, aggres
sive rats showed more pronounced anxious behavior as compared to the
tame ones. This observation is in good agreement with the data obtained
by Albert and colleagues, who concluded that there is a difference in
anxiety between the strains in the open-field test and dark-light box test
[1]. It has been reported that the aggressive rats manifest an enhanced
response in an acoustic startle reflex test as compared to the tame ones
[50], indicating greater fear in this strain of rats. It should be noted that
STEP knockout mice demonstrate deficits in pre-pulse inhibition in this
test in comparison to the wild type [77]. It is well known that fluoxetine is a selective serotonin reuptake in
hibitor and increases the 5-HT level in the synaptic cleft thereby stim
ulating 5-HT receptors coupled with Gs proteins (5-HT4, 5-HT6, or 5-
HT7), increases cAMP concentration in the neuron, activates protein
kinase A, and increases BDNF synthesis. The synthesized BDNF in turn
interacts with receptor TrkB and activates kinases ERK1/2, thus stimu
lating neurogenesis and neuronal survival [14]. Notably, recently, it was
demonstrated that fluoxetine directly binds to TrkB and facilitates its
activation by BDNF [7]. On the other hand, TC-2153 selectively inhibits
STEP and in this way can facilitate ERK1/2 activation [48]. Addition
ally, TC-2153 upregulates BDNF in the brain [37] and elevates the level
and metabolism of serotonin [39] as well as influences serotonergic
receptors [35,40]. In the current study, we evaluated the impact of drugs
on male rats, whereas the influence of sex on the response to antide
pressants was examined earlier [79]; the effects of TC-2153 and fluox
etine on aggressive female rats are a good subject for future
investigation. Nevertheless, our results indicate that TC-2153 and
fluoxetine affect the regulation of fear-induced aggression. i In the hope to completely eliminate aggression in aggressive rats, we
investigated the effect of chronic administration of TC-2153 on this type
of behavior. 4. Discussion the mechanisms of fear-induced aggression and domestication [30,50,
65–68,85], here we were the first to employ this model to elucidate the
involvement of two STEP isoforms in these types of behavior. In this study, we researched the association between STEP isoforms
and fear-induced aggression using a unique model based on Norway rats
selected for more than 90 generations for enhancement of aggression
toward humans. The tame rats selected for the elimination of an
aggressive reaction to humans have been suggested as a model of
domestication [49] and were chosen in this project as an opposite
phenotype relative to the aggressive rats. Although these rat strains have
been extensively used to investigate the role of the brain 5-HT system in The main approach in this work was the evaluation of the effects of
acute and chronic treatment with a STEP inhibitor, benzopentathiepine
TC-2153, on the behavior as well as on Ptpn5 mRNA expression and
amounts of STEP protein isoforms in the brain of aggressive rats. We showed that acute TC-2153 administration attenuates fear-
induced aggressive behavior in aggressive rats, while exerting no ef
fect on the friendly behavior of tame rats as assessed by the glove test. 8 Biomedicine & Pharmacotherapy 147 (2022) 112667 V.S. Moskaliuk et al. Biomedicine & Pharmacotherapy 147 (2022) 112667
Fig. 9. Effects of chronic treatment with vehicle (control), TC-2153, or fluoxetine on the STEP46 protein level in the midbrain, hippocampus, and hypothalamus of
aggressive rats. The protein amounts are presented as a percentage of the GAPDH protein level. *p < 0.05 as compared to the control group (7–10 animals per group). An example of a western blot for the measurement of STEP46 protein concentration in aggressive rats. Groups: C, Control; TC, TC-2153; and F, fluoxetine. V.S. Moskaliuk et al. Fig. 9. Effects of chronic treatment with vehicle (control), TC-2153, or fluoxetine on the STEP46 protein level in the midbrain, hippocampus, and hypothalamus of
aggressive rats. The protein amounts are presented as a percentage of the GAPDH protein level. *p < 0.05 as compared to the control group (7–10 animals per group). An example of a western blot for the measurement of STEP46 protein concentration in aggressive rats. Groups: C, Control; TC, TC-2153; and F, fluoxetine. Based on the report that TC-2153 selectively inhibits STEP [88], it is fair
to assume that STEP can participate in the mechanisms underlying
fear-induced aggression. 4. Discussion Moreover, we compared the effect of chronic TC-2153
administration on the behavior of aggressive rats with that of a typical
selective serotonin reuptake inhibitor: fluoxetine. The activity of
fluoxetine has not previously been studied in this model of fear-induced
aggression; however, this antidepressant is known to decrease aggres
sion [13,18]. First of all, we showed that the basal level of aggression in
control rats did not alter throughout the treatment period, thereby l
According to the significant changes of aggressive behavior after the
administration of the STEP inhibitor (TC-2153), we hypothesized the
involvement of the STEP protein in this mechanism; the second aim of
the present study was to elucidate the association of STEP with fear- 9 Biomedicine & Pharmacotherapy 147 (2022) 112667 V.S. Moskaliuk et al. of kinases ERK1/2 can diminish 5-HT and BDNF signaling [38], thereby
enhancing anxiety and fear-induced aggression [8,12,55,58,70,76]. It is
well known that the elevation of STEP amounts is associated with neu
rodegeneration [17,27]. Consequently, these data may indicate an
impairment of neuronal plasticity in these two strains of rats. i induced aggression. For this purpose, we intended to measure the levels
of two transcripts, which code for isoforms STEP61 and STEP46, as well
as the levels of these proteins in the whole hippocampus, hypothalamus,
and midbrain. It is well known that all the brain structures under study
are involved in the regulation of aggressive behavior and anxiety [6,10,
21,23,55,76,81]. Isoforms STEP61 and STEP46 are located on the
plasma membrane and in the cytosol, respectively [5]. To investigate the
Ptpn5 mRNA expression, we utilized two sets of primers. The first one,
Ptpn5_ex8, anneals to exon 8, which is specific to the STEP61 isoform. The second set of primers, Ptpn5_ex16, binds to exon 16 and detects the
expression of both isoforms, STEP61 and STEP46. Thus, in this study, we for the first time demonstrated that striatal-
enriched protein tyrosine phosphatase is involved in fear-induced
aggression. The acute and chronic administration of the STEP inhibi
tor TC-2153 significantly diminished the aggression of aggressive rats. Moreover, the acute TC-2153 treatment produced a strong anxiolytic
effect. Chronic administration of the classic antidepressant fluoxetine
decreased the aggression and influenced the STEP46 expression in
aggressive rats. This finding implies an interesting similarity between
the mechanisms of action of TC-2153 and fluoxetine. Conflict of interest statement None. membrane joint isoform. This hypothesis requires additional studies. Nonetheless, chronic treatment with TC-2153 did not affect STEP46
concentration in all the assayed brain structures in aggressive rats. Nowadays, the only known direct target of the drug TC-2153 is STEP
[88], whereas it has been shown that acute as well as chronic admin
istration of TC-2153 also affects the serotoninergic system [35,39,40]. The observed unexpected absence of the effect of chronic TC-2153
treatment on the STEP protein with the significant antiaggressive ef
fect of acute and chronic administration of this drug could be explained
by some compensatory mechanisms or, perhaps, the effect on behavior
could be affected by a direct or indirect influence of TC-2153 on another
system, for instance, the serotonergic one. At the same time, we found
that chronic fluoxetine treatment downregulated STEP46 in the hippo
campus. The impact of fluoxetine on STEP46 expression can be attrib
uted to the recent discovery that fluoxetine directly influences receptor
TrkB [7] and through this receptor can reduce the STEP46 level [63,74]. Furthermore, the hippocampus is responsible for learning, memory,
and neuroplasticity [10,56]. Earlier, it was found that in the Morris
water maze test, the aggressive rats exhibit a reduction in learning
abilities and memory as compared to the tame animals [59]. The
aggressive rats are characterized by an elevated level of proapoptotic
proBDNF in the hippocampus and midbrain [25] and changes in the
serotoninergic system [30,51,66,68,69,86]. Moreover, in the present
study, we demonstrated that STEP46 expression is significantly
increased in aggressive animals in the hippocampus, midbrain, and
hypothalamus. It can be hypothesized that STEP via dephosphorylation Nonetheless, chronic treatment with TC-2153 did not affect STEP46
concentration in all the assayed brain structures in aggressive rats. Nowadays, the only known direct target of the drug TC-2153 is STEP
[88], whereas it has been shown that acute as well as chronic admin
istration of TC-2153 also affects the serotoninergic system [35,39,40]. The observed unexpected absence of the effect of chronic TC-2153
treatment on the STEP protein with the significant antiaggressive ef
fect of acute and chronic administration of this drug could be explained
by some compensatory mechanisms or, perhaps, the effect on behavior
could be affected by a direct or indirect influence of TC-2153 on another
system, for instance, the serotonergic one. At the same time, we found
that chronic fluoxetine treatment downregulated STEP46 in the hippo
campus. 4. Discussion This research
brings us one step closer to the understanding of the mechanisms of fear-
induced aggression as well as to the development of new strategies for
aggressiveness correction. We did not notice any difference in Ptpn5_ex8- and Ptpn5_ex16-
measured mRNA levels in all the studied brain structures between
aggressive and tame rats. Moreover, neither acute nor chronic TC-2153
treatment affected the levels of these transcripts in these brain regions. The TC-2153 treatment did not influence the STEP61 level in the
hippocampus, midbrain, and hypothalamus either. STEP61 is a
membrane-associated STEP isoform with two transmembrane domains
[5] and can dephosphorylate and inactivate neuronal substrates,
including kinases ERK1/2 and p38 [48], FYN [57], PYK2 [89], and
subunits of NMDA receptor [42] and AMPA receptor [90]. We did not notice any difference in Ptpn5_ex8- and Ptpn5_ex16-
measured mRNA levels in all the studied brain structures between
aggressive and tame rats. Moreover, neither acute nor chronic TC-2153
treatment affected the levels of these transcripts in these brain regions. The TC-2153 treatment did not influence the STEP61 level in the
hippocampus, midbrain, and hypothalamus either. STEP61 is a
membrane-associated STEP isoform with two transmembrane domains
[5] and can dephosphorylate and inactivate neuronal substrates,
including kinases ERK1/2 and p38 [48], FYN [57], PYK2 [89], and
subunits of NMDA receptor [42] and AMPA receptor [90]. Acknowledgments The English language was corrected and certified by shevchuk-
editing.com. CRediT authorship contribution statement Vitalii S. Moskaliuk: Conceptualization, Investigation, Formal
analysis, Visualization, Writing – original draft preparation, Writing –
review & editing. Rimma V. Kozhemyakina: Investigation, Resources. Elena Terenina: Investigation. Darya V. Bazovkina: Investigation. Tatyana M. Khomenko: Resources. Konstantin P. Volcho: Resources. Nariman F. Salakhutdinov: Resources. Alexander V. Kulikov:
Conceptualization, Formal analysis, Software, Writing – review & edit
ing. Vladimir S. Naumenko: Conceptualization, Investigation, Writing
– review & editing. Elizabeth A. Kulikova: Conceptualization, Inves
tigation, Formal analysis, Writing – original draft preparation, Writing –
review & editing, Supervision. Data Availability Data will be made available on request. Funding This work was supported by Russian Science Foundation project
number 21-15-00051. Unlike STEP61, the STEP46 isoform is still poorly studied. STEP46 is
a cytosolic isoform and can dephosphorylate kinases ERK1/2 and p38
[48]. Until now, it has been found only in the striatum, amygdala, optic
nerve, and retina [19,78]. We are the first to find the STEP46 protein in
the midbrain, hippocampus, and hypothalamus. Control aggressive rats
feature higher concentration of this isoform in all studied brain struc
tures than do the control tame animals. Furthermore, acute treatment
with TC-2153 in the dose of 20 mg/kg downregulated the STEP46
protein in the midbrain, hippocampus, and hypothalamus of aggressive
rats but not tame rats. Such an evident difference in STEP46 protein
expression means that the selection for high and low aggression toward
humans strongly affected this protein’s expression. The influence on the
protein level and not on the mRNA may indicate the effect of the drugs
on a translation mechanism rather than on transcription. Moreover, it is
known that TC-2153 can block the activity of recombinant STEP by
forming a trisulfide bond with cysteine residues in the catalytic domain
[88]. Perhaps, the complex of TC-2153 and STEP46 could be eliminated
by some yet unknown molecular mechanism. Moreover, it is possible
that TC-2153 more easily affects the cytosolic isoform STEP46 than the
membrane joint isoform. This hypothesis requires additional studies. Conflict of interest statement The impact of fluoxetine on STEP46 expression can be attrib
uted to the recent discovery that fluoxetine directly influences receptor
TrkB [7] and through this receptor can reduce the STEP46 level [63,74]. Appendix A. Supporting information Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.biopha.2022.112667. Furthermore, the hippocampus is responsible for learning, memory,
and neuroplasticity [10,56]. Earlier, it was found that in the Morris
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abilities and memory as compared to the tame animals [59]. The
aggressive rats are characterized by an elevated level of proapoptotic
proBDNF in the hippocampus and midbrain [25] and changes in the
serotoninergic system [30,51,66,68,69,86]. Moreover, in the present
study, we demonstrated that STEP46 expression is significantly
increased in aggressive animals in the hippocampus, midbrain, and
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English
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Are medium‐scale farms driving agricultural transformation in sub‐Saharan Africa?
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Agricultural economics
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cc-by
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T. S. Jayne1
Milu Muyanga1
Ayala Wineman2
Hosaena Ghebru3
Caleb Stevens4
Mercedes Stickler5
Antony Chapoto6
Ward Anseeuw7,8
Divan van der Westhuizen9
David Nyange10 T. S. Jayne1
Milu Muyanga1
Ayala Wineman2
Hosaena Ghebru3
Caleb Stevens4
Mercedes Stickler5
Antony Chapoto6
Ward Anseeuw7,8
Divan van der Westhuizen9
David Nyange10 T. S. Jayne1
Milu Muyanga1
Ayala Wineman2
Hosaena Ghebru3
Caleb Stevens4
Mercedes Stickler5
Antony Chapoto6
Ward Anseeuw7,8
Divan van der Westhuizen9
David Nyange10 1Department of Agricultural, Food and
Resource Economics, Michigan State
University, East Lansing, MI, USA
2University of Washington, Seattle,
Washington, USA
3International Food Policy Research Institute,
Washington, DC, USA
4USAID, Washington, DC, USA
5World Bank, Washington, DC, USA
6Indaba Agricultural Policy Research
Institute, Lusaka, Zambia
7International Land Coalition, Rome, Italy
8Agricultural Research Centre for
International Development (CIRAD)
9University of Pretoria, Hatfield, Pretoria,
South Africa
10ASPIRES Project, Dar es Salaam, Tanzania
Correspondence
T.S.Jayne,University Foundation Professor of
Agricultural,Food and ResourceEconomicsat
Michigan StateUniversity,East Lansing,MI,
USA. Email:jayne@msu.edu 1Department of Agricultural, Food and
Resource Economics, Michigan State
University, East Lansing, MI, USA
2University of Washington, Seattle,
Washington, USA
3International Food Policy Research Institute,
Washington, DC, USA
4USAID, Washington, DC, USA
5World Bank, Washington, DC, USA
6Indaba Agricultural Policy Research
Institute, Lusaka, Zambia
7International Land Coalition, Rome, Italy
8Agricultural Research Centre for
International Development (CIRAD)
9University of Pretoria, Hatfield, Pretoria,
South Africa
10ASPIRES Project, Dar es Salaam, Tanzania
Correspondence
T.S.Jayne,University Foundation Professor of
Agricultural,Food and ResourceEconomicsat
Michigan StateUniversity,East Lansing,MI,
USA. Email:jayne@msu.edu Abstract
This study presents evidence of profound farm-level transformation in parts of sub-
Saharan Africa, identifies major sources of dynamism in the sector, and proposes an
updated typology of farms that reflects the evolving nature of African agriculture. Repeat waves of national survey data are used to examine changes in crop production
and marketed output by farm size. Between the first and most recent surveys (generally
covering 6 to 10 years), the share of national marketed crop output value accounted
for by medium-scale farms rose in Zambia from 23% to 42%, in Tanzania from 17% to
36%, and in Nigeria from 7% to 18%. The share of land under medium-scale farms is
not rising in densely populated countries such as Kenya, Uganda, and Rwanda, where
land scarcity is impeding the pace of medium-scale farm acquisitions. Medium-scale
farmers are a diverse group, reflecting distinct entry pathways into agriculture, encour-
aged by the rapid development of land rental, purchase, and long-term lease markets. The rise of medium-scale farms is affecting the region in diverse ways that are dif-
ficult to generalize. Correspondence Correspondence
T.S.Jayne,University Foundation Professor of
Agricultural,Food and ResourceEconomicsat
Michigan StateUniversity,East Lansing,MI,
USA. Email:jayne@msu.edu K E Y W O R D S
agricultural transformation, farm size distribution, farm surveys, land tenure, medium-scale farms agricultural transformation, farm size distribution, farm surveys, land tenure, medium-scale farms J E L C L A S S I F I C AT I O N
O13, Q12, Q15, Q18, Q24, R14 Agricultural Economics. 2019;50:75–95. T. S. Jayne1
Milu Muyanga1
Ayala Wineman2
Hosaena Ghebru3
Caleb Stevens4
Mercedes Stickler5
Antony Chapoto6
Ward Anseeuw7,8
Divan van der Westhuizen9
David Nyange10 Findings indicate that these farms can be a dynamic driver of
agricultural transformation but this does not reduce the importance of maintaining a
clear commitment to supporting smallholder farms. Strengthening land tenure secu-
rity of local rural people to maintain land rights and support productivity investments
by smallholder households remains crucial. wileyonlinelibrary.com/journal/agec Abstract 2University of Washington, Seattle,
Washington, USA 7International Land Coalition, Rome, Italy Received: 7 August 2019
Revised: 5 October 2019
Accepted: 21 October 2019
DOI: 10.1111/agec.12535 Received: 7 August 2019
Revised: 5 October 2019
Accepted: 21 October 2019
DOI: 10.1111/agec.12535 Received: 7 August 2019
Revised: 5 October 2019
Accepted: 21 October 2019 “Medium-scale”
farm landholdings of five to 100 ha now account for a sub-
stantial and growing share of farmland in many African coun-
tries (Jayne et al., 2016).1 Perhaps ironically, the amount of
land acquired by this category of African farmer since 2000
far exceeds the amount of land acquired by foreign investors
(Jayne et al., 2014a). This might be considered a surprising
development, but in retrospect, perhaps it should not have
been. The dramatic rise in global food prices after 2007 ini-
tiated major foreign investment in African farmland. Why
should not African investors have done the same? Parallel to these developments, the region is witnessing
changes in land tenure institutions that influence who is
acquiring land (Boone, 2014; Knapman, Silici, Cotula, &
Mayers, 2017). Parts of the region are experiencing a notable
shift in the allocation of customary land, moving from a
rights-based approach that secures access to land for local-
born members of the community to market-based approaches
in which land becomes a commodity for rent or sale. Although
SSA’s rural areas contain 20.3 million km2 of land, only
25% of the region is arable (CIA 2019). With an estimated
rural population of 620 million people in 2017, the region is
sparsely populated at 31 persons per km2. However, roughly
72% of SSA’s rural population resides on only 10% of its rural
areas (Jayne, Chamberlin, & Headey, 2014b). For this major-
ity of the region’s rural population, the average population
density is 223.2 persons per km2. Hence, even though most
of SSA might be considered “land abundant” and sparsely
populated, a relatively large proportion of rural Africans face
land scarcity, rapidly rising land prices, and perceptions of
tenure insecurity (Knapman et al., 2017; Lawry et al., 2014;
Wineman & Jayne, 2018). As population densities rise and
land becomes scarcer in many areas, tenure security is becom-
ing increasingly important, as research evidence shows that
security of tenure generally promotes long-term land invest-
ments and agricultural productivity (Atwood, 1990; Gold-
stein, Houngbedji, Kondylis, O’Sullivan, & Selod, 2015;
Holden, Deininger, & Ghebru, 2009; Place, 2009). The causes and consequences of changing farm structure
and the rise of medium-scale farms are discussed in Sec-
tions 4 and 5. Though the literature remains thin, emerging
evidence indicates that medium-scale farms generate mostly
positive spillover effects on smallholder farmers. size distributions are beneficial for small-scale farm house-
holds, who still constitute the vast majority of rural house-
holds in Africa, and whether they are promoting or retarding
equitable forms of economic transformation in Africa. This
study reviews the evidence on these policy issues. Revolution (Hayami & Ruttan, 1971; Johnston & Kilby,
1975). Because small-scale farms also constitute the vast
majority of farms in Africa, agricultural economists have gen-
erally accepted that a smallholder-led strategy also holds the
best prospects for agricultural development in Africa (e.g.,
Hazell, Poulton, Wiggins, & Dorward, 2010; Mellor, 1995). y
p
y
To address these questions, we focus on the causes and
consequences of the rise of medium-scale farms in Africa. This literature remains highly limited by the fact that accurate
data on farms over 20 ha is not available in the majority
of African countries. We therefore collected new primary
data on medium-scale farms that are considered statistically
representative of farms operating between 5 and 100 ha for
particular districts or comparable administrative units in
Malawi, Nigeria, and Senegal. While most of the studies
attempting to analyze farm structure in Africa utilize Living
Standards Monitoring Surveys (LSMS) or similar nationwide
farm data sets, it is increasingly acknowledged that almost
all of these datasets provide highly imprecise and most
likely under-reported estimates of the numbers of farms
operating over 10 ha of land. Evidence of this is provided in
Section 2. However, even when utilizing these datasets, as we
do for Ghana, Nigeria, Tanzania, and Zambia in Section 3,
it is shown that medium-scale farms are accounting for a
rising proportion of national farmland and the value of crop
production and marketed output. However, in other countries,
especially those that are relatively densely populated, the data
suggest that the number of medium-scale farms has grown rel-
atively slowly or not at all, but we cannot tell with confidence
whether this is a valid conclusion or an artifact of sampling
designs that almost certainly under-report relatively large
farms. However, parts of sub-Saharan Africa (SSA) are witness-
ing rapid changes in farm size distributions. 1 This paper defines “small-scale farms” as those between zero and five
hectares of farmland. Medium-scale farms are defined as farms between 5
and 100 hectares, and large farms those over 100 hectares. These definitions
may not correspond exactly to those used by all national governments in the
region. 1
INTRODUCTION smallholder-led growth strategy would also be the pathway for
achieving economic transformation and mass poverty reduc-
tion in Africa. Over 90% of farms in South and East Asia
were smaller than two hectares at the beginning of the Green Ever since the critical acclaim given to the Asian green revo-
lution starting in the 1980s, it has been widely accepted that a © The Authors. Agricultural Economics published by Wiley Periodicals, Inc. on behalf of International Association of Agricultural Eco Agricultural Economics. 2019;50:75–95. 76 JAYNE ET AL. 76 2
DATA AND METHODS To ascertain the potential bias
associated with using LSMS data to understand farm size dis-
tributions, a comparison of Tanzania’s 2008 NPS and 2008
ASCS is presented in These population-based surveys may be considered appro-
priate for studying the small-scale sector because historically
90% or more of the farm households in most African coun-
tries have been between zero and five hectares. However,
population-based data sets such as the LSMS may be less than
ideal for understanding the distribution of farmland owner-
ship and use patterns if larger farms constitute a low propor-
tion of the population (and hence a low probability of being
included in the sample) but a sizeable proportion of national
farmland. For example, the 2008 Tanzania LSMS contains
3,265 households according to our computations, but only 15
have landholdings over 20 ha. The Uganda LSMS contains
12 farms between 20 and 50 ha and none over 50 ha. The Malawi 2010/11 LSMS contains one farm observation
between 10 and 20 ha, one farm between 20 and 50 ha, and
none over 50 ha. These surveys obviously do not contain a
sufficient sample size of farms over 20 ha to make confi-
dent statements about their rate of growth.2 In Kenya, despite
widespread anecdotal evidence that large farms connected to
the three Presidential families of Kenya may account for up
to 20% of Kenya’s agricultural land (e.g., Namwaya, 2004),
we found that the 2006 KIHBS (the most recent population-
based large-scale household data set in Kenya) contains only
four households with landholdings over 100 ha. These find-
ings raise obvious concerns about the ability of population-
based surveys to generate reliable estimates of the numbers of The results in Table 1 show that LSMS and ASCS produce
very similar estimates of farmland held and under operation
among small-scale farms between 0 and 5 ha; the two surveys
produce nearly identical estimates of land under operation. For medium-scale holdings between 5 and 100 ha, the results
diverge substantially, with ASCS revealing 51.4% more land
being controlled by medium-scale farms at the national level
than indicated by LSMS. The results diverge even more so
in terms of national land held by large-scale holdings over
100 ha, with ASCS indicating 60% more land under the con-
trol of large-scale farms than indicated by LSMS. 2
DATA AND METHODS medium- and large-scale farms, the areas under cultivation by
farms of this size, and the characteristics of these farmers. We use data on farm size distributions from two kinds of
sources: (i) available national population-based surveys (in
Ghana, Rwanda, Nigeria, Tanzania, and Zambia); and (ii) pri-
mary data collected in collaboration with local agricultural
policy research institutes or universities in Senegal, Nigeria,
Zambia, and Malawi. From the first category, data on land-
holding sizes, area cultivated, and the value of crops pro-
duced and marketed by small-scale (0–5 ha) and medium-
scale farms (5–100 ha) come from the following sources:
the 1999, 2005, and 2013 Ghana Living Standards Surveys
(GLSS), implemented by the Ghana Statistical Service; the
1994 Kenya Welfare Monitoring Survey I and the 2006 Kenya
Integrated Household Budget Survey (KIHBS), implemented
by the Kenya National Bureau of Statistics; the National Panel
Surveys (NPS/LSMS) 2009, 2011, and 2013, implemented
by the Tanzania National Bureau of Statistics; the Uganda
LSMS surveys of 2006 and 2014, implemented by the Uganda
Bureau of Statistics; and the Crop Forecast Surveys, of 2001,
2008, and 2015, implemented by the Zambia Central Statisti-
cal Office. Most of these data sets are supported by the World
Bank’s LSMS unit. A recent study by Lowder, Skoet, and Raney (2016) has
shown that where it is possible to compare farmland own-
ership and distribution from LSMS and national agricultural
censuses (as they did for several Latin American countries),
the former tends to show an under-reporting of large farms
and operated area under large farms, and more tightly clus-
tered and less skewed distributions. Fortunately for our purposes, Tanzania’s National Bureau
of Statistics implemented a survey of 10% of all farms listed in
their 2008 Census (the Agricultural Sample Census Survey or
ASCS), containing a sample size of 53,600 households. The
ASCS over-samples medium and large-scale households and
then uses statistical weights derived from the Census to gen-
erate nationally representative estimates of farm area in each
size category. For these reasons, the ASCS is more likely to
be representative of large farms than typical population-based
surveys. We compare the estimates provided by LSMS and
the ASCS regarding the numbers of farms and area controlled
by farms categorized as small-scale, medium-scale and large-
scale according to our definitions (holdings of 0–5, 5–100,
and over 100 ha, respectively). 2 This conclusion is also acknowledged in the World Bank’s recent 2018
Myths and Facts book relying on the use of LSMS data (Christiaensen &
Demery, 2018, p. 10). 77 JAYNE ET AL. 77 In Section 6,
we examine the characteristics of medium-scale farmers and
the various pathways to becoming a medium-scale farmer. Section 7 examines how medium-scale farmers are acquiring
their land and how these pathways differ from how small-scale
farm household tend to acquire land. Section 8 reviews the
evidence on changes in land tenure systems and security and
how medium-scale farms may be indirectly influencing tenure
systems. A summary of the main findings and policy impli-
cations of the study are presented in Section 9. In the process,
we propose an updated typology of farms that reflects recent
changes in the relative importance of different farm categories
and sheds light on the heterogeneity found even among small-
holder farms. Section 9 also addresses how land tenure secu-
rity by members of local communities and vulnerable groups
in particular may be enhanced even while evolving land insti-
tutions are encouraging market-based land transfers and the
“commodification” of land in rural Africa. African policy makers and development organizations are
increasingly interested in whether these new trends in farm 2
DATA AND METHODS 0%
10%
20%
30%
40%
Small, on-farm
focus, not com
Small, on-farm
focus, com
Small, off-farm
focus, not com
Small, off-farm
focus, com
Medium
Large
2008
2010
2012
2014
F I G U R E 1
Distribution of land cultivated by
farm category, Tanzania, 2008, 2010, 2012, and 2014
[Color figure can be viewed at wileyonlinelibrary.com]
Source. National Panel Survey / LSMS, Tanzanian
National Bureau of Statistics, Dar es Salaam. F I G U R E 1
Distribution of land cultivated by
farm category, Tanzania, 2008, 2010, 2012, and 2014
[Color figure can be viewed at wileyonlinelibrary.com]
Source. National Panel Survey / LSMS, Tanzanian
National Bureau of Statistics, Dar es Salaam. consultation with local district agricultural offices, national
farmer unions, and/or village authorities. The population lists
serve two purposes. First, they enabled the generation of ran-
dom samples within selected districts/divisions to obtain sta-
tistically representative analysis of medium-scale farms in
these areas. Surveys of medium-scale farmers included mod-
ules on the sociodemographic characteristics of these farmers,
where they reside, and the tenure type of their land, and ret-
rospective life history modules that make it possible to under-
stand how, why, and when in their lives they acquired their
medium-scale holdings. A second purpose of the population
lists is to assess the robustness of our numbers of farms in spe-
cific size categories with those indicated by population based
and agricultural census data in the same divisions/districts. cultivation, the value of production and marketed crop out-
put on farms under five hectares is generally declining over
time with corresponding increases in shares among medium-
scale farms (Figures 1 and 2, Tables 2 and 3). In countries
with substantial unutilized land, as in Zambia, Tanzania, and
parts of Ghana and Nigeria, the share of farm production
and marketed output accounted for by farms in the 5–100 ha
category is rising quite rapidly. In Ghana, for example, the
share of national cropped area under medium-scale farms is
close to 50%, and medium-scale farms account for over half
of all nationally marketed oilseeds and horticultural crops,
even with the caveats noted in Section 2 about the under-
representation of medium-scale farms. This trend is not happening everywhere. 2
DATA AND METHODS In densely popu-
lated countries such as Kenya, Malawi, Uganda, and Rwanda,
land scarcity and high land values are impeding the pace
of medium-scale farm acquisitions, and the share of land
under medium-scale farms is growing slowly if at all. How-
ever, as we have established in Section 2, LSMS data
may under-represent medium and large-scale farm holdings,
based on comparisons of larger farm censes and LSMS
data from the same year. Therefore, the share of cultivated
land, farm production and marketed output accounted for by
medium-scale farms as reported in this review are most likely
underestimated. 2
DATA AND METHODS In terms of
land under operation (defined as land cultivated, in fallow and
under pasture), the ASCS reports 35.8% and 16.5% greater
operated area under medium-scale and large-scale farms than
LSMS. Based on this comparison of agricultural census versus
population-based surveys, we utilize LSMS and comparable
nation data sets cautiously, understanding that they may rep-
resent a lower bound estimate of their share of national farm-
land, cultivated area, and farm production. Finally, we draw upon recent surveys of medium-scale
farms conducted by the Federal University of Agriculture at
Abeokuta in Nigeria, the Tegemeo Institute of Egerton Uni-
versity in Kenya, the Sokoine University in Tanzania, and
by the Indaba Agricultural Policy Research Institute in Zam-
bia. These exercises involved the compilation of lists of the
full population of 5 to 100 ha farms in selected districts in JAYNE ET AL. 78 TA B L E 1
Comparison of farmland owned and land under cultivation in Tanzania, 2008/09 season TA B L E 1
Comparison of farmland owned and land under cultivation in Tanzania, 2008/09 season
Farm land controlled
Land under operation
LSMS
Ag Sample
Census Survey
% difference
LSMS
Ag Sample
Census Survey
% difference
By holdings of:
Million hectares
Million hectares
0–5 ha
8.246
8.595
+4.2
8.117
8.130
+0.002
5–100 ha
3.872
5.861
+51.4
3.816
5.181
+35.8
Over 100 ha
0.809
1.294
+60.0
0.809
0.942
+16.5
N t
Land nder operation
c lti ated + fallo
+ other ses p
+
+
Source. Tanzania National Bureau of Statistics 2008/09 Agricultural Sample Census Survey and 2008/09 National Panel Survey/LSMS. 0%
10%
20%
30%
40%
Small, on-farm
focus, not com
Small, on-farm
focus, com
Small, off-farm
focus, not com
Small, off-farm
focus, com
Medium
Large
2008
2010
2012
2014
F I G U R E 1
Distribution of land cultivated by
farm category, Tanzania, 2008, 2010, 2012, and 2014
[Color figure can be viewed at wileyonlinelibrary.com]
Source. National Panel Survey / LSMS, Tanzanian
National Bureau of Statistics, Dar es Salaam. p
Source. Tanzania National Bureau of Statistics 2008/09 Agricultural Sample Census Survey and 2008/09 National Panel Survey/LSMS. 3
CHANGES IN THE
DISTRIBUTIONS OF FARM
SIZE, CROP PRODUCTION,
AND MARKETED OUTPUT The size distributions of farms in many African countries
are rapidly changing. In most of the countries for which
LSMS/ISA or similar national rural household surveys exist,
and particularly those with substantial potential for cropland
expansion, it is no longer true that the vast majority of farm-
land in Africa is small-scale. The national shares of area under The rise of medium-scale farms has occurred during a
period when the rate of agricultural production growth in JAYNE ET AL. 7
0
10
20
30
40
50
60
Grains/roots/tubers
Industrial cash crops
Horticuture
Legumes/oil seeds
Grains/roots/tubers
Industrial cash crops
Horticuture
Legumes/oil seeds
Grains/roots/tubers
Industrial cash crops
Horticuture
Legumes/oil seeds
Grains/roots/tubers
Industrial cash crops
Horticuture
Legumes/oil seeds
Ghana [1992-2013]
Nigeria [2011-2016]
Tanzania [2008-2014]
Rwanda [2006-2014]
Percentage share
Period 1
Period 2 79 JAYNE ET AL. F I G U R E 2
Medium-scale (5–100 ha) farms’ share of national value marketed crop output [Color figure can be viewed at Medium-scale (5–100 ha) farms’ share of national value marketed crop output [Color figure can be viewed at wileyonlinelibrary.com] wileyonlinelibrary.com]
Source. Computed from national household survey data as described in Section 2. All data sets are considered nationally representative official data
collected by national statistical services. Source. Computed from national household survey data as described in Section 2. All data sets are considered nationally representative official data
collected by national statistical services. Source. Computed from national household survey data as described in Section 2. All data sets are considered nationally representative official da
collected by national statistical services. hanges in the shares of national crop production value by farm size category TA B L E 2
Changes in the shares of national crop production value by farm size category Countries with relatively sparsely populated areas
Relatively densely populated areas
Zambia
Tanzania
Ghana
Nigeria
Uganda
% share of national value of crop output
1999
2015
2009
2015
1999
2013
2011
2016
2006
2014
0–5 ha
79.6
66.3
82.0
70.7
78.0
56.0
93.9
88.0
84.2
95.3
5–10 ha
13.7
18.9
12.1
17.8
11.8
25.9
5.1
6.8
7.6
3.0
10–20 ha
5.1
12.0
2.1
9.3
6.4
12.3
0.7
4.9
3.3
1.6
20–100 ha
1.4
2.8
2.3
2.1
3.5
5.5
0.3
0.3
4.3
0.1
+ 100 ha
0.0
0.0
1.3
0.1
0.3
0.3
0.1
0.0
0.6
0.0
100
100
100
100
100
100
100
100
100
100
Source. National household surveys as described in Section 2. Ghana Note. Value of farm output includes both crop and livestock production. The Tanzania surveys are found to underestimate area cultivated of farms 5–20 ha and 20+ ha by
roughly 50% (Jayne et al., 2016), so Tanzania figures most likely underestimate the shares of farm output growth. Zambia data considered statistically representative of
farms 0–20 ha, so they do not account for the contributions of farm growth and cultivated area of this farm size category. Source. Computed from national household survey data as described in Section 2. Note. Value of farm output includes both crop and livestock production. The Tanzania surveys are found to underestimate area cultivated of farms 5–20 ha and 20+ ha by
roughly 50% (Jayne et al., 2016), so Tanzania figures most likely underestimate the shares of farm output growth. Zambia data considered statistically representative of
farms 0–20 ha, so they do not account for the contributions of farm growth and cultivated area of this farm size category. Source. Computed from national household survey data as described in Section 2. majority of farms in the country (92% in Tanzania and 90% in
Zambia), they accounted for only slightly more than 50% of
the additional value of farm production in Tanzania between
2008/2009 and 2014/2015 and in Zambia between 2001
and 2015. In Zambia, note that farms cultivating over 20 ha
were not included in the sampling frame – if they were, the
share accounted for by small-scale farms would certainly be
lower while that of medium-scale farms would be higher. Small-scale farms contributed only 40% of the additional
value of farm production in Ghana between 2005 and 2013. Farms cultivating between 5 and 10 ha contributed 51% while
farms over 10 ha contributed 8.7% of Ghana’s additional
farm output between 2005 and 2013. In short, medium-scale was 4.35%, 0.61%, and 3.50% per year. Table 4 disaggregates
the changes in agricultural production growth based on the
available large-scale farm surveys available in each country
for two points in time. The results in Table 4 show that medium-scale farms
accounted for 47% of the additional value of farm output
produced nationally between 2008/2009 and 2014/2015. Of
these medium-scale farms, 26% of the additional value of
farm output was contributed by farms cultivating between
5 and 10 ha, whereas 20.7% was contributed by farms over
10 ha (despite being substantially under-reported in this
LSMS dataset as noted in Section 2). 3
CHANGES IN THE
DISTRIBUTIONS OF FARM
SIZE, CROP PRODUCTION,
AND MARKETED OUTPUT sub-Saharan Africa has been the highest of any region in
the world. Sub-Saharan Africa has achieved 4.6% inflation-
adjusted annual mean increases in agricultural growth
between 2000 and 2016 (World Bank, 2017), roughly double
that of the prior three decades. The region’s per capita GDP
increased by almost 35% in real terms over this period, dou-
bling in some countries (Barrett, Christiaensen, Sheahan, &
Shiferaw, 2017). Poverty rates have declined significantly for
the region as a whole since 2000.1 Nutritional indicators also show gradual but clear improvement (Masters, Rosenblum, &
Alemu, 2018). At the same time, the pace of transformation
has been highly uneven across the region. sub-Saharan Africa has been the highest of any region in
the world. Sub-Saharan Africa has achieved 4.6% inflation-
adjusted annual mean increases in agricultural growth
between 2000 and 2016 (World Bank, 2017), roughly double
that of the prior three decades. The region’s per capita GDP
increased by almost 35% in real terms over this period, dou-
bling in some countries (Barrett, Christiaensen, Sheahan, &
Shiferaw, 2017). Poverty rates have declined significantly for
the region as a whole since 2000.1 Nutritional indicators also Given sub-Saharan Africa’s impressive rate of agricultural
production growth since 2000, it would be important to under-
stand the extent to which medium-scale farms have con-
tributed to agricultural production growth in these countries. The inflation-adjusted growth rates of agricultural value addi-
tion in Tanzania, Zambia, and Ghana between 2001 and 2016 JAYNE ET AL. 80 TA B L E 3
Changes in the shares of national marketed crop production value by farm size category TA B L E 3
Changes in the shares of national marketed crop production value by farm size category
Relatively sparsely populated areas
Relatively densely populated areas
Zambia
Tanzania
Ghana
Nigeria
Uganda
% share of national value of marketed crop output
2001
2015
2009
2015
1999
2013
2011
2016
2006
2014
0–5 ha
74.2
52.9
80.2
67.1
79.9
56.6
92.2
80.7
77.1
88.9
5–10 ha
15.0
22.6
12.6
22.0
11.7
22.9
6.8
9.5
10.3
7.3
10–20 ha
8.3
19.6
4.0
8.7
5.6
13.1
0.7
9.2
5.4
3.6
20–100 ha
2.5
4.8
2.4
3.0
2.8
7.0
0.2
0.8
6.3
0.2
+ 100 ha
0.1
0.0
0.8
0.1
0.1
0.4
0.2
0.0
0.9
0.0
100
100
100
100
100
100
100
100
100
100
Source. National household surveys as described in Section 2. 3
CHANGES IN THE
DISTRIBUTIONS OF FARM
SIZE, CROP PRODUCTION,
AND MARKETED OUTPUT A B L E 4
Contributions to farm output and cultivated area by farm size category, Tanzania, Zambia, and Ghana TA B L E 4
Contributions to farm output and cultivated area by farm size category, Tanzania, Zambia, and Ghana
Farm size category (area cultivated)
Tanzania
Unit
0–4.99 ha
5–9.99 ha
10 and over
National
(all farms)
Change in value of farm output
(2014/2015 minus 2008/2009)
Billions of 2015 real
Tanzanian shillings
1,756
858
682
3,296
Share of growth in value of farm
output (2014/2015 minus
2008/2009), by farm size category
53.3%
26.0%
20.7%
Zambia
0–4.99 ha
5–9.99 ha
10–20 ha
All farms,
0–20 ha only
Change in value of farm output (2015
minus 2001)
Millions of real 2015
rebased Kwacha
4,014
1,901
1,498
7,413
Share of growth in value of farm
output (2015 minus 2001), by farm
size category
54.1%
25.6%
20.3%
Ghana
0–4.99 ha
5–9.99 ha
10 and over
National
(all farms)
Change in value of farm output (crop
only) (2013 minus 2005)
Millions of real 2013
Ghana Cedis
1,166.64
1,513.47
254.40
2,934.52
Share of growth in value of farm
output (2013 minus 2005), by farm
size category
39.7%
51.6%
8.7%
Note. Value of farm output includes both crop and livestock production. The Tanzania surveys are found to underestimate area cultivated of farms 5–20 ha and 20+ ha by
roughly 50% (Jayne et al., 2016), so Tanzania figures most likely underestimate the shares of farm output growth. Zambia data considered statistically representative of
farms 0–20 ha, so they do not account for the contributions of farm growth and cultivated area of this farm size category. Source. Computed from national household survey data as described in Section 2. 4.2
An era of high global food prices Food prices in Africa rose substantially after the global food
price surge of 2007/2008.3 This has fueled an increase in
demand for farmland as both global and domestic investors
recognized that quality farmland in parts of Africa was under-
valued.4 The sustained agricultural growth that many African
countries have experienced in the recent period of high local
and world food prices also suggests that new land acquisitions
during this period reflect perceptions of the profitability of
agriculture as a business for those able to mobilize sufficient
land, capital, and management expertise. farms contributed over 45% of the farm production growth
experienced in each of the three countries over the specified
periods, which is particularly noteworthy considering that the
shares attributed to farms over 10 ha as reported in Table 4
are likely to be under-reported, and given that farms over 20
ha in Zambia are excluded from the analysis. tory tenure with freehold or long-term lease titles (Knapman
et al., 2017). One likely outcome of such trends is that custom-
ary lands are being privatized more quickly with less being
available as a birthright of future generations of rural-born
youth. 4.3
Policy reforms An under-appreciated contributory factor has been the
contentious agricultural market and economy-wide policy
reforms undertaken during the 1980s and 1990s. These policy
reforms removed major barriers to private trade. The effects
of the reforms were mostly dormant until the mid 2000s when
world food prices suddenly skyrocketed, enabling thousands
of small, medium and large-scale private firms to rapidly
respond to profitable incentives, thereby rapidly building up
the region’s agri-food systems during this period (Jayne,
Mather, & Mghenyi, 2010). Reductions in trade barriers and
state control of agricultural markets has enabled domestic
food prices to become better aligned with import parity con-
ditions compared with earlier years. Small-, medium-, and
large-scale private firms have invested all along agricultural
value chains in response to these policy reforms, which are
part of the agri-food systems transformations that the region
is now experiencing. Today, land sales markets constitute a major pathway
for the expansion of medium-scale farms. There are several
types of land sales markets. In some African countries, the
purchase and sale of land is now legal. According to the
2014/2015 LSMS in Tanzania, for example, purchased land
accounts for 29.6% of all plots held by farm households and
36.5% of all cultivated land. Qualitative surveys indicate that
relatively wealthy rural people as well as urban-based people,
and even relatively successful smallholder farmers are buying
land in areas of favorable market access conditions from other
households who are relocating to urban or more hinterland
rural areas (Anseeuw, Jayne, Kachule, & Kotsopoulos, 2016;
Knapman et al., 2017; Muyanga et al., 2019). Also common
is the privatization and sale of land by traditional authorities. Historically, chiefs and headmen were seen as custodians of
unutilized land, allocating it to members of their local com-
munities as their numbers swell, but the rising acquisition of
landholdings by non-resident people indicate the possibility
that customary land is being “sold” based on willingness to
pay criteria. Transfers of land from customary tenure to for-
mal or informally privatized land appear to be associated with
the rise of domestic investor farmers in at least some countries
such as Zambia and Malawi. Where customary land institu-
tions still prevail, they are increasingly utilized by wealthy
outsiders as a means to acquire land (Knapman et al., 2017). This often results in a transfer of land from customary tenure
(under the authority of chiefs or their representatives) to statu- 4
CAUSES OF CHANGING FARM
STRUCTURE There are four main causes of changing farm size distributions
in Africa: the rise of land markets, the recent era of relatively
high global food prices, greater agricultural policy reforms,
and the actions of farm lobbies. 4 Rural land values in favorable market access areas of Tanzania have doubled
in real terms between 2009 and 2013, rising more rapidly than wage rates or
other inputs into agricultural production (Wineman & Jayne, 2018). 4.1
The rise of land markets Rapid rural population growth has transformed settled areas
from land-abundant areas where rural-born people would
receive land as a birthright and where even migrants from
different regions could often easily acquire land to areas of
land scarcity where the value of land has skyrocketed in recent
years. Land purchase/sales markets are developing rapidly in
countries where they were considered illegal not more than a
generation ago. 3 The international prices of maize, rice, and wheat over the 2006–2018
period, adjusted by two different global deflators (the US GDP deflator
and the global Manufacturing Unit Values Index), are on average 49%,
46%, and 39% higher than their inflation-adjusted 1990–2005 averages,
based on World Bank Pink Sheet (https://www.worldbank.org/en/research/
commodity-markets , last accessed July 19, 2019). Ghana While small-scale
farms cultivating between 0 and 5 ha account for the vast 81 JAYNE ET AL. 81 5
CONSEQUENCES OF CHANGING
FARM STRUCTURE This is a fairly new area of research and the evidence base
remains thin. Based on the few studies undertaken so far, we
highlight both positive and potentially negative effects from
the rapid acquisition of land by medium-scale farms. One the positive side, medium-scale farms are pulling in
major new private investment in value chains that improve
market access conditions for nearby smallholders. For exam-
ple, farming areas with a high concentration of medium-scale
farms attract greater investment by large-scale grain buyers
in Kenya, Zambia, and Tanzania (Sitko, Burke, & Jayne,
2018). Small-scale farmers are significantly more likely
to sell to large grain trading firms if they are located in
districts with a high concentration of medium-scale farms,
even after controlling for agro-ecological and market access
conditions (Burke, Jayne, & Sitko, 2019). Though the
large-scale grain traders are initially attracted to invest in
an area by the large marketable surpluses of medium-scale
farms, once they establish their buying stations, they improve
market access conditions for all farms in the area. Using
LSMS data from Tanzania, Van der Westhuisen et al. (2018)
find that small-scale farms are much more likely to rent
mechanization services in areas with a high concentration
of medium-scale farms. Although only 3% of small-scale
farms rent tractors in the 21 regions of mainland Tanzania
with the lowest concentration of medium-scale farms, 23%
of smallholders rent mechanization services in the 5 regions
with the highest concentration of medium-scale farms. Mechanization rental services have sprung up in areas with a
high concentration of medium-scale farms, catering to their
demand for tractor services, which has made it more feasible
for small-scale farms to rent tractors, reduce their labor
input into farming, and reallocate their labor to higher-return
non-farm activities, while still deriving income from farming. Wineman, Jayne, Isinika-Modamba, and Kray (2018) also
show important “spillover benefits” whereby the presence of
medium-scale farms tends to improve small-scale farmers’
access to agricultural inputs and services. Other studies in
this emerging “spillovers” literature tend to find positive
synergies in some cases and no clear statistical relationship
in others (Ali, Deininger, & Harris, 2019; Lay, Nolte, &
Sipangule, 2018; Deininger & Xia, 2016). regained control over the policy agenda starting in the early
2000s, often in an environment of nascent multiparty polit-
ical systems. Parties often adopted populist stances such as
offering free inputs and support prices for local farmers to pro-
mote food self-sufficiency. These developments enhanced the
voice and influence of national farmer unions that ostensibly
lobbied for the interests of the farming community. However,
farmers are not a homogeneous group and particular policies
affect them in different ways. Farmer unions in some coun-
tries lobby forcefully for a system of agricultural subsidies
and land allocation that channel the majority of public expen-
ditures to agriculture to relatively large farms (Binswanger,
Deininger, & Feder, 1995). Most national farmer unions in
the region support policies that raise food prices (rewarding
farms that produce the largest marketable surpluses), promote
the conversion of land from customary tenure to statutory land
to promote access to land through market transactions, farm
block programs, and input and credit subsidy programs that
allow bigger farms to participate disproportionately in the pro-
grams. Common rhetoric used to justify this position is that
public support should go to “progressive” farmers who view
“farming as a business” and have entrepreneurial experience
to transform African agriculture. These positions reflect the
interests of relatively capitalized “emergent” farmers, and at
a minimum suggest the possibility that some of the national
farmer unions have been captured by these interests (Sitko &
Jayne, 2014).5 Because investor farmers tend to be more edu-
cated and have more extensive social connections with tra-
ditional and state authorities, they also tend to have advan-
tages with respect to navigating both customary and statutory
land institutions to access land. For these reasons, and espe-
cially since the rise of world food prices in the mid 2000s, the
profitability of commercial farming has increased and this has
been associated with the increase in medium-scale land acqui-
sitions in the region. public agricultural budgets and policies, much as large farm
interests have in other parts of the world. 5 For example, about 50% of the Zambian government’s agricultural bud-
get goes into subsidy programs benefitting the most privileged 5% of farm-
ers (Jayne, Mather, & Mghenyi, 2010). Similarly, government preoccupation
with clearing the way for land market transactions, despite extensive rhetoric
to the contrary, is largely focused on trying to create processes whereby large
investors can gain access to land (Sitko & Chamberlin, 2016). 5 For example, about 50% of the Zambian government’s agricultural bud-
get goes into subsidy programs benefitting the most privileged 5% of farm-
ers (Jayne, Mather, & Mghenyi, 2010). Similarly, government preoccupation
with clearing the way for land market transactions, despite extensive rhetoric
to the contrary, is largely focused on trying to create processes whereby large
investors can gain access to land (Sitko & Chamberlin, 2016). 4.4
Farm lobbies and political capture After roughly a decade of intense struggle between African
governments and international lenders over the course of
agricultural policy between 1985 and 1995, local interests 82 JAYNE ET AL. 82 medium-scale (5–100 ha) farms in Kenya using relatively
large samples shows a distinct advantage to medium-scale
farms in three alternative measures of productivity includ-
ing TFP, net value of agricultural output per hectare, and
agricultural labor productivity (Muyanga & Jayne, 2019). The productivity advantage of medium-scale farms were due
to differences in technical choice related to mechanization,
which substantially reduces labor input per hectare, and from
greater intensity of cash input use. Medium-scale farms are
also more likely to be early adoptors of, and comply with the
protocols of, new farm technical innovations and practices. indicates that the rise of medium-scale farms may be con-
tributing strongly to the development of agricultural output
and factor markets, investment incentives by small-, medium-
, and large-scale agribusiness firms, and general equilibrium
multiplier effects associated with the region’s dynamism
over the past decade (Jayne, Chamberlin, & Benfica, 2018). As described in Section 4, causality between economic
dynamism and the rise of commercialized medium-scale
farms clearly runs in both directions. However, there are some warning signs as well. The rising
acquisition of land by outside investors certainly reduces the
stock of land under customary tenure that will be accessible
to current and future generations of local people. If traditional
authorities are selling off land to outside investors, it will
raise the price of land. Young people from these communities
will find access more difficult, which will increase their
likelihood of exiting farming and/or migrating out of the
area (Bezu & Holden, 2014; Chimhowu & Woodhouse,
2006; Knapman et al., 2017; Kocec, Ghebru, Holtemeyer,
Mueller, & Schmidt, 2018). Ghebru and Girmachew (2017),
Ghebru and Lambrecht (2017), and Ghebru and Girmachew
(2019) indicate that smallholders’ perceived tenure security
in Ghana, Nigeria, and Mozambique is negatively correlated
with the degree of local land market activity. Households
residing in communities with more vibrant land markets
perceive greater risk of losing land due to private dispute
or expropriation by the government. As the customary land
tenure system comes under greater stress as land becomes
increasingly commodified, the protection that traditional sys-
tems have historically provided to safeguard individual rights
to land are starting to break down. Badiane (2019) noted that
one of the major historical differences between poor rural
households in Africa and Asia was that at least most of those
in Africa had some rights to land. Unfortunately, in recent
years, researchers are detecting increasing signs of rural land-
lessness in much of Africa too (e.g., Mueller & Chan, 2015). Evidence to date from Tanzania, Kenya and Nigeria sug-
gests that the cropping patterns of medium-scale farms does
not differ greatly from those of small-scale farms (Muyanga
& Jayne, 2019; Muyanga et al., 2019; Wineman et al.,
2018). However, in most cases, small-scale farms do utilize a
higher proportion of their land. Many medium-scale farmers
acquired their land relatively recently and state an intention
to bring a progressively higher proportion of their land under
cultivation over time (Muyanga & Jayne, 2019). y
g
y
Chamberlin and Jayne (2018) using LSMS data from Tan-
zania find that districts with a high concentration of farmland
under medium-scale farms are associated with significantly
higher farm and nonfarm incomes of small-scale and non-
farm households. Exploiting inter-district variation in farm-
land distribution patterns in Tanzania, their study finds that
household incomes from farm, agricultural wage, and non-
farm sources are positively and significantly associated with
the share of land in the district controlled by 5–10 ha farms
(after controlling for market access, rainfall, soils, and other
local conditions). These positive spillover benefits are smaller
and less statistically significant in districts with a relatively
high share of farmland controlled by farms over 10 ha in size. Anecdotal interviews of key informants in rural areas suggest
that medium-scale farms, particularly those in the 5–20 ha
range, share many social and economic ties with small-scale
farm households, participate in the same rural institutions, and
hence may be more likely to have mutually beneficial eco-
nomic synergies. By contrast, many large farms are controlled
by people of other ethnic backgrounds or reside outside the
area, and hence may not share strong social ties in the local
community. It would be oversimplifying matters to conclude that
medium-scale investor farmers are the main source of tenure
insecurity for local rural people. The empirical evidences
show that members of the local community, often family
members, are a major source of land insecurity of indigenous
rural people. Ali, Deininger, and Goldstein (2011), Lawry
et al. 5
CONSEQUENCES OF CHANGING
FARM STRUCTURE We believe that a small-scale farm-led agricultural transfor-
mation strategy could have succeeded, and could still succeed
in Africa, as it did in much of Asia, if African governments
provide sustained support for smallholders through policies
and public expenditures targeted toward them. Ethiopia and
Rwanda, for example, appear to be pursuing such a strategy
with reasonable success. Yet as the political importance of
commercialized medium-scale farmers continues to rise, as it
has in many but not all of the countries examined, their inter-
ests may increasingly influence the composition and design of Direct comparisons of farm productivity between small-
scale and medium-scale farms are scarce because almost all
of the existing farm survey datasets from Africa (including
LSMS) contain very few observations of farms over 10
ha. New evidence that matches small-scale (0–5 ha) vs. 83 JAYNE ET AL. 83 6
STEPPING UP” VS. “STEPPING
IN”: CHARACTERISTICS OF
MEDIUM-SCALE FARMERS? Studies were conducted in Zambia, Kenya, Ghana, Nige-
ria, and Malawi to understand the characteristics of these
medium-scale farms and how they became medium-scale
farms (Anseeuw et al., 2016; Chapoto, Mabiso, & Bonsu,
2013; Jayne et al., 2016; Muyanga et al., 2019). We were
particularly interested in understanding the extent to which
current medium-scale farms started out as small-scale farms,
acquired more land and expanded their farm operations
(“stepping up”) or were formerly primarily engaged in non-
farm jobs, invested in land and began farming either as an
owner−operator or by hiring a farm manager to run the farm
(“stepping into” medium-scale farming). We highlight three
insights from these studies. We hypothesize that the importance of non-farm invest-
ment into medium-scale farming in the 2005–2013 period
was at least partially driven by the unusually high world food
prices that prevailed during this time. Many wealthy people in
both rural and urban areas found that food production could be
an attractive investment, especially in areas where traditional
authorities were willing to allocate relatively large tracts of
customary land at low cost to the investor. A sizeable propor-
tion of medium-scale farms – particularly those who acquired
land from non-farm income sources – started their farms after
2005 when world food prices rose dramatically. In Zambia, for
example, 63% of these farms were started after 2005. Hence,
as might have been expected, substantial resources appeared
to flow into agriculture from outside the sector, not only by
international investors but by local investors as well (Jayne
et al., 2016). The first few studies of medium-scale farmers (covering
recall periods between 2005 and 2013 and summarized
in Jayne et al., 2016) indicated that fewer than 25% of
them started out as small-scale farmers who were primarily
engaged in farming for their livelihoods and who successfully
expanded their operations into medium-scale status. Ghana
was the lone exception to this, where the majority of MS
farms did start out as small-scale farms (Chapoto et al., 2013). In Zambia, Kenya, and Tanzania, the majority of medium-
scale farmers were initially engaged primarily in non-farm
activities; they used their savings to invest in relatively
large landholdings to begin farming or expand their farming
operations. many medium-scale farms is reinforced by data in nationally
representative Demographic and Health Surveys (DHS),
which ask questions of both rural and urban households about
whether they own land and if so, how much land. As reported
in Table 6, urban-based individuals control a significant
proportion of total national land in the five African countries
examined, and in almost all cases, the proportion of land
owned by urban people has increased between the first and
most recent survey. For example, in Ghana, the proportion
of national land owned by urban residents increased from
26.8% to 31.9% between 2008 and 2014. In Kenya and
Zambia, urban-based individuals and families control close
to one-third of all national land controlled by individuals and
families. In Malawi, by contrast, the share of national land
controlled by urban households was only 3% in 2004, and it
rose only to 6.5% by 2010. However, taken together, it seems
that an important segment of farms in the 5–100 ha category
were owned by people who invested into agriculture using rev-
enues from non-farm employment, acquiring land from local
authorities or from increasingly active and sanctioned land
markets. informally to others, and become dependent on the local non-
farm economy for their livelihoods (Mueller & Chan, 2015). These land purchase market developments produce winners
and losers in the short run, and the evidence is not fully clear
whether the short-run losers become winners in the long run
through processes of economic transformation and growth. Policy makers will need guidance on how to minimize these
hardships – protecting those who are most vulnerable as the
processes of economic transformation gradually raise living
standards for the majority of the population. (2014), and Ghebru and Girmachew’s (2019) work on
land tenure systems indicate that the interests of vulnera-
ble groups such as women and youth will often require spe-
cial interventions in areas where land markets are developing
rapidly and that these interventions will need to be context
specific and hence vary by location. Also noteworthy is that the rise of commercialized
medium-scale farms is likely to facilitate means by which
governments may raise taxes or contributions that can be re-
invested into public goods in relevant rural areas. This has
yet to occur in many cases, but the surplus production of
commercialized medium-scale farms represents in principle a
future opportunity for Ministries of Finance and local com-
munities to raise revenues for reinvestment in local public
goods. Therefore, the development of land purchase/sale markets
is part of more wholesale changes in social systems, in some
ways uprooting the traditional social fabric and creating new,
new power structures. The rise of land markets is creating a
new class of landless workers in Africa, who sell their land Although this nascent literature requires additional evi-
dence before robust conclusions can be made, initial evidence JAYNE ET AL. 84 6
STEPPING UP” VS. “STEPPING
IN”: CHARACTERISTICS OF
MEDIUM-SCALE FARMERS? As shown in Table 5, roughly 60% of randomly
sampled medium-scale farms in four districts of Zambia
and Kenya “stepped in” to medium-scale farming using
revenues from nonfarm sources to buy land and start farming. Many of these farmers were relatively wealthy or privileged
rural people (civil servants, rural businesspeople, extension
agents, religious leaders, traditional headmen or chiefs, etc.)
or urban-based people. Emergent farmers who reside in urban
areas and hire managers to run their farms have become com-
mon enough in the region that the term “telephone farmers”
has emerged to describe them. The urban-based residences of However, more recent surveys indicate that the pace of land
investment by urban and rural elites may have slowed in recent
years. This might have been anticipated as world food prices,
while still relatively high compared to the 1990s and early
2000s, have declined from their unusually high levels between
2006 and 2012, and as land prices in favorable farming areas
have continued to rise due to population growth and commer-
cial interest. Recent studies in Zambia, Senegal, and Nige-
ria indicate that perhaps 60% of medium-scale farmers sur-
veyed in 2017 and 2018 have “stepped up” from small-scale
status. For example, the 2018 survey of medium-scale farms
in Ogun and Kaduna States by Muyanga et al. (2019) shows
that “stepping up” from small-scale to medium-scale farm-
ing as a pathway to agricultural commercialization in Nige-
ria, was more predominant than directly “stepping in” into
medium scale farming from nonfarm activities (Table 5). The 85
B L E 5
Characteristics of medium-scale farmers: “Stepping up” vs. 7
HOW ARE MEDIUM- AND
SMALL-SCALE FARMERS
ACQUIRING THEIR LAND? Medium-scale farms are growing most rapidly in areas where
land is still relatively cheap (e.g., Zambia, Tanzania, north-
ern Ghana) and least so in areas where additional land is
scarce and land prices are highest (Rwanda, Kenya, South-
ern Ghana). Small-scale and medium-scale farm households
acquire land via four main forms: (i) inheritance; (ii) renting
land; (iii) obtaining land from traditional authorities, either
allocated for free as a social right, or via purchase or long-
term lease; and (iv) purchasing land or obtaining a long-term
lease from another owner/household. Inheritance: Historically, inheritance has been a major
form of land access for rural–born people. However, inher-
itance is declining in most countries, especially those with
already high population densities. In the 2014/2015 Tanzania
LSMS survey, for example, inheritance accounts for 33.2% of
all plots and 38.3% of all area under cultivation. Inheritance
accounts for 40% of plots in Ethiopia, 14% in Nigeria, 70%
in Niger, and 62% in Uganda. Because of rising life expectan-
cies in sub-Saharan Africa – from 48 years in 1980 to 60 years
in 2015 – rural youth will need to wait longer to inherit land
(Jayne et al., 2014b). Continued subdivision and fragmenta-
tion will continue to limit the amount of land to be inherited
by today’s rural youth (Yamano, Place, Nyangena, Wanjiku,
& Otsuka, 2009). For these reasons, it is increasingly unlikely
that rural African youth will inherit land in the future. For
those who do, they tend to inherit much smaller parcels than
in the past and at a later stage in their lives. Lack of inheritance
options is a major reason for youth outmigration (Holden &
Bezu, 2014; Kocec et al., 2018; Muyanga et al., 2019). However, even the medium-scale farmers who “stepped
up” from small-scale might be considered atypical of most
small-scale farm households, given that their initial landhold-
ing size at the time they started farming was 4.0 ha in Nigeria,
14 ha in Kenya, and 29 ha in Zambia (Table 5). They may have
been farming less than five hectares when they started out,
but very few small-scale farm households possess landhold-
ings of that size in Kenya or Zambia, pointing to rather unique
circumstances of the small-scale farms who tend to “step-up”
into medium-scale status. 6
STEPPING UP” VS. “STEPPING
IN”: CHARACTERISTICS OF
MEDIUM-SCALE FARMERS? TA B L E 6
Extent of urban household control of national agricultural land Notes. For Zambia, Malawi, and Kenya, landholdings over 95 ha were reclassified as 95 ha. For Ghana, landholdings over 95 acres were reclassified as 95 acres (38 ha). These caps on the reported landholding size may result in underestimates of the land controlled by urban households. Source. Demographic and Health Surveys, https://www.dhsprogram.com/Data/. fully out of farming than to become medium-scale farmers in
the years to come. But much will still depend on government
policies and the composition of their spending on agriculture. study of medium-scale farmers in Senegal by Bourgoin et al. (in press) similarly shows that the majority of randomly sur-
veyed medium-scale farms started out as small-scale farm-
ers who utilized land markets and other forms of land
acquisition to expand their farming operations. The Senegal
case study does show major investment by people primarily
involved in non-farm businesses in areas close to major urban
centers such as Niayes, but in most other areas, most medium-
scale farms started out as small-scale farms primarily engaged
in farming. In Nigeria, land accessibility and mechanization
rental markets were found to be the most important factors
that enabled small-scale farms to “step up” into medium-
scale status, pointing to the importance of land and mecha-
nization markets for the “stepping up” process. These results
from Nigeria, Senegal, and Zambia point to a small but grow-
ing class of entrepreneurial commercialized African farmers
who are using proceeds from farming to expand their opera-
tions into the 5–20 ha category. The growing dynamism and
upward mobility among some segments of smallholder farm-
ers is associated both spatially and temporally with increas-
ingly active land, labor, agricultural input, output, and finance
markets, which is making it easier for individuals to overcome
binding constraints on farmland expansion and productivity. 6
STEPPING UP” VS. “STEPPING
IN”: CHARACTERISTICS OF
MEDIUM-SCALE FARMERS? “Stepping in”
Nigeria, 2018
Kenya, 2013
Zambia, 2013
Primary mode of entry into medium-scale farming (i.e., operating between 5 and 100 ha)
Stepping up:
Growth from
small-scale
farming
(n = 476)
Stepping in:
Acquisition of land
from non-farm
employment
(n = 534)
Stepping up:
Growth from
small-scale
farming
(n = 120)
Stepping in:
Acquisition of land
from non-farm
employment
(n = 180)
Stepping up:
Growth from
small-scale
farming
(n = 118)
Stepping in:
Acquisition of land
from non-farm
employment
(n = 164)
of cases
47.1
52.9
40
60
42
58
men
95.4
98.1
82.5
80.0
92.9
91.4
ar of birth
1974
1963
1945
1947
1966
1960
ars of education of head
7.0
8.0
7.5
12.7
8.2
11.0
ve held a job other than
as a farmer (%)
19.2
88.9
17.5
83.3
32.9
100.0
merly or currently employed
by the public sector (%)
13.5
53.3
12.5
56.7
5.8
59.6
ial landholding size when
tarted farming (ha)
4.0
11.7
14.0
22.6
28.8
106.6
rrent landholding size (ha)
7.8
25.6
32.7
50.1
38.2
74.9
of land currently under
ultivation
78.8
62.3
54.1
46.6
46.9
24.7
cade when land was acquired
by individual:
979 or earlier (%)
32.1
43
24
10.6
6.2
980–1989 (%)
20
20
14.8
7.4
990–2005 (%)
26.7
14
32
32.2
23.8
2005 or later (%)
41.2
23
25
42.0
63.4
. The “stepping-in” category includes individuals who purchased over 5 ha of land to initiate their farming activities. e. Expanded from Jayne et al. (2016) to include 2018 data from Nigeria as reported in Muyanga et al. (2019). 85 JAYNE ET AL. 85 JAYNE ET AL. 86 TA B L E 6
Extent of urban household control of national agricultural land
Ghana
Kenya*
Malawi*
Tanzania
Zambia*
2008
2014
2009
2014
2004
2010
2005
2010
2007
2014
n = households
11,777
11,835
9,057
36,430
13,664
24,825
9,735
9,623
7,164
15,920
% of national landholdings held by
urban households
26.8
31.9
22.0
32.1
3.0
6.5
11.8
32.7
16.8
22.0
% of landholdings of >20 ha held
by urban households
36.9
42.7
34.3
41.2
1.2
7.6
17.2
78.9
21.7
29.3
Notes. For Zambia, Malawi, and Kenya, landholdings over 95 ha were reclassified as 95 ha. For Ghana, landholdings over 95 acres were reclassified as 95 acres (38 ha). These caps on the reported landholding size may result in underestimates of the land controlled by urban households. Source. Demographic and Health Surveys, https://www.dhsprogram.com/Data/. 8
CHANGES IN LAND TENURE
SYSTEMS AND SECURITY markets is rising. Most studies of land rental markets indi-
cate that they are welfare-enhancing, by transferring land
from labor-deficit, land-rich households to land-constrained
households with available labor to work the land (Cham-
berlin & Ricker-Gilbert, 2016; Deininger, Savastano, & Xia,
2017). Since 1980s, researchers have documented the changing
dynamics between customary and statutory tenure systems
wrought by informal land sales.6 The processes by which
investor farmers have acquired customary land has been
highly variable, but early observers noted that a common
motivation of such acquisitions has been to undertake
commercialized crop production.7 However, the formal legal
status of customary tenure and the scale of these interactions
give the contemporary urban investor farmer phenomenon
unique features. Although in prior decades almost all SSA
resisted conferring legal state recognition of customary
tenure systems, since the 1990s the trend has been towards
formal recognition of customary tenure. Until the 1990s
most SSA countries had an official or unofficial policy
to extinguish customary tenure by promoting individual
titling. These efforts generally failed because of titling costs
and the striking resilience of customary tenure systems. Meanwhile, informal transfers in accordance with customary
norms, including to investor farmers, accelerated especially
during the past decade of relatively high world food prices
(Chimhowu & Woodhouse 2006; Lawry et al., 2014). Allocation of land by traditional authorities: In some areas,
allocation of new land is not possible because all land under
customary tenure has already been allocated. In areas where
significant amounts of unallocated land remain under custom-
ary tenure systems, the rise of investor farmers is competing
with future generations of rural youth for land (Jayne et al.,
2016). This is where land registration and certification may
provide the greatest benefits in terms of securing access to
land for locally-born rural people (Ghebre and Lambrecht,
2017; Gebhre and Girmachew, 2017). Land markets for purchase and long-term lease: Because
of growing scarcity, land is increasingly recognized as hav-
ing value. Over the past 20 years, land purchase markets have
sprung up rapidly, even in customary tenure areas where it
has traditionally been considered taboo (Chimhowu & Wood-
house, 2006; Sitko & Chamberlin, 2016; Woodhouse, 2003). Purchase and long-term leases provide better leverage for the
farmer to undertake permanent improvement on the land such
as irrigation and soil conditioning as compared to short-term
tenancy that is not amenable to land development and con-
ditioning. 7
HOW ARE MEDIUM- AND
SMALL-SCALE FARMERS
ACQUIRING THEIR LAND? Although these recent studies indicate that half or more
of the current MS farms were formerly small-scale farmers,
an exceedingly small fraction of small-scale farm households
ever become medium-scale farmers. The probability that a
small-scale farmer (0–5 ha cultivated) will become a medium-
scale farmer (5–100 ha cultivated) is on average about 4–5%. Many more small-scale farms are expected to exit partially or Land rental markets: Most of the LSMS surveys indi-
cate that rural household participation in land rental JAYNE ET AL. 87 TA B L E 7
Changes in the concentration of farmland ownership
Period
Movement in Gini
coefficient
Ghana (cultivated area) (GLSS)
1992 →2013
0.54 →0.69
Kenya (cultivated area) (KIHBS)
1994 →2006
0.51 →0.55
Tanzania (landholdings) (LSMS)
2008 →2012
0.63 →0.69
Tanzania (area controlled) (ASCS)
2008
0.89
Zambia (landholding) (CFS)
2001 →2012
0.42 →0.49
Source. Computed from National Household Survey data as described in Section 2. All data sets are considered nationally representative official data collected by national
statistical services. TA B L E 7
Changes in the concentration of farmland ownership TA B L E 7
Changes in the concentration of farmland ownership
Period
Movement in Gini
coefficient
Ghana (cultivated area) (GLSS)
1992 →2013
0.54 →0.69
Kenya (cultivated area) (KIHBS)
1994 →2006
0.51 →0.55
Tanzania (landholdings) (LSMS)
2008 →2012
0.63 →0.69
Tanzania (area controlled) (ASCS)
2008
0.89
Zambia (landholding) (CFS)
2001 →2012
0.42 →0.49
Source. Computed from National Household Survey data as described in Section 2. All data sets are considered nationally representative official data collected by national
statistical services. Source. Computed from National Household Survey data as described in Section 2. All data sets are considered nationally representative official data collected by national
statistical services. 6 USAID Country Profiles of Land Tenure, 1986; Is Indigenous Tenure A
Development Constraint?, Bruce, 1986.
7 Ibid. 6 USAID Country Profiles of Land Tenure, 1986; Is Indigenous Tenure A
Development Constraint?, Bruce, 1986. Based on their
analysis of six household datasets collected across rural farm-
ing areas of Ethiopia, Guinea, Liberia, and Zambia, Stickler,
Huntington, and Ewing (under review) find that a clear major-
ity of respondents, most of whom possess no documentation
of their land rights, do not perceive a significant risk of land
appropriation by either internal or external actors.9 This is an
important finding, since many land registration policies are
based on the premise that unregistered rights are inherently
insecure. Still, significant minorities in Liberia and Zambia
(10–30%) reported that the land they currently possess could
be appropriated by internal or external actors in the near future
(1–5 years). Given the scale of the acquisitions, one question is how
investor farmers are viewed by those in customary commu-
nities – as sources of economic dynamism and employment
or as exploiter? Historically, the nature of land conflict in
SSA centers on dynamics between autochthonous members
of a community (i.e., those with a real or mythic link to a
community’s original inhabitants or settlers) and strangers, or
newcomers (Boone, 2014). As with emergent investor farm-
ers, strangers have been attracted to certain lands because of
the potential for commercial crops (e.g. cocoa in Ghana and
Cote d’Ivoire; rice in Tanzania; maize in Zambia). Conflicts
with long-established customary communities would invari-
ably follow. However, as Boone argues, whether the conflicts
remained localized or impacted, security at the national level
depended on whether the central government consistently
favored autochthonous communities or the stranger, newcom-
ers. An open question is how the emergent investor farmer
phenomenon maps onto these well-documented dynamics
across SSA. Will African investor farmers be regarded as
strangers or not depending on the extent they rely on per-
sonal and familial connections to acquire land vs. outright
purchase from traditional authorities? Whom will SSA gov-
ernments favor in a context of rising land scarcity and pal-
pable tenure insecurity? One the one hand, many SSA gov-
ernments embrace, to greater or lesser degrees, the need to
respect and protect customary land rights. On the other hand,
investor farmers represent an opportunity for significant gains
in crop production and economic growth, but perhaps at the
expense of customary tenure holders. Across all datasets, female-headed households were signif-
icantly more likely to report a risk of internal appropriation
than male-headed households were. (Huntington et al., under review), with only 20% of respon-
dents reporting that encroachment on their land is likely and
most with relatives and neighbors within the village posing
the most important threat. Further, households reported
significant concerns about land reallocation by chiefs and
village headmen. This reallocation appears to be motivated
not primarily by demand for land from elite external actors,
but when another member of the village or a family member
needs land to farm, especially if the plot of land in question
is not under cultivation at the time of reallocation. ownership (Stevens, 2014). The onerous statutory process
means in practice most acquisitions stopped at the Tribal
Certificate, which has become a de facto deed of ownership
for many (Ibid). In some parts of Liberia, Tribal Certificates
comprise as much as 50% of the land area.8 Although more
research is needed, Tribal Certificates can cover hundreds,
or even thousands, of acres, and in at least some cases are
used to secure farms with cash crops and for high value
land in proximity to cities and major transit routes. Similar
approaches have been pursued in other African countries to
make it legal for governments or traditional authorities to sell
of land to investors (Boone, 2014). ownership (Stevens, 2014). The onerous statutory process
means in practice most acquisitions stopped at the Tribal
Certificate, which has become a de facto deed of ownership
for many (Ibid). In some parts of Liberia, Tribal Certificates
comprise as much as 50% of the land area.8 Although more
research is needed, Tribal Certificates can cover hundreds,
or even thousands, of acres, and in at least some cases are
used to secure farms with cash crops and for high value
land in proximity to cities and major transit routes. Similar
approaches have been pursued in other African countries to
make it legal for governments or traditional authorities to sell
of land to investors (Boone, 2014). Evidence to date indeed points to some degree of displace-
ment of indigenous small-scale farm households in areas of
favorable commercial potential. There is little evidence of dis-
placement in more remote areas, primarily because relatively
few investor farmers are locating in such areas. 9 Although the response categories were standardized within datasets and
grouped into standard categories across datasets, we note that respondents
may nevertheless have reported threats differently according to their percep-
tions or interests. For example, an urban family member could be reported as
either a family member (internal threat) or an urban elite/investor (external
threat). 8 This data was collected by USAID’s Land Tenure Office. 88 JAYNE ET AL. 88 8
CHANGES IN LAND TENURE
SYSTEMS AND SECURITY The rising importance of land purchase markets
may therefore be a source of productivity improvement for
those able to secure long-term rights to land. Moreover, grow-
ing participation in legal and clandestine land purchase/sale
markets are a major source of rising land prices in parts of
rural Africa. Using LSMS data from Tanzania, Wineman and
Jayne (2018) identify the following factors as correlates of
land values in Tanzania: the net value of crop output per
acre, soil quality, and market access conditions – all prox-
ies for a region’s agricultural commercialization potential. The growth of small and medium-sized towns are improving
market access conditions in farming areas once considered
remote, thereby raising land values in such areas. Because
of all these land allocation processes, farmland ownership is
becoming more concentrated, as measured by the Gini coef-
ficient of owned land (Table 7). With the advent of formal recognition of customary
tenure, investor farmers must obtain otherwise customary
land through a combination of familial or personal connec-
tions and quasi-legal documentation. In many SSA countries,
transfers of customary land are strictly controlled, at least
on paper. The informal or vernacular land market responds
to this regulatory burden by adapting available formal
legal instruments to secure the transfer, such as reassigning
significant amounts of land under a nebulous third tenure
category, neither customary nor statutory. For example, a
significant amount of land in Liberia is under “Tribal Cer-
tificates.” Under the now repealed Public Lands Law, Tribal
Certificates were issued as part of the first steps in transfer-
ring land from customary tenure to private individualized 7 Ibid. It is therefore impor-
tant to consider the effectiveness of alternative types of inter-
ventions to register land rights with respect to impacts on
(i) perceptions of tenure security, (ii) long- and short-term
productivity-enhancing investments; (iii) land productivity,
and (iv) youth behavior, including decisions to migrate and/or
pursue farming. Lawry et al. (2014) stress that attempts to
implement a new tenure system may not necessarily pro-
duce the intended benefits of improved tenure security – local
context and the performance of existing land institutions are
decisive. It is generally accepted that interventions to regis-
ter individual farmland rights in Africa through private, free-
hold titles have failed may have decreased tenure security in
cases where formal land administration systems proved less
capable than customary systems of protecting rights (Atwood,
1990; Carter & Wiebe, 1990; Lawry et al., 2014; Migot-
Adholla et al. 1994; Place, 2009). There is also evidence
suggesting that individual land titling may have had partic-
ularly negative impacts on the rights of secondary and vul-
nerable land users, such as women and the poor (Meinzen-
Dick & Mwangi, 2008). However, in other cases, formaliza-
tion of land rights, e.g., through certificates, has improved
tenure security and land-based investments on affected plots
(Goldstein et al., 2015; Holden et al., 2009; Melesse & Bulte,
2015). In Burkina Faso, a rural land governance pilot involv-
ing participatory land use planning, the development of com-
munity land use charters, and capacity building for dispute
resolution reduced the predicted probability of serious land
conflicts by more than half (56%) and of severe land con-
flicts by 96% (Linkow, 2018). These findings are significant
given that earlier research suggested that high levels of per-
ceived concern about land conflicts in Burkina Faso was asso-
ciated with a reduction in agricultural productivity of over
40% (Linkow, 2016). Similarly, a randomized control trial
(RCT) in Zambia found that beneficiaries of a pilot to map
and register customary land rights and strengthen customary
land governance institutions felt their fields are now more The evidence on the impact of different land interventions
on land rental markets is somewhat mixed. Research on
Ethiopia’s farmland certification program also found that
the program increased land rental market activity, including
for women, suggesting that landholders felt more confident
in their ability to uphold their rights to rented out land
(Holden et al., 2009). In contrast, very few respondents (1–2%) in any dataset
reported having actually lost land to involuntary reallocation,
and land conflict is reportedly relatively rare, ranging from
19% of households situated near to the town of Chipata in
Zambia to just 1% in Guinea and Zambia’s remote Luangwa
Valley (Stickler et al., under review). Boundary disputes are
reportedly the most common kind of land conflict across all
study sites, suggesting that some form of boundary clarifica-
tion and/or recording process could strengthen tenure secu-
rity. There were no significant differences between female-
and male-headed households with respect to past experience
of land conflict. secure from reallocation or unauthorized appropriation by
both internal and external actors (Huntington et al., under
review). On the question whether land security interventions have
increased short- and long-term productivity-enhancing invest-
ments and agricultural productivity in Africa, the evidence is
generally in support. For example, an RCT in Benin found
that the first stage of a government intervention to map and
register customary land rights increased beneficiary invest-
ment in perennial cash crops and trees by roughly 40% and
increased fallowing by female-headed households (Goldstein
et al., 2015). Similarly, Ethiopia’s program to map and reg-
ister use rights to farmland parcels increased investments in
trees and soil conservation structures and led to productivity
increases of 35–45% (Holden et al., 2009; Melesse & Bulte
2015), whereas Rwanda’s pilot farmland use rights registra-
tion program led to 10 percentage point increase in benefi-
ciaries’ investment in soil conservation and a 19 percentage
point increase for females (Ali et al., 2011). In Zambia, early
RCT evidence finds that customary land registration increased
investment in long-term productivity-enhancing practices by
both the average beneficiary household (e.g., planting basins
increased 7% and manure increased 6%) and by vulnerable
subgroups, including households headed by youth and elders,
as well as poor and land-constrained households (Huntington
et al., under review). A number of recent studies suggest that customary tenure
systems, which confer undocumented rights to users, provide
a greater degree of tenure security than previously thought
(Childress, Spievack, Varela, & Ameyaw, 2018; Lawry et al.,
2014; Stickler et al., under review). No such differences were
found for risk of land appropriation from external sources;
in fact, in two areas of Ethiopia and Zambia, male-headed
households were more likely to report external sources of
land insecurity. The greatest threat to tenure security also
differed across country datasets, ranging from threats within
the community to those deriving from outside authorities or
investors. Respondents in Guinea reported family members
as the most likely threat to their tenure security (9% of
at-risk plots), whereas the largest proportion of respondents
in Liberia (23%) feared appropriation by neighbors. By
contrast, farmers in the Chipata District of eastern Zambia
indicated local authorities were the greatest threat (23%
of fields). Only in the primarily pastoral region of Afar in
northern Ethiopia did outside investors rank as the largest
threat, albeit still relatively small (6% of respondents; Stickler
et al., under review). In rural areas of favorable market access and commercial-
ization potential (e.g. Zambia’s Copperbelt), investor farmers
may represent a significant source of tenure insecurity for
customary landholders as they leverage their connections
with traditional leaders and state government to wrest control
of customary land (Huntington, Stickler, & Stevens, under
review). In other areas less attractive to investor farmers,
the main sources of tenure insecurity tend to be internal
to communities. For example, in relatively remote areas
of Zambia’s Eastern Province, tenure is relatively secure 8 This data was collected by USAID’s Land Tenure Office. 89 JAYNE ET AL. 89 90
9
POLICY IMPLICATIONS Africans
with the resources to respond to this demand are doing so. Many Africans with money and resources found farming to
be a lucrative investment opportunity – especially during this
sustained period of high global food prices since the mid
2000s. Second, policy reforms in the 1990s removed major
barriers to private trade and improved the conditions for pri-
vate investment in African agri-food systems. One example of
this was the removal of restrictions on private movement of
food commodities across district borders. The effects of these
reforms exploded after world food prices suddenly skyrock-
eted. They enabled thousands of small, medium and large-
scale private firms to rapidly respond to profitable incen-
tives. Third, as land became more highly valued in response
to growing rural population density and land scarcity, both
formal and informal land markets have developed, making it
possible for individuals with money and resources to acquire
land. Fourth, medium-scale “emergent” farmers have become
a powerful political force in many African countries with
growing influence over government agricultural and land
policies. Medium-scale farmers are a diverse group. Many are
relatively
wealthy
and
influential,
often
professionals,
entrepreneurs, or retired civil servants. Many accumulated
wealth from nonfarm jobs, invested in land and became either
part-time or full time farmers. Many are based in rural areas
and have political or social influence with local traditional
authorities. Others are urban “telephone farmers” who retain
jobs in the cities, hire managers to attend to their farms and
occasionally visit on weekends. In more recent years, since
investor interest in agriculture may have subsided somewhat
as food prices have subsided from their 2006–2012 high, the
composition of entry into medium-scale farming appears to
have shifted, with most of them being formerly small-scale
farm households who successfully expanded their operations. The increasing dynamism of agricultural land, labor, finance,
and agro-input markets for mechanization and inputs may
be supporting the aspirations of entrepreneurial smallholder
farmers to successfully expand their farming operations. In
the study of medium-scale farms in Ogun and Kaduna States
by Muyanga et al. (2019) for example, roughly 60% of those
interviewed stated that they were formerly primarily engaged
as smallholder farmers who were able to save from their
farm sales to rent, buy or lease new land to expand their
operations into medium-scale status. 90
9
POLICY IMPLICATIONS JAYNE ET AL. 90 small-scale farms, only a very small proportion of currently
small-scale farmers will ever step-up into medium-scale
operations. The vast majority of small-scale farm households
will remain small-scale and their share of the total population
will dwindle over time as the majority of their children
increasingly move into off-farm sources of employment. Medium-scale farms have become a major force in many
African countries’ agricultural sectors. Since 2000, the
amount of agricultural produce that these farms contribute to
countries’ national food supplies has risen rapidly. In some
countries, like Tanzania and Zambia, medium-sized farms
now account for roughly 40% of the country’s marketed agri-
cultural produce. This is not uniformly true across Africa. In land-constrained, densely populated areas like Kenya and
Rwanda, small-scale farms still account for most of the agri-
cultural output. Medium-scale farms are on the rise mainly
where there is substantial, undeveloped land. Medium-scale farmers bring new sources of capital and
know-how to African agriculture. They have in some coun-
tries become a politically powerful group that are well rep-
resented in farm lobbies and national agricultural strategies. They have solidified African governments’ commitments to
support agriculture. They get their land from traditional chiefs
or by purchasing land from others, including small-scale farm
households. Displaced smallholders, especially young peo-
ple, tend to move off farm in search of other sources of
employment. While much remains unknown and the story is still unfold-
ing, we believe that medium-scale farms are an important
driver of rural transformation in much of Africa – with
mostly positive results. The prolonged surge in global food
prices starting in 2006 ushered in major and much publi-
cized investment in African farmland by foreign investors. What happened largely under the radar were huge farmland
investments by African professionals, entrepreneurs, civil ser-
vants, retirees, augmented by millions of relatively success-
ful small-scale farmers who expanded into the lower-end of
the medium-scale farm category (operating from 5 to 20 ha). In Ghana, Zambia, Tanzania, and Kenya, the amount of land
acquired by these medium-scale African farmers since 2000
has far exceeded the amount acquired by foreign investors
(Jayne et al., 2014a). We identified four reasons for the recent growth of African
medium-scale farms. First, rapid population growth, urban-
ization, and rising incomes have contributed to massive
growth in demand for food in African countries. Likewise, in Zambia, beneficiaries of
customary land registration were 1.67 times more likely to
report borrowing or renting-in land compared to those in the
control group (Huntington et al., under review). However, in
Benin, Goldstein et al. (2015) found a 1.6 percentage point
decline in the proportion of parcels rented or sharecropped
out in villages benefiting from the first phase of customary
land registration. The authors hypothesize this finding
indicate landowners may have reclaimed parcels they had
previously rented or shared-cropped out and/or delayed land
transactions to assert their land rights prior to the issuance of
land certificates in the next phase. There is very limited evidence on the impact of land inter-
ventions on land sales markets, which may be at least partly
explained by the fact that many countries in Africa only
recognize use rights to farming parcels and continue to pro-
hibit or heavily restrict land sales. Nonetheless, evidence from
Rwanda’s pilot land registration program found that land
market activity actually declined, suggesting that the risk of
distress sales leading to landlessness may be overestimated
(Ali et al., 2011). 9.1
Implications for agricultural policies Small-scale 0–5 ha farms, with much
greater access to productive assets and therefore higher levels
of productivity. They are contributing to rural transformation,
and are better off than Group 1 because they possess educa-
tion and entrepreneurial skills that enable them to devote some
of their labor time to viable off-farm forms of employment. This allows Group 2 to diversify and increase their incomes,
and effectively capitalize their farm operations compared to
Group 1. Some are relatively productive and well above the
poverty line; some of them may eventually “step up” to Group
3 below. Off-farm income is an important feature that distin-
guished Group 2 from Group (1). This group constitutes 15–
20% of households in rural farm surveys and tends to be rising. However, in densely populated areas where small-scale
farms predominate and where there remains limited additional
land for area expansion, the priority is clear: focus on promot-
ing the productive potential of small farms, realizing that over
time success in this endeavor will lead to progressive move-
ments of individuals and households out of farming and into
off-farm jobs as part of the structural transformation process. In short, a successful smallholder-led agricultural strategy
will result in a declining share of the labor force in farming. Are there examples of successful smallholder-led agricul-
tural growth? Possibly Ethiopia comes closest to this. The
country has registered 6.0% real average annual agricultural
production growth from 2000 to 2015 (World Bank Devel-
opment Indicators, last accessed November 2018), and it is
largely a smallholder-led growth story. This was made pos-
sible by strong government support for smallholder farming,
including improved infrastructure, agricultural R&D, exten-
sion support, diverse farmer support services such as soil
testing. Other key ingredients of an effective smallholder-
led strategy include a hospitable environment for private
sector investment and competition (which might have enabled
Ethiopia to progress even faster). y
g
Group 3: Farmers “stepping up” from small-scale to
medium-scale
holdings. Commercialized
medium-scale
farmers operating 5–100 ha who were formerly small-scale
farmers, successfully expanding their operations (“stepping
up”) and contributing to rural transformation processes. Most
of these former small-scale farmers are now operating in the
5–20 ha range, and their sociodemographic characteristics
are similar to Group 2. A higher share of this group’s children
remain in farming because of relatively superior returns
compared to Group 1. into the local economy through their expenditures, stimulat-
ing off-farm employment opportunities for many rural peo-
ple who were formerly dependent on subsistence farming
(Chamberlin & Jayne, 2018). Medium-scale farms have also
contributed a significant portion of the additional growth in
agricultural output in selected countries where comparable
nationally representative data is available. rural people in the process. Land registration and certifica-
tion procedures – in sync with customary social norms and
institutions – will be needed to provide such protection
(Holden et al., 2009; Lawry et al., 2014; Huntington et al.,
under review). We conclude with an updated “typology of African farms,”
arguing that old perceptions of unimodal smallholder-based
agricultural systems are increasingly obsolete: Group 1: Traditional semi-subsistence farm households. Small-scale 0–5 ha, primarily in semisubsistence farming,
devoting most of their labor to farming because of limited
off farm opportunities, low levels of education, and highly
constrained productive assets. They remain close to or below
poverty line. Up until 1990 or so, this group constituted the
vast majority of farm households in sub-Saharan Africa. They
are still the majority in many African countries and their
numbers continue to grow, albeit slowly, because the chil-
dren of these households are rapidly getting out of farming
and entering into off-farm employment. In areas experienc-
ing sustained economic dynamism, this group will continue
to decline slowly as a proportion of the population over time. This group constitutes 60–70% of the rural population in most
rural household surveys and this share has clearly declined
over time in most countries. 9.1
Implications for agricultural policies A major policy question for African governments and inter-
national development partners concerns the future role of
smallholder farms in Africa. The dynamic role of medium-
scale farms does not reduce the importance of agricultural
ministries maintaining a clear commitment to supporting
smallholder farms as a vehicle to accelerate agricultural and
economic transformation with poverty reduction. The evi-
dence presented earlier indicates that where competition for
land is not intense, new investment in medium-scale farms can
attract private sector investments in input and output markets
that improve market access conditions and commercialized
potential of small-scale farms. There appear to be strong syn-
ergies between small and relatively large farms in such areas,
and therefore questions of “either/or” might be somewhat
misplaced. Group 2: Relatively commercialized and productive small-
holder farm households. Small-scale 0–5 ha farms, with much
greater access to productive assets and therefore higher levels
of productivity. They are contributing to rural transformation,
and are better off than Group 1 because they possess educa-
tion and entrepreneurial skills that enable them to devote some
of their labor time to viable off-farm forms of employment. This allows Group 2 to diversify and increase their incomes,
and effectively capitalize their farm operations compared to
Group 1. Some are relatively productive and well above the
poverty line; some of them may eventually “step up” to Group
3 below. Off-farm income is an important feature that distin-
guished Group 2 from Group (1). This group constitutes 15–
20% of households in rural farm surveys and tends to be rising. Group 3: Farmers “stepping up” from small-scale to
medium-scale
holdings. Commercialized
medium-scale
farmers operating 5–100 ha who were formerly small-scale
farmers, successfully expanding their operations (“stepping
up”) and contributing to rural transformation processes. Most
of these former small-scale farmers are now operating in the
5–20 ha range, and their sociodemographic characteristics
are similar to Group 2. A higher share of this group’s children
remain in farming because of relatively superior returns
compared to Group 1. This group typically constitutes 5–10%
of the rural farm population in rural household surveys and is
rising. The purchasing power of groups 2 and 3 are expanding
the demand for nonfarm and off-farm goods and services,
thereby expanding employment and earnings in the rural
nonfarm economy. Group 2: Relatively commercialized and productive small-
holder farm households. 90
9
POLICY IMPLICATIONS However, although the
majority of medium-scale farms may have formerly been With the rise of the medium-scale farms, legitimate con-
cerns have been raised whether their land acquisitions are
marginalizing small-scale farmers. The evidence to date is
somewhat mixed. Medium-scale farms are clearly providing
access to markets and services for nearby smallholder farms. Medium-scale farms have attracted tractor rental providers,
who now provide mechanization services to smallholders. This allows them to farm their land with much less labor input,
freeing up opportunities to work in off-farm pursuits. Large
trading firms have established buying depots in areas with a
high concentration of medium-scale farms, thereby improv-
ing access to output markets for smallholders too (Sitko et al.,
2018). We also found that the medium-scale farms inject cash 91 JAYNE ET AL. 91 able impacts on perceived tenure security and agricultural
development. Group 4: New entrants “stepping in” to commercial-
ized medium-scale operations. Urban-based and rural people
who relied primarily on non-farm incomes and then diversi-
fied their way into commercialized medium-scale (5–100 ha)
farming operations. They generally have the education, con-
nections, and access to finance to be productive farmers,
and contributors to rural dynamism and transformation. This
group constitutes an unknown share of the total number of
farm households in Africa because they tend to be grossly
under-represented in otherwise nationally representative rural
household surveys in Africa, as detailed in Section 2. Based
on the one country in Africa where this groups has most
likely been reasonably well captured (the Tanzanian Agricul-
tural Sample Census Survey of 2008/2009), they constituted
roughly two to three percent of all farms but up to 25% of total
farmland under cultivation. Their major challenge to expan-
sion is knowledge and trust – finding trusted managers who
will effective manage farm operations on their behalf while
they continue to work in the city. This group also contains
many retirees who invest in land and return to farming when
they have the time to relocate to the rural area and oversee
their farm operations. Based on interviews of this group, a
relatively large share of these farmers’ children are entering
into farming, inheriting their parents’ operations and/or using
capital from the family to expand into their own medium- or
large-scale farming operations. Where customary land institutions remain relevant, as in
most rural contexts in Africa, land policies that either for-
mally recognize the authorities of these institutions (subject
to conformity with national policy principles and objectives,
for example with respect to strengthening women’s rights)
or incorporate them into more comprehensive formal land
administration institutions are most likely to succeed. For
example, Zambia recognizes the authority of customary insti-
tutions to manage customary rural land rights, and a number
of other land policies in the region have proscribed legal roles
for customary leaders in land registration and administration
(e.g., Botswana, Côte d’Ivoire, Ghana, Kenya). These policies
represent a more “fit for purpose” approach to land adminis-
tration that seeks to address current land tenure realities rather
than attempting to replace existing customary institutions or
introduce onerous technical standards that go beyond what is
currently required to secure land rights. Given the diverse nature of customary land tenure systems
in Africa and threats to tenure security facing smallholder
farmers, policies to strengthen tenure security and regulate
land transactions in Africa will need to be carefully tailored
to the local tenure context and needs of different landholders
to affect perceived tenure security and agricultural outcomes. Where land rights derive primarily from community mem-
bership, customary tenure systems effectively regulate within-
community transactions, and external actors pose the primary
threat to land rights, land registration at the community level
accompanied by formal recognition of customary tenure insti-
tutions may be sufficient to reduce insecurity. However, com-
munity rights registration may be expected to have more lim-
ited impacts on agricultural investment and productivity as
compared to registration of rights and transactions on indi-
vidual farm parcels, as the latter would strengthen the rights
of those who invest in productivity-enhancing investments to
future yields and/or rents. Thus, where land rights are already
individualized and internal actors pose the greatest tenure
security threat, and especially where informal transactions
involving outsiders are common, the more costly and time-
consuming investment of registering individual land rights
and transactions may be needed to secure existing rights and
avoid conflicts that are beyond the capacity of customary insti-
tutions to manage (Stickler et al., under review). Group 5: Large-scale farms operating over 100 ha. This
group is not well surveyed in otherwise nationally represen-
tative datasets such as the LSMS and General Living Stan-
dards surveys, but including foreign-controlled, descendants
of European settler farmers, African elites, state supported
farm blocks, etc. Their role in African agriculture varies
widely across countries based largely on historical and cur-
rent policies of government. 9.1
Implications for agricultural policies This group typically constitutes 5–10%
of the rural farm population in rural household surveys and is
rising. The purchasing power of groups 2 and 3 are expanding
the demand for nonfarm and off-farm goods and services,
thereby expanding employment and earnings in the rural
nonfarm economy. Our view about the role of medium-scale farms is that
they should be allowed to develop under a land tenure policy
that does not conflict with land tenure security of indigenous
rural people or foreclose area expansion opportunities for
small-scale farm households. They appear to be a source of
rural dynamism as long as they are not displacing indigenous 92 92 JAYNE ET AL. Smallholders have high marginal propensities
to consume and spend their money in the local rural econ-
omy, thereby stimulating growth linkages between farm and
nonfarm sectors (Mellor, 1995). If a few large-scale farmers
dominate production and spend their money outside the local
rural economy, then local growth multipliers may be weaker
than in areas with more egalitarian land distributions (John-
ston & Kilby, 1975). As shown earlier in this report, a sizeable
and rising share of national agricultural land is controlled by
urban-based households. To the extent that many of them are
medium-scale investor farmers, they may be altering the rela-
tionship between the location of agricultural growth and the
strength and location of growth multipliers with the nonfarm
economy. Another issue for future research concerns the broader
effects of factor market development in Africa on changes in
farmland ownership and use. It is noted that rural financial
markets and financial inclusion are improving in the region;
are they enabling people with access to these markets to pur-
chase or lease land, invest in irrigation and soil conditioning,
and intensify their use of cash inputs such as fertilizers and
improved seeds, in ways that alter the distribution of farm
sizes and scale. General equilibrium effects are almost certain
to be important. 9.3
Implications for national statistical
agencies We do not yet know how generalizable these trends are across
the region. However, it is probably safe to say that existing
population-based data collection platforms are systematically
under-reporting a very dynamic segment of African agricul-
ture: the medium-scale farms. While this omission is under-
standable, it has profound implications. Under the status quo,
African governments cannot monitor, much less understand,
how farm structure is changing over time. Similarly, policy
makers cannot adequately address such routine questions as
the magnitude and location of marketed agricultural surplus. These questions are certainly important for guiding strate-
gic policy decisions aimed at stimulating agricultural growth,
reducing rural poverty, and managing strategic food reserves
and trade policies. We have just begun to scratch the surface in our understand-
ing of this important new development in Africa. African
policy makers will benefit from immediate investment in
improved data collection on medium- and large-scale farms as
well as in-depth analysis to understand their potentially wide-
ranging impacts on African economies. Redressing this informational blind spot will require new
modes of data collection and will certainly not be cost-free. We advocate for the expansion of agricultural sample cen-
sus surveys, as was recently done in Tanzania, to better cap-
ture the magnitude, location, and other characteristics of this
growth of medium- and large farms that cannot be adequately
captured via population-based LSMS-type surveys. We also
advocate for the systematic collection of data on nonlocal land
control, that is, ownership or other usufruct rights over rural
agricultural land held by urban or other nonlocally residing
households. This will require new approaches to sampling,
listing, and enumeration, as well as questionnaire designs that
explicitly capture nonlocal holdings. (both fiscally and in terms of their ability to reflect land
transactions), land administration must be decentralized to
a level that balances user accessibility (and demand for land
services) with recurrent administrative costs. (This remains
an elusive objective for most land administration systems in
Africa and calls into question the long-term sustainability of
recent land registration interventions.) Finally, it is increas-
ingly acknowledged that secure land rights are a necessary but
insufficient condition to promote on-farm investment, pro-
ductivity growth, broader multiplier/growth linkages between
on-farm and off-farm development, and rural economic trans-
formation. To accomplish these broader policy objectives,
land administration systems must be linked to complementary
institutions and services (Lawry et al., 2014). These include
customary and formal dispute resolution mechanisms, as well
as rural finance, utility services, and agricultural value chains,
for example by linking service provision to recognized land
rights and ensuring that land registration beneficiaries have
access to agricultural input and output markets. location of farm, off-farm, and nonfarm growth linkage multi-
pliers that influence rural incomes, economic growth, and the
pace of rural poverty reduction? A stylized fact from Asia’s
agricultural development experience is that relatively uncon-
centrated land distribution patterns may stimulate rural devel-
opment more effectively than highly concentrated landhold-
ing patterns. Smallholders have high marginal propensities
to consume and spend their money in the local rural econ-
omy, thereby stimulating growth linkages between farm and
nonfarm sectors (Mellor, 1995). If a few large-scale farmers
dominate production and spend their money outside the local
rural economy, then local growth multipliers may be weaker
than in areas with more egalitarian land distributions (John-
ston & Kilby, 1975). As shown earlier in this report, a sizeable
and rising share of national agricultural land is controlled by
urban-based households. To the extent that many of them are
medium-scale investor farmers, they may be altering the rela-
tionship between the location of agricultural growth and the
strength and location of growth multipliers with the nonfarm
economy. location of farm, off-farm, and nonfarm growth linkage multi-
pliers that influence rural incomes, economic growth, and the
pace of rural poverty reduction? A stylized fact from Asia’s
agricultural development experience is that relatively uncon-
centrated land distribution patterns may stimulate rural devel-
opment more effectively than highly concentrated landhold-
ing patterns. 9.2
Implications for land tenure policies The evidence available suggests that the wholesale conver-
sion of customary land rights and traditional land adminis-
tration systems into private, individual, freehold titles admin-
istered solely by formal government authorities is unlikely
to contribute to much greater tenure security or land invest-
ments, in most cases. Instead, more flexible interventions to
document customary land rights and strengthen customary
or hybrid government-customary land administration insti-
tutions are needed to increase landholders’ perceived tenure
security and agricultural investment and productivity out-
comes. Although direct comparisons of different approaches
to strengthening land tenure is complicated by the varying
nature of the interventions and institutional arrangements,
it is clear that land policies in Africa need to recognize
the continued existence of customary land rights and cus-
tomary land administration institutions to be have sustain- Regardless of the institutional form (customary/govern-
ment/hybrid) and level of land registration (individual/
community), experience suggests that land administration
systems must adhere to a number of other principles to
positively impact tenure security and rural transformation. First, to be effective in increasing perceived tenure security,
land administration systems must be seen as legitimate by all
stakeholders, including the most vulnerable landholders, as
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The strategic case for establishing public-private partnerships in cancer care
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© 2015 Holden et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Holden et al. Health Research Policy and Systems (2015) 13:44
DOI 10.1186/s12961-015-0031-x Open Access Abstract Background: In 2007, the National Cancer Institute (NCI) launched the NCI Community Cancer Centers Program
(NCCCP) as a public-private partnership with community hospitals with a goal of advancing cancer care and
research. In order to leverage federal dollars in a time of limited resources, matching funds from each participating
hospital were required. The purpose of this paper is to examine hospitals’ level of and rationale for co-investment in
this partnership, and whether there is an association between hospitals’ co-investment and achievement of
strategic goals. Methods: Analysis using a comparative case study and micro-cost data was conducted as part of a comprehensive
evaluation of the NCCCP pilot to determine the level of co-investment made in support of NCI’s goals. In-person or
telephone interviews with key informants were conducted at 10 participating hospital and system sites during the
first and final years of implementation. Micro-cost data were collected annually from each site from 2007 to 2010. Self-reported data from each awardee are presented on patient volume and physician counts, while secondary data
are used to examine the local Medicare market share. Results: The rationale expressed by interviewees for participation in a public-private partnership with NCI included
expectations of increased market share, higher patient volumes, and enhanced opportunities for cancer physician
recruitment as a result of affiliation with the NCI. On average, hospitals invested resources into the NCCCP at a level
exceeding $3 for every $1 of federal funds. Six sites experienced a statistically significant change in their Medicare
market share. Cancer patient volume increased by as much as one-third from Year 1 to Year 3 for eight of the sites. Nine sites reported an increase in key cancer physician recruitment. Conclusions: Demonstrated investments in cancer care and research were associated with increases in cancer
patient volume and perhaps in recruitment of key cancer physicians, but not in increased Medicare market share. Although the results reflect a small sample of hospitals, findings suggest that hospital executives believe there to
be a strategic case for a public-private partnership as demonstrated through the NCCCP, which leveraged federal
funds to support mutual goals for advancing cancer care and research. Keywords: Cancer care, Partnerships, Program evaluation, Strategic case United States, not all patients have access to these cen-
tres. Rather, the majority of cancer patients in the United
States receive care in their local communities. Abstract To pro-
mote access to the same high-quality care for patients,
regardless of residence, the National Cancer Institute
(NCI), one of the 27 institutes of the National Institutes
of Health, designed the Community Cancer Centers Pro-
gram (NCCCP) pilot as a public-private partnership to
accelerate the diffusion of the latest science to local
communities. United States, not all patients have access to these cen-
tres. Rather, the majority of cancer patients in the United
States receive care in their local communities. To pro-
mote access to the same high-quality care for patients,
regardless of residence, the National Cancer Institute
(NCI), one of the 27 institutes of the National Institutes
of Health, designed the Community Cancer Centers Pro-
gram (NCCCP) pilot as a public-private partnership to
accelerate the diffusion of the latest science to local
communities. The strategic case for establishing public-
private partnerships in cancer care Debra J. Holden1*, Kristin Reiter2, Donna O’Brien3 and Kathleen Dalton1 Debra J. Holden1*, Kristin Reiter2, Donna O’Brien3 and Kathleen Dalton1 * Correspondence: debra@rti.org
1RTI International, 3040 E Cornwallis Rd, PO Box 12194, Research Triangle
Park, NC 27709, USA
Full list of author information is available at the end of the article Data collection and secondary sources The success of public-private partnerships such as the
NCCCP rests with the commitment of executive leaders
at private sector (non-government) organizations to in-
vest time and resources into the required activities. However, challenges exist when resources are con-
strained, and financial benefits cannot be demonstrated. Leatherman et al. [6] demonstrated the effects of finan-
cing misalignments on quality initiatives, defining the
concepts of the business, economic, and social cases for
quality of care. They argued that private sector organiza-
tions were unlikely to sustain quality improvement activ-
ities in the absence of a demonstrated business case:
either a short-term, positive financial return on an activity
or the expectation of positive changes to organizational
function and sustainability. Data collection and secondary sources
The
evaluation
collected
data
on
NCCCP-related
spending as well as qualitative data from interviews
with hospital executive management to gain insights
into how executives view the financial and other (stra-
tegic) benefits associated with NCCCP participation
and how they weigh those benefits against the costs. RTI conducted initial and follow-up interviews with
Chief Financial Officers (CFOs) by phone and with
Chief Executive Officers (CEOs) or Chief Operating
Officers (COOs) during site visits each year to partici-
pating hospitals. Selected anonymous quotes from these
interviews are presented in italics and identified as
being stated by a CFO or a CEO/COO during the
specified year of program implementation.a Micro-cost data were collected each year on an Excel-
based input document called the Cost Assessment Tool
[20-22]. The tool was customized to capture spending
across three cost domains for each year of program im-
plementation: NCI-funded subcontract costs (invoiced
costs), costs underwritten by the sponsoring organization
(matching costs), and a valuation of donated time (do-
nated costs).b In addition to these primary data sources,
numerous secondary data sources were used to examine
elements of the context within which each site was operat-
ing. In this paper, we present findings from the following
secondary sources. Previous studies examining quality improvement initia-
tives in cancer have focused on the business case [7,8],
and researchers have struggled to find hard evidence of a
quantifiable financial return on investment. However,
leaders continue to invest in clinical programs such as
cancer care, citing indirect benefits such as reputational
effects and the ability to provide access to cutting-edge
care. Data collection and secondary sources These sorts of qualitative considerations have been
shown to play a significant role in decision making around
investments by not-for-profit hospitals [8-11]. As a result,
the concept of a business case may not fully capture all of
the perceived benefits of an investment to improve a
cancer program. Therefore, for this study, we defined the
“strategic case” for NCCCP participation. Background Cancer is a complex disease that requires broad engage-
ment of various clinical specialists and organizations to
develop a patient-centered delivery system that inte-
grates scientific discovery and provides highly coordi-
nated, evidence-based multi-specialty care across the
care
continuum. While
these
systems
are
well-
established in many of the largest cancer centres in the * Correspondence: debra@rti.org
1RTI International, 3040 E Cornwallis Rd, PO Box 12194, Research Triangle
Park, NC 27709, USA
Full list of author information is available at the end of the article Page 2 of 9 Holden et al. Health Research Policy and Systems (2015) 13:44 In a public-private partnership, government agencies
and private sector organizations share in the financing
and operations of a program or project to accomplish
important public goals [1,2]. In the case of the NCCCP,
the federally-funded NCI partnered with not-for-profit
community hospitals to share risk and cost, as well as
knowledge and capabilities to advance cancer care in six
key areas: disparities, clinical trials, biospecimens, infor-
mation technology, quality of care, and survivorship and
palliative care [3-5]. Through the NCCCP, the NCI
provided funding to eight community hospitals and two
multi-hospital systems, each operating a cancer program
and caring for at least 1,000 new cancer patients annu-
ally. Each site was awarded approximately US$ 500,000
per year to cover costs for specific deliverables related to
the NCCCP award. In addition to receiving federal
funds, each organization was expected to contribute in-
stitutional
dollars
(i.e. matching
funds)
to
cover
NCCCP-related activities including redesigning care pro-
cesses
and
organizational
structures;
however,
the
amount of matching funds was not specified. management decisions in how best to allocate program-
specific resources. This paper presents study findings
that explore (1) the extent to which hospital executive
management believed there to be a strategic case for
participation and the specific outcomes that comprised
the strategic case, (2) the level of investments made by
the participating hospitals, and (3) whether the total in-
vestments were associated with changes in the strategic
outcomes specified by hospital executive management. Methods NCI contracted with RTI International (RTI) to conduct
a comprehensive evaluation of the NCCCP pilot [12], in-
cluding a micro-cost study and a comparative case study
[5]. Although a total of 16 hospitals implemented the
NCCCP, only the 10 lead sites are the focus of the evalu-
ation findings reported herein. In addition to these com-
ponents of the independent evaluation, research projects
were conducted by leaders from the program [13-17], or
were added as evaluation methods [18,19]. Medicare market share (i.e. increases in Medicare market share, cancer patient
volume, and key cancer physicians) relative to hospital
investment levels. The market of the NCCCP hospitals was defined as the
Core Based Statistical Area in which the hospital is
located and identification of other hospitals located
within the NCCCP markets obtained from the Annual
Survey of the American Hospital Association data. For
all hospitals in each NCCCP market, the total number of
Medicare discharges in the hospital during Fiscal Years
(FYs) 2006 and 2011 was determined using the “bills”
variable from the annual Medicare Inpatient Prospective
Payment System.d For each FY and each hospital market,
total Medicare discharges from market hospitals were
summed to determine the total number of Medicare
discharges. The NCCCP hospital’s share of Medicare
discharges was calculated as the proportion of the
market’s discharges that were from that hospital. Assessment surveys In our definition, the strategic case encompasses the
business, economic, and social cases and includes those
outcomes of greatest interest to the hospital executive
management. Strategic outcomes are those that drive Annual surveys completed by each participating hospital
that collected variables such as unduplicated cancer
patient volume c and the number of key cancer physi-
cians that operate in the cancer centre or program. Page 3 of 9 Page 3 of 9 Holden et al. Health Research Policy and Systems (2015) 13:44 Holden et al. Health Research Policy and Systems (2015) 13:44 Relevant hospital characteristics During the process of site selection for the NCCCP
pilot, NCI purposefully selected hospitals that had vary-
ing levels of development of their cancer programs or
service lines [3]. Several sites had limited capacity or
history with conducting clinical trials research, working
with disparate populations, and/or coordinating care
across the cancer continuum to ensure that patients
received evidence-based, timely care. Across the partici-
pating hospitals, the position of the cancer program
within the organizational structure also differed (Holden,
et al., 2012). For example, some hospitals had independ-
ent cancer service lines with a physician director report-
ing directly to hospital executive, whereas others were
much more fragmented organisationally. Eight of the
lead sites reported having a physician director for the
cancer centre or program as of 2007, with varying
degrees of time devoted to this position. All but two of
the 10 lead sites relied primarily on cancer physicians
working in private practices. All lead sites had to have at
least 1,000 new cancer patients in CY 2006 in order to
qualify for NCCCP [12]. Analysis strategy
Q
li
i
d Qualitative data were coded and analysed using state-
of-the-art procedures to identify thematic findings
characterising the strategic outcomes valued by hos-
pital executives [23]. To assess levels of hospital co-
investment, micro-cost data were analysed using Stata
software and the methods described in the Economic
Evaluation Report [20]. To assess whether there were
changes over time in any of the three strategic out-
comes – patient volume, market share, and physician
recruitment – identified through the interviews, we
collected secondary data from the sources described
above and tested for statistically significant differences
between FY 2006 and FY 2011 using
t-tests for
samples of equal variance. Finally, to assess whether
levels of investment were associated with performance
on any of the strategic outcomes, data for the three
outcomes
were
ranked
according
to
the
percent
change for a hospital site, specific to each variable. This allowed consideration of the variation in starting
points for the hospitals. The percent change from FY
2006 to FY 2011 was rank ordered such that those
hospitals
that
were
among
the
highest
achievers
(compared to the other nine NCCCP sites) for that
outcome received a rank of 1, and the lowest received
a rank of 10. These ranks were then categorized as
either “high” for the top five sites or “low” for the
bottom five sites to explore patterns across sites on
how they performed on each outcome relative to their
total investments in the NCCCP. P <0.01.
aSites have been randomly numbered to ensure confidentiality of data.
b Cancer patient volume Becoming a market leader may not, in and of itself, be
sufficient to maintain the NCCCP beyond the funding
period, especially in the face of fiscal challenges. Respon-
dents noted that the strategic case for NCCCP participa-
tion also involved increasing patient volume, along with
enhancing their payer mix: d1 = highest positive change; 10 = lowest positive change or highest negative change.
e Overview of top hospital management interview findings
and changes in outcomes g
CEOs/COOs and CFOs tended to stress different rea-
sons for NCCCP participation. For example, CEOs/
COOs tended to emphasize how the program would
help them improve quality of cancer care and enhance
their image in cancer research and care, whereas CFOs
tended to emphasize how the program would help them
to become a leader in their local market. However, all
consistently agreed that there were four primary motiva-
tions that provide a strategic case for their involvement:
(1) increases in their market share for cancer care, par-
ticularly through the affiliation with NCI; (2) increases
in their patient volume and enhancement of their payer
mix; (3) recruitment of key cancer physicians; and (4)
increases in access to care and/or research for local
cancer patients. Although many sites found it difficult
to separate the effects of the NCCCP from other
efforts
related
to
cancer
care,
virtually
all
CFOs
recognized the strategic fit of the NCCCP with qual-
ity improvement goals, and the reputational value of
formal affiliation with NCI. For example, two separate
CFOs commented: In this paper, we first present perceptions of the stra-
tegic case as described by CFOs and CEOs or COOs,
and descriptive analysis of changes over time in the
three strategic outcomes. We then present overall cost
study data for each site across the 3 years of the pilot
program. Finally, we examine the extent to which the
three key strategic outcomes were achieved by the sites “Volume growth, contribution to margin increase, and
recruitment of physicians and retention are things that Page 4 of 9 Holden et al. Health Research Policy and Systems (2015) 13:44 we are tracking for our strategic program and for an
entire service line.” (CFO, Year 1) Medicare market share, although the increases cannot be
causally linked to the NCCCP. “We also saw the NCI connection as a way to
differentiate ourselves from other community hospitals
out there who are competitors…” (CFO, Year 1) “We also saw the NCI connection as a way to
differentiate ourselves from other community hospitals
out there who are competitors…” (CFO, Year 1) aSites have been randomly numbered to ensure confidentiality of data.
b eH = high degree of change; L = low degree of change. ata Source: Medicare Provider of Service file.
ata Source: Medicare Provider of Service file. y
bData Source: Medicare Provider of Service file. rovider of Service file.
ovider of Service file. eH = high degree of change; L = low degree of change. e: Medicare Provider of Service file.
positive change; 10 = lowest positive change or highest negative change.
f h
L
l
d
f h y numbered to ensure confidentiality of data.
rovider of Service file rovider of Service file.
ge; 10 = lowest positive change or highest negative change.
ge; L = low degree of change cData Source: Medicare Provider of Service file.
d Market share
h As shown in Table 1, sites varied in the extent to which
they led their local market for Medicare patients as of
2006, ranging from 1.72% (Site 2), indicating a highly
competitive market, to 80.57% (Site 8), a market leader. CFOs across the sites consistently viewed the NCCCP as
providing them with a mechanism for becoming a
market leader for cancer care: “[The NCCCP] has been a catalyst for us to raise
the bar. It drives growth. We want to have more
patients go through our program to show those
outcomes, because greater numbers prove a lot
more…” (CEO/COO, Year 1) “We are one of the leaders in the market from a
cardiology standpoint, surgical standpoint, and
orthopedic standpoint, but in oncology we are not. That is one of the reasons why we are interested in
this particular program…We view these as important
value-added activities and view the payoff [as] coming
from the additional business that is provided…and
attracting the payer mix that will provide the funding
to do these kinds of things.” (CFO, Year 1) “Patients choosing to come here – because we offer
complete and state-of-the-art cancer care services –
is critical… We benefit financially from that,
because it is one of our more profitable service
lines.” (CFO, Year 1) “Patients choosing to come here – because we offer
complete and state-of-the-art cancer care services –
is critical… We benefit financially from that,
because it is one of our more profitable service
lines.” (CFO, Year 1) Although enhanced reputation is difficult to assess,
the overall evaluation did capture the extent to which
hospitals changed their cancer patient volume. Al-
though a causal link to NCCCP participation cannot
be established, data reported by sites in their initial
proposals for funding compared to their Final Assess-
ment Survey (specific to NCCCP) suggest the undu-
plicated cancer patient volume increased by about
one-third from Year 1 to Year 3 for four sites (percentage
increases of 29.1% to 36.8%); the remaining four sites
reported increases ranging from 1.9% to 20.1% (Table 2). aSites have been randomly numbered to ensure confidentiality of data.
bData Source: Medicare Provider of Service file bData Source: Medicare Provider of Service file.
cD
S
M di
P
id
f S
i
fil d1 = highest positive change; 10 = lowest positive change or highest negative change.
eH = high degree of change; L = low degree of change h degree of change; L = low degree of change. 1 = highest positive change; 10 = lowest positive change or highest negative change.
eH = high degree of change; L = low degree of change. “I think reputation and market share: those are
measures that show we are doing something right in
the community…” (CFO, Year 1) “I think reputation and market share: those are
measures that show we are doing something right in
the community…” (CFO, Year 1) “I think reputation and market share: those are
measures that show we are doing something right in
the community…” (CFO, Year 1) As shown in Table 1, Medicare market share in 2011 for
the 10 lead sites had changed to some extent, ranging from
1.54% (Site 2) to 80.01% (Site 8). Over the 5-year period, six
sites experienced a statistically significant change in their Table 1 Results of Medicare market share at baseline and follow-up of NCCCP implementation
Site numbera
Medicare market
share (2006)b %
Medicare market
share (2011)c
Differences
Ranking of
differencesd
Degree of
changee
Site 1
30.97
31.21
0.24
3
H
Site 2
1.72
1.54*
−0.18
5
H
Site 3
46.54
45.60
−0.94
9
L
Site 4
50.61
54.60*
3.99
1
H
Site 5
15.45
14.69*
−0.76
7
L
Site 6
8.85
8.93
0.08
4
H
Site 7
7.51
5.05*
−2.46
10
L
Site 8
80.57
80.01
−0.56
6
L
Site 9
48.22
47.43
−0.79
8
L
Site 10
25.20
26.42*
1.22
2
H
*P <0.01. Table 1 Results of Medicare market share at baseline and follow-up of NCCCP implementation dicare market share at baseline and follow-up of NCCCP implementation d1 = highest positive change; 10 = lowest positive change or highest negative change. e Holden et al. Health Research Policy and Systems (2015) 13:44 Page 5 of 9 Page 5 of 9 Table 2 Results of cancer patient volume at baseline and follow-up of NCCCP implementation
Total cancer patient volume
Site numbera
Baseline (CY 2006)b
Follow-up (CY 2009)c
Net change
Percent change (%)
Ranking of changed
Degree of changee
Site 1
1,057
1,400
343
32.4
3
H
Site 2
1,527
1,577
50
3.3
7
L
Site 3
2,591
2,116
475
−18.3
10
L
Site 4
1,429
1,424
−5
−0.3
9
L
Site 5
2,290
3,133
843
36.8
1
H
Site 6
2,863
3,133
280
9.8
6
L
Site 7
1,075
1,464
389
36.2
2
H
Site 8
1,379
1,781
402
29.1
4
H
Site 9
1,236
1,484
248
20.1
5
H
Site 10
2,595
2,645
50
1.9
8
L
aSites have been randomly numbered to ensure confidentiality of data. bData Source: Initial Request for Proposals responses from sites for Calendar Year (CY) 2006. cData Source: Final Assessment Survey collected for CY 2009. Micro-cost data findings Micro-cost data findings Sites 3 and 4 experienced a decrease in their cancer
patient volume during the course of the pilot (−18.3%
and −0.3%, respectively). Sites 3 and 4 experienced a decrease in their cancer
patient volume during the course of the pilot (−18.3%
and −0.3%, respectively). As revealed through the hospital executive management
interviews, site leaders recognized the need to invest in
the program and the components that were important
to NCI, committing to co-investment at the time of
program initiation. Across the 3 years of the pilot, the
10 lead sites reported a total of nearly US$ 37 million of
their own investment as matched to less than US$ 15
million received from NCI. In terms of donated costs,
sites reported a total of 78,634 hours. Reported donated
time varied across sites from less than 400 hours (Site 6)
to more than 24,000 hours (Site 8). The following
sections provide a summary of variations in the costs by
year and site. Costs across pilot implementation Years 1–3 Costs across pilot implementation Years 1 3
The NCCCP was very successful in leveraging govern-
ment dollars, both by direct site investment and by
donated services (Table 4). We define leverage as the
value of these
co-investments (i.e. private funds)
expressed relative to the value of NCCCP funding (i.e. public funds). Per dollar of NCCCP funds distributed
to sites, the overall leverage was US$ 36.8 million
across all 3 years of pilot implementation. Findings
indicate further that for every US$ 1 received by NCI
each year, sites invested a total of US$ 3.47 in Year 1,
US$ 3.66 in Year 2, and US$ 4.00 in Year 3, including
both matching and donated costs. “The connection to NCI is very important. [It] helps us
recruit new physicians and helps us recruit excellent
nurses. People want to be a part of a special program. The linkage is pretty compelling.” (CEO/COO, Year 3) While full attribution to the NCCCP is not possible,
all but one site (Site 7) reported an increase in the
number of key cancer physicians available to their
program, with additions for the nine remaining hospi-
tals ranging from as high as 35 to as low as 1 (Table 3). Anecdotally, we know that two hospitals recruited new
cancer physician directors during the first year of the
program, each of whom told us in later years that they
came to their hospital largely because of the NCI
connection and the NCCCP. “I think reputation and market share: those are
measures that show we are doing something right in
the community…” (CFO, Year 1) d1 = highest positive change; 10 = lowest positive change or highest negative change. eH = high degree of change; L = low degree of change. Table 2 Results of cancer patient volume at baseline and follow-up of NCCCP implementation
Total cancer patient volume Table 2 Results of cancer patient volume at baseline and follow-up of NCCCP implementation Key cancer physician recruitment As the hospitals grew in their local reputations for cancer
care through NCCCP participation, the consistent hope
among hospital executive management was that this would
also enhance their ability to recruit key cancer physicians. This enhanced recruitment would be performed through
their association with NCI and the obvious priority they
were placing on their cancer service line as well as through
their added capacity to conduct cancer research, something
that many noted was important to their physicians: “The ability to recruit specialists is really important to
us, and we haven’t really tried to quantify that, but
that is important.” (CFO, Y1) Co-investment by site bSites have been randomly numbered to ensure confidentiality of data. cData Source: Baseline Assessment Survey (BAS) collected for Calendar Year (CY) 2006. dData Source: FAS collected for CY 2009. e1 = highest positive change; 10 = lowest positive change or highest negative change. Note that three hospitals reported a positive change of five additional key
cancer physicians so are ranked as ties, skewing the count to seven “high” ranked sites. volume, and/or number of key cancer physicians. As
shown in Table 5, cancer patient volume seemed to be
more closely associated with total investments than
the other two outcomes, with four sites reporting the
highest degree of change in cancer patient volume
paired with the highest level of investments and four
other sites reporting the opposite (low volume and
low investments relative to other sites). There did not
seem to be an association between positive Medicare
market share change and investments. Findings spe-
cific to increases in number of key cancer physicians
are difficult to determine because all but one site
experienced an increase. spending patterns over time than others. As shown in
Table 4, sites ranged from investing as little as US$ 0.88
for every US$ 1 received from NCI (both matching and
donated costs) to as high as US$ 7.47, with an average
additional investment across all 3 years of US$ 4.14. aRanking is across the sites, by total additional investments, from highest (1) to lowest (10). aRanking is across the sites, by total additional investments, from highest (1) to lowest (10). urce: RTI Analysis of Completed Cost Assessment Tools, Years 1–3. Source: RTI Analysis of Completed Cost Assessment Tools, Years 1–3. y
g
of Completed Cost Assessment Tools, Years 1–3. es, by total additional investments, from highest (1) to lowest (10). Co-investment by site The biggest component of both matching and donated
costs reported by sites every year was physician time. Although the share of total spending by cost domain
varied across sites, some sites showed more consistent Page 6 of 9 Holden et al. Health Research Policy and Systems (2015) 13:44 Table 3 Results of key cancer physician recruitment at baseline and follow-up of NCCCP implementation
Number of key cancer physiciansa
Site numberb
Baseline (CY 2006)c
Follow-up (CY 2009)d
Change
Ranking of change
Degree of changee
Site 1
8
11
+3
8
L
Site 2
4
39
+35
1
H
Site 3
32
37
+5
5
H
Site 4
10
11
+1
9
L
Site 5
9
18
+9
3
H
Site 6
25
30
+5
5
H
Site 7
12
10
−2
10
L
Site 8
10
16
+6
4
H
Site 9
17
38
+19
2
H
Site 10
19
24
+5
5
H
aSites were asked to provide the “number and type of clinical staff” that “operate in the cancer centre [as of a given date] as noted in the definition provided.”
For this variable, sites were to indicate the number and type of staff such as medical oncologists, radiation oncologists, and clinical research nurses. From this
information, the number of physicians was extrapolated. The count presented excludes physician assistants, physicists, dosimetrists, therapists, technicians,
geneticists, phlebotomists, or any other role clearly not a physician. bSites have been randomly numbered to ensure confidentiality of data. cData Source: Baseline Assessment Survey (BAS) collected for Calendar Year (CY) 2006. dData Source: FAS collected for CY 2009. e1 = highest positive change; 10 = lowest positive change or highest negative change. Note that three hospitals reported a positive change of five additional key
cancer physicians so are ranked as ties, skewing the count to seven “high” ranked sites. 3 Results of key cancer physician recruitment at baseline and follow-up of NCCCP implementation aSites were asked to provide the “number and type of clinical staff” that “operate in the cancer centre [as of a given date] as noted in the definition provided.”
For this variable, sites were to indicate the number and type of staff such as medical oncologists, radiation oncologists, and clinical research nurses. From this
information, the number of physicians was extrapolated. The count presented excludes physician assistants, physicists, dosimetrists, therapists, technicians,
geneticists, phlebotomists, or any other role clearly not a physician. Ranking is across the sites, by total additional investments, from highest (1) to lowest (10).
Source: RTI Analysis of Completed Cost Assessment Tools, Years 1–3. y
ource: RTI Analysis of Completed Cost Assessment Tools, Years 1–3. Association of co-investment with evidence of reaching
desired strategic goals We explored whether there was an association be-
tween levels of co-investment and the extent to which
hospitals experienced changes in three strategic out-
comes identified during interviews with executives:
increases in Medicare market share, cancer patient Table 4 Additional spending per dollar of total NCCCP funding, by site Table 4 Additional spending per dollar of total NCCCP funding, by site
Site
Invoiced dollar
(US$)
Matching dollar
(US$)
Donated time dollar
(US$)
Total additional investment
(US$)
Rankinga
Degree of change
Site 1
1.00
2.91
2.33
5.24
2
H
Site 2
1.00
3.05
0.52
3.57
5
H
Site 3
1.00
1.81
1.10
2.91
7
L
Site 4
1.00
0.84
0.04
0.88
10
L
Site 5
1.00
1.95
1.05
3.00
6
L
Site 6
1.00
1.18
0.05
1.23
9
L
Site 7
1.00
4.25
0.46
4.72
4
H
Site 8
1.00
6.06
1.41
7.47
1
H
Site 9
1.00
4.67
0.42
5.09
3
H
Site 10
1.00
2.35
0.12
2.46
8
L
Average, all sites
1.00
3.29
0.85
4.14
a
k
h
b
l dd
l
f
h h
( )
l
(
) Holden et al. Discussion One of the most innovative aspects of the NCCCP is the
fact that it was established as a public-private partner-
ship between the federal government and numerous
not-for-profit hospitals [1,3]. With significant decreases
in funding from both the private and public sectors, the
need for such collaborations is paramount to improving
health quality while also reducing costs [1,3,24]. To
establish this partnership, NCI provided each selected
hospital with a relatively small amount of funding (ap-
proximately US$ 500,000 per year for each of the 10
funded sites), and any remaining resources needed to
meet NCCCP deliverables were required as matching or
donated funds from participating hospitals. Micro-cost
study findings indicate that, by the end of Year 3, on
average, for every US$ 1 provided by NCI, participating
hospitals had spent more than US$ 3 on NCCCP imple-
mentation. We were able to demonstrate that invest-
ments in cancer care and research appeared to be
associated with at least one of three strategic goals that
hospital executive management valued most from their
affiliation with NCI: increased cancer patient volume. We were unable to demonstrate an association between
investments and changes in market share or recruitment
of key cancer physicians. Although the NCCCP evaluation was a comprehen-
sive, mixed-method design, there are limitations to the
data used for the analyses that should be considered. One obvious challenge is the limited number of sites, al-
though extensive data were collected and several sources
summarized for the 10 sites presented here. While find-
ings from the evaluation of the NCCCP are limited to a
sample size of these 10, our findings provide a unique
opportunity to assess whether public-private partner-
ships, as vehicles to leverage local investment, can mutu-
ally benefit NCI, the participating institutions, and the
communities they serve. Timing of data collection was
also a factor since presented data were collected at
slightly different points in time, making explanations of
findings challenging (e.g. cost data were collected at a
different time of year than interviews were conducted). Another element of time that is important to consider is
that each hospital’s cancer program was in a different
phase of their development, with some being clear
leaders in their local markets at the start of NCCCP
while others were not. Association of co-investment with evidence of reaching
desired strategic goals Health Research Policy and Systems (2015) 13:44 Page 7 of 9 Table 5 Examination of the association between site total co-investments in NCCCP and changes in desired outcomes:
increased market share, cancer patient volume, and number of key cancer physicians
Total investments
(matching + donated costs)
Medicare market share
(change from year 1 to year 3)
Cancer patient volume
(change from year 1 to year 3)
Number of key cancer physicians
(change from year 1 to year 3)
Low
High
Low
High
Low
High
Matching + Donated costs: LOW TOTAL 2
4
4
1
1
4
Matching + Donated: HIGH TOTAL
2
2
1
4
2
3 Table 5 Examination of the association between site total co-investments in NCCCP and changes in desired outcomes:
increased market share, cancer patient volume, and number of key cancer physicians
Total investments
(matching + donated costs)
Medicare market share
(change from year 1 to year 3)
Cancer patient volume
(change from year 1 to year 3)
Number of key cancer physicians
(change from year 1 to year 3)
Low
High
Low
High
Low
High
Matching + Donated costs: LOW TOTAL 2
4
4
1
1
4
Matching + Donated: HIGH TOTAL
2
2
1
4
2
3 reputations as a direct result of being a NCCCP site, and
two rural hospitals and six suburban hospitals were able
to recruit subspecialists (e.g. breast surgeon, gynaeco-
logical oncologist) to their area for the first time. These
respondents, who were new to the hospital, shared in
interviews that the hospital’s partnership with NCI and
their clear commitment to the cancer service line were
the primary reasons they had opted to accept a position
there. In addition, most hospitals reported having more
physicians engaged in ongoing care coordination be-
cause of the extra efforts being made through NCCCP
implementation (e.g. multidisciplinary care conferences). Conclusions The NCCCP was a pilot program implemented on a
relatively small scale (in 16 hospitals) to test the feasi-
bility of the concept of the NCCCP as a public-private
partnership to more quickly diffuse the rapidly chan-
ging science around cancer care into community
settings. The overall evaluation was intended to pro-
vide a formative evaluation of what seemed to work
well and what should be improved upon to enhance
the NCCCP model. In this way, the evaluation find-
ings, along with the program, had to evolve over time
because so many lessons were learned during each
phase of implementation. Importantly, implementation of the NCCCP occurred
within the context of the worst economic downturn
since the Great Depression, which added to the stress of
hospitals investing in this public-private partnership. Moreover, it coincided with the early years of the
Affordable Care Act, when significant market shifts were
occurring and reimbursement methods were changing. The multiple dimensions of the turbulent environment
faced by NCI, as well as the management and clinicians
in the participating community hospitals were a chal-
lenge. Yet, despite these challenges, the participating
hospitals found value sufficient to justify their invest-
ment, thus illustrating the potential of public-private
partnerships with aligned goals to leverage federal funds
to achieve important policy objectives. The scientific landscape (e.g. advances in genomics),
healthcare reform, and policy and socioeconomic issues
all influenced the rationale for creating the NCCCP as a
public-private partnership [1]. NCI leveraged its funding
to fulfil important research goals through partnerships
with community hospitals. These partnerships benefited
the hospitals through their affiliations with NCI, and the
access to NCI staff and resources that these affiliations
afforded. In exchange, the NCI realized the benefits of
hospitals’ co-investments and efforts to support mutually
beneficial goals. The partnerships required collaboration
with the management of the participating hospitals,
since management controls the resources of the institu-
tion and must make decisions to navigate competitive
and policy-driven environments. Clinician participation
in the NCCCP was clearly important, but gaining and
sustaining hospitals’ commitment relied critically on the
existence of a strategic case for involvement in the
NCCCP. This took the form of expected patient volume
and market share increases, as well as enhanced oppor-
tunities for physician recruitment. In the years ahead, initiatives to advance value-based
care will increase given the major changes underway as
a result of the Affordable Care Act’s passage in 2009. Discussion Similarly, some hospitals had fully
integrated cancer service lines with a medical director
and clinical trials program while others did not have that
capacity at the start. Therefore, we assessed the percent
change in each outcome for each individual hospital (i.e. comparing their post-measure to their own pre-measure,
not comparing them to other hospitals) and rank
ordered them within two groups, producing a relatively
crude estimate of change over time. With a larger sam-
ple size, we would have been able to perform more
sophisticated analyses to explore patterns in investments
and the three outcomes. Finally, because all hospitals in
the study were participating in the NCCCP, and many Increased cancer patient volume seemed to be the
outcome with the clearest association to total NCCCP
investments. This measurable change could be because
it is the outcome most likely to change in the shortest
period of time. Since the time period examined was only
three years, it is unlikely that sufficient time had passed
to expect a change to be demonstrated in data specific
to Medicare market share. Increases in key cancer physi-
cians affiliated with the participating hospitals were
reported for nine of the 10 lead sites. Because all but
two hospitals participating in the NCCCP pilot rely
primarily on private practice physicians, this increase
represents substantial growth, particularly for specific
hospitals (e.g. increases of as many as 19 or 35 physi-
cians). Without knowing the extent to which changes in
physician numbers had occurred prior to NCCCP, it is
impossible to link the program’s efforts to this particular
outcome. However, we know anecdotally that two subur-
ban hospitals recruited new cancer centre directors to
their areas who were also physicians with national Page 8 of 9 Holden et al. Health Research Policy and Systems (2015) 13:44 levels of financial investment were only associated with
increases in cancer patient volume over the 3-year
period. Increasing market share may take more time as
it is highly dependent on the competitive environment. Recruitment of key cancer physicians was seen across all
but one of the participating hospitals, regardless of levels
of matching investment. Results of qualitative interviews
suggest that physician recruitment may be driven more
by the NCI affiliation and the perceived commitment of
the hospital to cancer care, than by actual levels of
spending, at least in the short-term. Discussion were making other cancer care changes contemporaneous
to the program, full attribution of outcomes to the
NCCCP is not possible. Still, these findings provide a first
look at the motivations of hospital executives to commit
to programs such as the NCCCP, and the types of changes
that might be expected in a relatively short period of time. Conclusions Based on the experience of the NCCCP, there is no
doubt that there is much that government can accom-
plish in collaboration with healthcare organizations to
manage disruptive change and to advance science and
improve healthcare in the community. Despite its small
sample size, the NCCCP evaluation provides an oppor-
tunity to document the lessons learned from the pro-
gram and ensure that future programs are informed by
these efforts. This study also contributes to existing
research on the business case for quality by expanding
the concept to the strategic case. Our study is a first step
in determining how to assess the ‘strategic case’ for such
a partnership, and helps to inform the types of outcomes
that can realistically be expected to occur in a relatively
short time-frame. Still, more needs to be understood
about how best to facilitate these public-private partner-
ships and under what circumstances they lead to out-
comes desired by all partners involved. Our cost study shows that private investment in can-
cer service lines translated into an average of US$ 3 in
hospital funding for every US$ 1 provided by NCI. This
level of investment alone demonstrates how much of a
catalyst the NCCCP was in driving the hospitals to focus
on advancing science in their local areas. We have
reported elsewhere that the evaluation results indicate
that most of the participating hospitals were able to
expand their cancer programs and research infrastruc-
tures [13-16], increase evidence-based care [17], increase
physician engagement [18,19], and invest significant
matching funds [20]. However, with regards to execu-
tives’ reported strategic goals, this study showed that Authors’ contributions DH served as Principle Investigator of the overall evaluation, conceived of
the topic, analyzed all the data presented, and wrote the majority of the
manuscript. KR and DO’B worked closely with DH in developing the concept of
this paper, interpreting results, writing sections, and reviewing drafts of the
manuscript. KD served as Study Leader for the micro-cost study, collected and
analyzed the cost data, and led the writing of the economic analysis report
from which this manuscript is drawn (she has since retired from RTI). All authors
read and approved the final manuscript. 13. St. Germaine, D, Denicoff, AM, Dimond, EP, Carrigan, A, Enos, RA, Gonzalez,
MM, Wilkinson, K, Mathiason, MA, Duggan, B, Einolf, S, McCaskill-Stevens, W,
Bryant, DM, Thompson, MA, Grubbs, SS, & Go, RS. Use of the National
Cancer Institute Community Cancer Centers Program Screening and Accrual
Log to Address Cancer Clinical Trials Accrual. J Onc Practice. 2014; 10(2). doi:10.1200/JOP.2013.001194. 14. Siegel RD, Castro KD, Eisenstein J, Stallings H, Hegedus PD, Bryant DM, et al. Quality improvement in the National Cancer Institute Community Cancer
Centers Program: the quality oncology practice initiative experience. J Oncol Pract. 2015;11(2):e247–54. Availability of supporting data y
The data set(s) supporting results of this article and
collected from the participating hospitals are not avail-
able due to constraints of our Data Use Agreements. Medicare market share data are readily available via
www.cms.gov (Accessed 10-1-15). Page 9 of 9 Page 9 of 9 Holden et al. Health Research Policy and Systems (2015) 13:44 p
g
The authors declare that they have no competing interests. The authors declare that they have no competing interests. Abbreviations
C
C l
d CY: Calendar year; CEO: Chief Executive Officer; COO: Chief Operating Officer;
CFO: Chief Financial Officer; FY: Fiscal year; NCCCP: National Cancer Institute’s
Community Cancer Centers Program; NCI: National Cancer Institute; RTI: RTI
International. 10. Straley PF, Schuster TB. Evaluation criteria: a framework for decision making. Top Health Care Financ. 1992;19(1):14–25. 11. Wheeler JR, Clement JP. Capital expenditure decisions and the role of the
not-for-profit hospital: an application of a social goods model. Med Care
Rev. 1990;47(4):467 Received: 3 March 2015 Accepted: 17 September 2015 20. Dalton K, Holden DJ, Wright A, Kane H, Allaire BT. NCCCP economic evaluation:
final report on the pilot program costs and the strategic case for participation. 2012. http://ncccp.cancer.gov/files/Economic-Evaluation-Report.pdf. Accessed 26 Jul 2013. Endnotes 5. Clauser SB, Johnson MR, O’Brien DM, Beveridge JM, Fennell ML, Kaluzny AD. Improving clinical research and cancer care delivery in community settings:
evaluating the NCI community cancer centers program. Implement Sci. 2009;4:63. doi:10.1186/1748-5908-4-63. aFor more details on the methods and data sources
used in the overall evaluation, see [12]. b bFor more details on the micro-cost study methodology,
see [20]. 6. Leatherman S, Berwick D, Iles D, Lewin LS, Davidoff F, Nolan T, et al. The
business case for quality: case studies and an analysis. Health Aff. 2003;22(2):17–30. cNote that the baseline patient volume count for
calendar year 2006 was provided by sites in their initial
proposals for funding. 7. Holler B, Forgione DA, Baisden CE, Abramson DA, Calhoon JH. Interactive
financial decision support for clinical research trials. J Health Care Finance. 2011;37(3):25–37. 8. Song PH, Reiter KL, Weiner BJ, Minasian L, McAlearney AS. The business case
for provider participation in clinical trials research: an application to the
National Cancer Institute’s community clinical oncology program. Health
Care Manage Rev. 2013;38(4):284–94. doi:10.1097/HMR.0b013e31827292fc. dAvailable from the Centers for Medicare & Medicaid
Services 9. Kleinmuntz CE, Kleinmuntz DN. A strategic approach to allocating capital in
healthcare organizations. Healthc Financ Manage. 1999;53(4):52–8. Acknowledgements Disclaimer: This project has been funded in whole or in part with federal
funds from the National Cancer Institute, National Institutes of Health, under
Contract No. HHSN261200800001E. The content of this publication does not
necessarily reflect the views of policies of the Department of Health and
Human Services, nor does mention of trade names, commercial products, or
organizations imply endorsement by the United States government. All
research presented in this manuscript received approval from the RTI
International Institutional Review Board, Research Triangle Park, NC, USA. The
authors would like to thank Joy Beveridge, from Leidos Biomedical Research,
for collection and analysis of data presented from the site’s Baseline and
Final Assessment Surveys, Heather Kane for collection and initial analysis of
the interview data, Alton Wright for the analysis of the micro-cost study data,
and Pamela Spain for analysis of the Medicare market share data. 15. Dimond EP, St. Germain D, Nacpil LM, Zaren HA, Swanson SM, Minnick C, et
al. Creating a culture of research in a community hospital: strategies and
tools from the National Cancer Institute Community Cancer Centers
Program. Clinical Trials. 2015;12(3):246–56. 16. Fennell ML, Das IP, Clauser S, Petrelli N, Salner A. The organization of
multidisciplinary care teams: modeling internal and external influences on
cancer care quality. J Natl Cancer Inst Monogr. 2010;40:72–80. 17. Friedman EL, Chawla N, Morris PT, Castro KM, Carrigan AC, Das IP, et al. Assessing the development of multidisciplinary care: experience of the
National Cancer Institute Community Cancer Centers Program. J Oncol
Pract. 2014. Ahead of print. 18. Abernethy AP, Locke SC. The relationship between participation in the
National Cancer Institute’s Community Cancer Centers Program (NCCCP)
and clinical trials activity. A special review as a part of the overall evaluation
of the NCCCP. 2012. http://ncccp.cancer.gov/files/Clinical-Trials-Analysis-
Report.pdf: Accessed 10-1-2015. Author details
1 1RTI International, 3040 E Cornwallis Rd, PO Box 12194, Research Triangle
Park, NC 27709, USA. 2Department of Health Policy and Management, The
University of North Carolina, Chapel Hill, NC 27599, USA. 3Strategic Visions in
Healthcare, LLC, New York, NY, USA. 19. Halpern MT, Spain P, Holden DJ, Stewart A, McNamara EJ, Gay G, et al. Improving quality of cancer care at community hospitals: impact of the
National Cancer Institute Community Cancer Centers Program Pilot. J Oncol Pract. 2013;9(6):e298–304. Competing interests 12. Holden DJ, Treiman K, Arena LC, Tzeng JA, Green S, Adams E, et al. Integrated evaluation report: overall findings for NCI’s Community Cancer
Centers Program Pilot. 2012. http://ncccp.cancer.gov/files/Integrated-
Evaluation-Report.pdf. Accessed 26 Jul 2013. References 1. Kaluzny AD, O'Brien DM. Managing disruptive change in healthcare: lessons
from a public-private partnership to advance cancer care research. New
York, NY: Oxford University Press; 2015. 1. Kaluzny AD, O'Brien DM. Managing disruptive change in healthcare: lessons
from a public-private partnership to advance cancer care research. New
York, NY: Oxford University Press; 2015. 21. Subramanian S, Ekwueme DU, Gardner JG, Trogdon J. Developing and
testing a cost-assessment tool for cancer screening programs. A J Prev Med. 2009;37(3):242–7. doi:10.1016/j.amepre.2009.06.002. 2. Mazzucato M. The entrepreneurial state: debunking public vs. private sector
myths. London: Anthem Press; 2013. 22. Subramanian S, Tangka F, Green J, Weir H, Michaud F. Economic assessment
of central cancer registry operations. Part II: developing and testing a cost
assessment tool. J Registry Manag. 2009;36(2):47–52. 3. Johnson MR, Clauser SB, Beveridge JM, O’Brien DM. Translating scientific
advances into the community setting: the National Cancer Institute
Community Cancer Centers Program pilot. Oncology Issues. 2009. http://
www.strategicvisionsinhealthcare.com/wp-content/uploads/2012/03/
CancerArticle-accc-pdf., accessed 10-1-2015. 23. Miles MB, Huberman AM. Qualitative data analysis: an expanded
sourcebook. 2nd ed. Thousand Oaks, CA: SAGE; 1994. 24. Portilla LM, Rohrbaugh ML. Leveraging public private partnerships to
innovate under challenging budget times. Curr Top Med Chem. 2014;14(3):326–9. 4. O’Brien D, Kaluzny AD. The role of a public-private partnership: translating
science to improve cancer care in the community. J Healthcare
Management. 2014;59(1):17–29.
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DETERMINAN KUALITAS LAPORAN KEUANGAN PEMERINTAH DAERAH: STUDI PADA OPD PEMERINTAH KOTA PEKANBARU
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Abstract This study aims to explain how Human Resources Competencies,
Utilization of Information Technology and The Application of
Internal Control System determine of the Quality of Financial
Reports of Pekanbaru City. This study uses a quantitative method. The population in this study were 33 OPD in Pekanbaru City
Government. The sample determination technique uses a purposive
sampling method, where questionnaires distributed to 99 respondents
were determined to the Head of Department, Head of Division/Sub-
Division/Financial Staff and PPTK of each OPD. The hypothesis test
used in this study uses t-Test. The statistical test aids used in this
study were the SPSS program ver. 20 for Windows and Ms. Excel
2013. The results show that the Competencies of Human Resources,
Utilization of Information Technology and the Application of Internal
Control System partially affected the Quality of Local Government
Financial Reports. Human Resources
Competencies,
Utilization of
Information
Technology, The
Application of Internal
Control System, The
Quality of Financial
Reports Received:
2020-05-11
Accepted:
2020-06-07
Available online:
2020-07-18 Program Studi Akuntansi, Fakultas Ekonomi dan Bisnis, Universitas Riau DETERMINAN KUALITAS LAPORAN KEUANGAN PEMERINTAH DAERAH:
STUDI PADA OPD PEMERINTAH KOTA PEKANBARU Justika Zebua1, Muhammad Rasuli2, Vera Oktari3*
123 Program Studi Akuntansi, Fakultas Ekonomi dan Bisnis, Universitas Riau, Pekanbaru
*E-mail: vera.oktari@lecturer.unri.ac.id Keywords
Human Resources
Competencies,
Utilization of
Information
Technology, The
Application of Internal
Control System, The
Quality of Financial
Reports
Article Information
Received:
2020-05-11
Accepted:
2020-06-07
Available online:
2020-07-18 Keywords
Human Resources
Competencies,
Utilization of
Information
Technology, The
Application of Internal
Control System, The
Quality of Financial
Reports
Article Information
Received:
2020-05-11
Accepted:
2020-06-07
Available online:
2020-07-18 E ISSN 2721-1819 | P ISSN 2721-2416 E ISSN 2721-1819 | P ISSN 2721-2416 Vol .1, No.2, Juli 2020, pp. 169-182 CURRENT
Jurnal Kajian Akuntansi dan Bisnis Terkini
https://current.ejournal.unri.ac.id CURRENT
Jurnal Kajian Akuntansi dan Bisnis Terkini
https://current.ejournal.unri.ac.id PENDAHULUAN Perubahan
sistem
penyelenggaraan
pemerintah
daerah
dari
sentralisasi
ke
desentralisasi membuat berlakunya otonomi daerah. Otonomi daerah sebagaimana dimaksud
dalam Undang-Undang No. 9 Tahun 2015 adalah hak, wewenang, dan kewajiban daerah
otonom untuk mengatur dan mengurus sendiri urusan pemerintahan dan kepentingan setempat
sesuai dengan perundang-undangan. Berlakunya otonomi daerah membuat pemerintah daerah
dapat mengelola keuangan daerah sepenuhnya. Berdasarkan Peraturan Pemerintah No. 12 Tahun 2019 tentang Pengelolaan Keuangan
Daerah adalah seluruh kegiatan yang terdiri dari perencanaan, penganggaran, pelaksanaan,
penatausahaan,
pelaporan,
pertanggungjawaban,
dan
pengawasan
keuangan
daerah. Pelaksanaan pengelolaan keuangan daerah diharuskan tertib, akuntabel dan transparan guna Justika Zebua, Muhammad Rasuli, Vera Oktari
DETERMINAN KUALITAS LAPORAN KEUANGAN PEMERINTAH DAERAH: STUDI PADA OPD PEMERINTAH KOTA
PEKANBARU Justika Zebua, Muhammad Rasuli, Vera Oktari 170 terwujudnya pemerintahan yang bersih. Bentuk pertanggungjawaban dalam penyelenggaraan
pemerintahan daerah adalah dengan mengadakan pelaporan keuangan sebagaimana diatur
dalam Undang Undang No. 17 Tahun 2003 tentang Keuangan Negara dan Undang Undang
No. 9 Tahun 2015 tentang Pemerintahan Daerah, sebagai tindakan untuk mewujudkan
transparansi dan akuntabilitas pengelolaan keuangan daerah. Laporan keuangan yang
dihasilkan harus sesuai dengan Peraturan Pemerintah No. 71 Tahun 2010 tentang Standar
Akuntansi Pemerintahan. Laporan keuangan pemerintah adalah struktur yang menyajikan posisi keuangan dan
kinerja keuangan dalam sebuah entitas (IAI, 2015:2). Laporan keuangan pemerintah daerah
bertujuan
untuk
mempertanggungjawabkan
pelaksanaan
fungsi
laporan
keuangan,
melaporkan hasil operasi, melaporkan kondisi keuangan dan melaporkan sumber daya jangka
panjang (GASB, 2009:54). Berdasarkan tujuan tersebut, diharapkan laporan keuangan
pemerintah daerah memiliki kualitas. Laporan keuangan yang berkualitas didasari oleh teori keagenan, yaitu suatu kontrak
antara pengelola (agent) yaitu pemerintah dan publik (principal) yaitu rakyat yang diwakili
dewan, dimana agen melakukan suatu jasa atas nama prinsipal serta memberi wewenang
kepada agen untuk membuat keputusan yang terbaik bagi prinsipal (Jensen dan Meckling,
1976 dan Lane, 2003). Maksudnya adalah pemerintah daerah sebagai agen bertugas untuk
memberikan jasa, dalam hal ini pengelolaan keuangan daerah yang akuntabel, yang dilakukan
atas kepentingan publik yang diwakilkan oleh DPRD sebagai wakil rakyat. Pemerintah daerah
merupakan agen bagi DPRD dan publik/rakyat (dual accountability) dan DPRD menjadi agen
bagi publik. Intinya adalah pertanggungjawaban laporan keuangan dilakukan atas nama rakyat
dan untuk kepentingan rakyat. Kualitas laporan keuangan adalah ukuran atau taraf seberapa mampu informasi
laporan keuangan pemerintah daerah memenuhi karakteristik kualitatif informasi keuangan
yang disyaratkan dalam pengungkapan secara penuh dan wajar. Program Studi Akuntansi, Fakultas Ekonomi dan Bisnis, Universitas Riau PENDAHULUAN Kualitas Laporan Keuangan
Pemerintah
Daerah
(LKPD)
penting,
sebab
membantu
pengguna
dalam
memenuhi
kebutuhannya atas informasi keuangan sekaligus memberikan perlindungan kepada pemilik
berdasarkan karakteristik kualitatif informasi keuangan dan pengungkapan secara penuh dan
wajar menjadi salah satu bentuk tata kelola pemerintahan yang baik (Yadiati, 2017). BPK setiap tahun memberikan penilaian berupa opini terhadap LKPD, yang mana
opini tersebut menjadi salah satu indikator penilaian kualitas laporan keuangan. Terdapat
jenjang penilaian laporan keuangan dari hasil pemeriksaan berupa enam jenis opini sesuai
dengan Peraturan BPK No. 1 Tahun 2017, yaitu Opini Wajar Tanpa Pengecualian (WTP), E ISSN 2721-1819 | P ISSN 2721-2416 CURRENT: Jurnal Kajian Akuntansi dan Bisnis Terkini . Vol .1, No.2, Juli 2020, pp. 169-182 CURRENT: Jurnal Kajian Akuntansi dan Bisnis Terkini .
Vol .1, No.2, Juli 2020, pp. 169-182 OPD sebagai organisasi pemerintahan daerah membutuhkan sumber daya manus Justika Zebua, Muhammad Rasuli, Vera Oktari
DETERMINAN KUALITAS LAPORAN KEUANGAN PEMERINTAH DAERAH: STUDI PADA OPD PEMERINTAH KOTA
PEKANBARU Justika Zebua, Muhammad Rasuli, Vera Oktari 172 yang kompeten. Pengetahuan dan keterampilan di bidang akuntansi dibutuhkan untuk dapat
mengelola informasi akuntansi. Menurut Juita (2013) dalam Karsana dan Suaryana (2017),
sumber daya manusia merupakan unsur yang sangat penting dalam organisasi, karena sumber
daya manusia ini dipastikan terlatih dengan baik agar kontribusi yang diberikan maksimal
dalam pencapaian tujuan organisasi. Oleh karena itu, kompetensi sumber daya manusia
diperlukan dalam mengelola dan menyajikan informasi keuangan sehingga laporan keuangan
yang disusun berkualitas. Hal ini sependapat dengan penelitian yang dilakukan oleh Indriasih
(2014), Tawaqal dan Suparno (2017), Evicahyani dan Setiawina (2016), Karsana dan
Suaryana (2017) serta Oktavia dan Rahayu (2019) mengatakan bahwa kompetensi sumber
daya manusia memiliki pengaruh positif terhadap kualitas LKPD. Namun, dalam penelitian
Sholohah dan Sulistyawati (2018) membantah pernyataan tersebut. Pemanfaatan teknologi informasi dalam manajemen data keuangan memberikan
keakuratan dan fleksibilitas dalam penyusunan laporan keuangan. Laporan keuangan dapat
disajikan tepat waktu karena teknologi informasi mendukung percepatan pemrosesan data
transaksi. Sehingga laporan keuangan dapat siap tepat waktu dan tidak kehilangan kualitasnya
(Wardani dan Nugroho, 2018). Salah satu bentuk pemanfaatan teknologi informasi dalam
bidang keuangan adalah penerapan Sistem Akuntansi Keuangan Daerah (SAKD) sesuai SAP
yang bertujuan menggalakkan akuntabilitas dan keandalan pengelolaan keuangan pemerintah
daerah melalui penataan dan pengembangan SAP berbasis akrual dalam menyusun LKPD. Hal ini sejalan dengan penelitian yang telah dilakukan oleh Tawaqal dan Suparno (2017),
Wardani dan Nugroho (2018), Oktavia dan Rahayu (2019) serta Hasanah, dkk. (2019)
mengatakan bahwa pemanfaatan teknologi informasi memiliki pengaruh positif terhadap
kualitas LKPD. Namun, dibantah oleh penelitian Sholohah dan Sulistyawati (2018). LKPD harus disusun berdasarkan Sistem Pengendalian Intern seperti yang tertera
dalam Pasal 56 Ayat (4) Undang-Undang No. 01 Tahun 2004 tentang Perbendaharaan Negara
yang menyebutkan bahwa kepala OPD selaku pengguna anggaran/barang memberikan
pernyataan bahwa pengelolaan APBD di lingkungan kerjanya telah menyelenggarakan Sistem
Pengendalian Intern secara memadai dan menyusun laporan keuangan sesuai dengan SAP. Tingkat SPI pada OPD Kota Pekanbaru masih dalam level 2 (berkembang) menuju level 3
(terdefinisi), artinya pemda Pekanbaru telah melaksanakan praktik pengendalian intern,
namun tidak terdokumentasi dengan baik dan pelaksanaannya sangat tergantung pada
individu dan belum melibatkan semua unit organisasi, sehingga masih terdapat kelemahan
yang cukup berarti (BPKP, 2018). CURRENT: Jurnal Kajian Akuntansi dan Bisnis Terkini .
Vol .1, No.2, Juli 2020, pp. 169-182 CURRENT: Jurnal Kajian Akuntansi dan Bisnis Terkini . Vol .1, No.2, Juli 2020, pp. 169-182 171 Wajar
Tanpa
Pengecualian
Dengan
Paragraf
Penekanan
Suatu
Hal,
Wajar
Dengan
Pengecualian (WDP) karena pembatasan ruang lingkup, Wajar Dengan Pengecualian (WDP)
karena adanya penyimpangan standar akuntansi, Opini Tidak Wajar (TP), dan Tidak Memberi
Pendapat (TMP). Wajar
Tanpa
Pengecualian
Dengan
Paragraf
Penekanan
Suatu
Hal,
Wajar
Dengan
Pengecualian (WDP) karena pembatasan ruang lingkup, Wajar Dengan Pengecualian (WDP)
karena adanya penyimpangan standar akuntansi, Opini Tidak Wajar (TP), dan Tidak Memberi
Pendapat (TMP). LKPD yang mendapat opini WTP menandakan secara umum tata kelola keuangannya
sudah baik. Pemberian opini WTP berdasarkan empat kriteria, yaitu kepatuhan pengelolaan
keuangan/aset
terhadap
peraturan
perundang-undangan,
efektifitas
pelaksanaan
sistem
pengendalian intern, kesesuaian dengan SAP dan pengungkapan informasi dalam LKPD yang
lengkap atau memadai. Namun, bukan berarti LKPD yang mendapatkan WTP bebas dari
kesalahan dan kelemahan (Treasury Indonesia, 2017). Peraturan Pemerintah No. 71 Tahun 2010 tentang Standar Akuntansi Pemerintahan
bagian Kerangka Konseptual Akuntansi Pemerintahan memuat kriteria dan unsur yang
membentuk kualitas laporan keuangan, yaitu: 1) Relevan, 2) Andal, 3) Dapat dibandingkan
dan 4) Dapat dipahami. LKPD Tahun 2018 Kota Pekanbaru mendapat opini WTP, pada Sidang Paripurna
Istimewa DPRD Riau. Pemerintah Kota Pekanbaru telah mendapat opini WTP sebanyak 3
kali berturut-turut terhitung sejak tahun pelaporan 2016-2018 (Ikhtisar Hasil Pemeriksaan
BPK atas LKPD Tahun 2016-2018). Konsistensi Pemerintah Kota Pekanbaru dalam mempertahankan kualitas laporan
keuangannya merupakan suatu topik yang menarik untuk diteliti. Walau sudah tiga kali
mendapat opini WTP, laporan keuangan pemerintah Kota Pekanbaru masih terdapat temuan
pemeriksaan. Adapun temuan tersebut berasal dari kelemahan sistem pengendalian intern dan
terkait dengan ketidakpatuhan terhadap perundang-undangan dalam penyusunan laporan
keuangan. Selain itu, pada awal tahun 2019 lalu terdapat 9 OPD di Pemerintah Kota
Pekanbaru yang tak kunjung menyelesaikan laporan keuangan tahun 2018. Deadline
diberikan pemko Pekanbaru sampai tanggal 19 Maret 2019 belum optimal, sehingga diberikan
waktu lebih kepada OPD yang harus menggesa pelaporan keuangannya. Artinya, penyusunan
laporan keuangan sebagian OPD di Pemko Pekanbaru masih tidak tepat waktu, yang mana
belum memenuhi karakteristik kualitatif yang disyaratkan; yaitu relevan (www.cakaplah.com). Berdasarkan fenomena-fenomena di atas, dapat dinyatakan bahwa LKPD Kota
Pekanbaru masih perlu dilakukan pembenahan terhadap berbagai aspek pada pemerintah
daerah terutama bagian penatausahaan keuangan agar hasil dari laporan keuangan yang
diterbitkan berkualitas serta bernilai informasi yang akurat. CURRENT: Jurnal Kajian Akuntansi dan Bisnis Terkini .
Vol .1, No.2, Juli 2020, pp. 169-182 Hal ini searah dengan penelitian yang telah dilakukan oleh
Indriasih (2014), Herawati (2014), Karsana dan Suaryana (2017) dan Muhammad (2019) yang E ISSN 2721-1819 | P ISSN 2721-2416 CURRENT: Jurnal Kajian Akuntansi dan Bisnis Terkini . Vol .1, No.2, Juli 2020, pp. 169-182 CURRENT: Jurnal Kajian Akuntansi dan Bisnis Terkini . Vol .1, No.2, Juli 2020, pp. 169-182 173 mengatakan bahwa penerapan sistem pengendalian intern memiliki pengaruh positif terhadap
kualitas LKPD. Sebaliknya, pernyataan tersebut dibantah oleh Wawo, dkk. (2013). Ketidakkonsistenan hasil di atas menyebabkan Peneliti ingin meneliti kembali
determinan
kualitas
laporan
keuangan
pemerintah
daerah. Penelitian
ini
merupakan
pengembangan dari penelitian Tawaqal dan Suparno (2017). Perbedaannya terletak pada:
pertama, variabel independen penerapan sistem informasi akuntansi tidak digunakan, yang
mana dalam penelitian ini menggunakan variabel independen pemanfaatan teknologi
informasi. Kedua, penelitian tersebut dilakukan di OPD Pemerintah Kota Banda Aceh,
sedangkan penelitian ini dilakukan di OPD Pemerintah Kota Pekanbaru. Berdasarkan latar belakang masalah di atas, membuat peneliti ingin mengetahui
pengaruh sebenarnya dari variabel-variabel independen tersebut terhadap kualitas laporan
keuangan pemerintah daerah. Program Studi Akuntansi, Fakultas Ekonomi dan Bisnis, Universitas Riau PENGEMBANGAN HIPOTESIS Pengaruh Kompetensi Sumber Daya Manusia terhadap Kualitas Laporan Keuangan
Pemerintah Daerah Pemerintah Daerah Peraturan Pemerintah No. 56 Tahun 2005 tentang Sistem Informasi Keuangan Daerah
menyebutkan bahwa untuk meneruskan terselenggaranya proses pembangunan yang sesuai
dengan prinsip tata kelola pemerintahan yang baik, pemerintah, serta pemerintah daerah wajib
mengembangkan dan memanfaatkan kemajuan teknologi informasi guna meningkatkan
kemampuan pengelolaan keuangan daerah, dan mendistribusikan informasi keuangan daerah
kepada pelayanan publik. Pemanfaatan kemajuan teknologi informasi perlu dioptimalisasikan
pemerintah daerah untuk membangun jaringan sistem informasi manajemen dan proses kerja
yang memungkinkan pemerintahan bekerja secara terpadu dengan menyederhanakan akses
antar unit kerja. Salah satu bentuk dari pemanfaatan teknologi informasi dalam hal penyusunan laporan
keuangan adalah penerapan sistem akuntansi keuangan daerah. Dalam Permendagri No. 59
Tahun 2007, Sistem Akuntansi Keuangan Daerah (SAKD) adalah serangkaian tahapan yang
dimulai dari proses pengumpulan data, pencatatan, pengikhtisaran, sampai pelaporan
keuangan dalam rangka pertanggungjawaban pelaksanaan APBD yang dilakukan secara
manual atau secara terkomputerisasi. Hal ini didukung oleh penelitian yang dilakukan oleh Tawaqal dan Suparno (2017),
Wardani dan Nugroho (2018), Oktavia dan Rahayu (2019) serta Hasanah, dkk. (2019) yang
menyebutkan bahwa pemanfaatan teknologi informasi berpengaruh positif terhadap kualitas
laporan keuangan daerah. Berdasarkan uraian tersebut, maka dirumuskan hipotesis: H2: Pemanfaatan Teknologi Informasi berpengaruh signifikan terhadap Kualitas
Laporan Keuangan Pemerintah Daerah. Justika Zebua, Muhammad Rasuli, Vera Oktari
DETERMINAN KUALITAS LAPORAN KEUANGAN PEMERINTAH DAERAH: STUDI PADA OPD PEMERINTAH KOTA
PEKANBARU Justika Zebua, Muhammad Rasuli, Vera Oktari
DETERMINAN KUALITAS LAPORAN KEUANGAN PEMERINTAH DAERAH: STUDI PADA OPD PEMERINTAH KOTA
PEKANBARU Justika Zebua, Muhammad Rasuli, Vera Oktari 174 manusia berpengaruh positif terhadap kualitas laporan keuangan daerah. Berdasarkan uraian
tersebut, maka dirumuskan hipotesis: manusia berpengaruh positif terhadap kualitas laporan keuangan daerah. Berdasarkan uraian
tersebut, maka dirumuskan hipotesis: H1: Kompetensi Sumber Daya Manusia berpengaruh signifikan terhadap Kualitas
Laporan Keuangan Pemerintah Daerah. Pemerintah Daerah Kompetensi sumber daya manusia adalah kemampuan yang dimiliki seseorang
pegawai yang bersangkutan dengan pengetahuan, keterampilan dan sikap yang secara
langsung dapat mempengaruhi kinerjanya dalam upaya mencapai tujuan yang diinginkan. Kompetensi sumber daya manusia sangat diperlukan agar laporan keuangan yang dihasilkan
dapat memenuhi karakteristik kualitatif laporan keuangan. Keberhasilan suatu entitas
dipengaruhi oleh kompetensi sumber daya manusia yang dimiliki untuk merumuskan,
melaksanakan, dan mengendalikan entitas tersebut. Sumber daya manusia yang akan
menjalankan sistem atau yang menghasilkan informasi tersebut, dituntut untuk memiliki
tingkat keahlian akuntansi yang memadai dan atau paling tidak memiliki keinginan untuk
terus belajar dan mengasah kemampuannya di bidang akuntansi. Apabila pegawai, khususnya
yang berhubungan dengan penyusunan laporan keuangan dari suatu organisasi ataupun
instansi tidak berkompeten, dalam arti mengerti akuntansi, maka kualitas dari laporan
keuangan daerah yang dikeluarkan akan mengalami penurunan atau kurang berkualitas. Namun sebaliknya, apabila pegawai dari suatu organisasi atau instansi itu berkompeten, maka
laporan keuangan daerah yang dikeluarkan akan baik atau berkualitas. Hasil penelitian ini didukung oleh penelitian yang dilakukan oleh Evicahyani dan
Setiawina (2016), Oktavia dan Rahayu (2019), Karsana dan Suaryana (2017), Tawaqal dan
Suparno (2017) serta Indriasih (2014) yang menunjukkan bahwa kompetensi sumber daya H3: Penerapan Sistem Pengendalian Intern berpengaruh signifikan terhadap Kualitas H3: Penerapan Sistem Pengendalian Intern berpengaruh signifikan terhadap Kualitas Laporan Keuangan Pemerintah Daerah. Program Studi Akuntansi, Fakultas Ekonomi dan Bisnis, Universitas Riau Pemerintah Daerah Weygandt et all (2005) mengungkapkan bahwa, jika pengendalian internal telah
diterapkan maka tiap kegiatan, sumber daya dan data akan dapat dipantau, diawasi, E ISSN 2721-1819 | P ISSN 2721-2416 CURRENT: Jurnal Kajian Akuntansi dan Bisnis Terkini . Vol .1, No.2, Juli 2020, pp. 169-182 CURRENT: Jurnal Kajian Akuntansi dan Bisnis Terkini . Vol .1, No.2, Juli 2020, pp. 169-182 175 tercapainya tujuan, kecilnya risiko, dan menghasilkan informasi yang berkualitas. Dengan
menerapkan pengendalian internal dalam sistem akuntansi, maka informasi akuntansi yang
dihasilkan akan lebih berkualitas (relevan, akurat, tepat waktu, dan lengkap), serta dapat
diaudit (Herawati, 2014). Keefektifan
penyelenggaraan
pengendalian
intern
dapat
dilihat
dari
tingkat
maturitasnya. Tingkat
maturitas
penyelenggaraan
SPIP
adalah
tingkat
kematangan/kesempurnaan penyelenggaraan sistem pengendalian intern pemerintah dalam
mencapai tujuan pengendalian intern sesuai dengan Peraturan Pemerintah No. 60 Tahun 2008
tentang Sistem Pengendalian Intern Pemerintah. Tingkat maturitas SPIP terdiri dari level 0
sampai
level
5. Semakin
tinggi
tingkat
maturitas
SPIP
pada
suatu
Kementerian/Lembaga/Pemda, maka pengendalian internalnya sudah dilakukan dengan baik
dan berkelanjutan, terintegarasi dalam pelaksanaan kegiatan yang didukung oleh pemantauan
otomatis menggunakan aplikasi komputer, sehingga meminimalkan kesalahsajian laporan
keuangan. Begitu
juga
sebaliknya,
jika
tingkat
maturitas
SPIP
pada
suatu
Kementerian/Lembaga/Pemda rendah, maka pengendalian internalnya buruk, sehingga
laporan keuangan yang dihasilkan tidak berkualitas. Herawati (2014), Indriasih (2014), Muhammad (2019) Karsana dan Suaryana (2017)
serta Tawaqal dan Suparno (2017) membuktikan secara empiris bahwa sistem pengendalian
internal akuntansi berpengaruh positif terhadap kualitas laporan keuangan pemerintah daerah
yang dinyatakan dengan ketepatwaktuan dan keterandalan. Berdasarkan uraian tersebut, maka
dapat dirumuskan hipotesis: Tabel 1 Tabel 1
Instrumen Penelitian
No
Variabel
Indikator
Skala
1
Kualitas Laporan
Keuangan Pemerintah
Daerah
1. Relevan
2. Andal
3. Dapat dibandingkan
4. Dapat dipahami
Likert
2
Kompetensi Sumber Daya
Manusia
1. Pengetahuan
2. Keahlian
3. Perilaku
Likert
3
Pemanfaatan Teknologi
Informasi
1. Memiliki komputer
2. Jaringan internet
3. Jaringan internet termanfaatkan dengan baik
4. Sistem terkomputerisasi
5. Software sesuai dengan UU
6. Integritas
7. Pemilihan peralatan
8. Perbaikan peralatan
Likert
4
Sistem Pengendalian
Intern
1. Lingkungan pengendalian
2. Penilaian risiko
3. Kegiatan pengendalian
4. Informasi dan komunikasi
5. Pemantauan
Likert
Sumber: Penelitian Terdahulu Instrumen Penelitian Sumber: Penelitian Terdahulu Teknik Pengumpulan Data Teknik pengumpulan data yang digunakan dalam penelitian ini menggunakan kuesioner
yang pernyataan-pernyataannya dibagi menjadi beberapa bagian dan merupakan kumpulan
dari beberapa penelitian terdahulu. Populasi dan Sampel Populasi dalam penelitian ini adalah seluruh Organisasi Perangkat Daerah di
Pemerintah Kota Pekanbaru. Sampel dalam penelitian ini ditentukan dengan metode
purposive sampling, yang mana kriteria sampel ditentukan atas dasar bahwa sampel tersebut
merupakan pihak yang terlibat secara langsung dalam rekapitulasi transaksi keuangan OPD
dan penyusunan laporan keuangan pemerintah daerah. Sampel dalam penelitian ini sebanyak
99 responden dari 33 OPD, yang terdiri dari Kepala Dinas masing-masing OPD, Kepala
Bagian/Sub Bagian/Staf Keuangan masing-masing OPD, Pejabat Pelaksana Teknis Kegiatan
masing-masing OPD. Justika Zebua, Muhammad Rasuli, Vera Oktari
DETERMINAN KUALITAS LAPORAN KEUANGAN PEMERINTAH DAERAH: STUDI PADA OPD PEMERINTAH KOTA
PEKANBARU Justika Zebua, Muhammad Rasuli, Vera Oktari
DETERMINAN KUALITAS LAPORAN KEUANGAN PEMERINTAH DAERAH: STUDI PADA OPD PEMERINTAH KOTA
PEKANBARU Justika Zebua, Muhammad Rasuli, Vera Oktari 176 Penentuan responden didasarkan pada alasan; (a) Membuat unit analisis bersifat
heterogen dan respon dari responden dapat tersebar merata di masing-masing OPD, (b)
Diasumsikan bahwa 3 responden ini pasti mengetahui tentang pelaporan keuangan pada
masing-masing OPD. Definisi Operasional dan Pengukuran Variabel Berikut adalah operasionalisasi variabel yang diukur menggunakan Skala Likert dengan
skor 5 poin, mulai dari poin 1 sangat tidak setuju sampai poin 5 sangat setuju, yang
menghasilkan data ordinal. Sumber: Penelitian Terdahulu Program Studi Akuntansi, Fakultas Ekonomi dan Bisnis, Universitas Riau Metode Analisis Data Hipotesis dalam penelitian ini diuji dengan menggunakan uji t yang menggunakan alat
bantu uji statistik program SPSS ver. 20 for Windows dan Microsoft Excel 2013 yang
termasuk dalam uji analisis linear berganda. Model uji analisis linear berganda adalah sebagai
berikut: E ISSN 2721-1819 | P ISSN 2721-2416 CURRENT: Jurnal Kajian Akuntansi dan Bisnis Terkini . Vol .1, No.2, Juli 2020, pp. 169-182 CURRENT: Jurnal Kajian Akuntansi dan Bisnis Terkini . Vol .1, No.2, Juli 2020, pp. 169-182 177 177 Y =
Keterangan: Keterangan: Keterangan: Y = Kualitas Laporan Keuangan Y = Kualitas Laporan Keuangan
α = Konstanta
β1 = koefisien regresi Kompetensi Sumber Daya Manusia
β2 = koefisien regresi Pemanfaatan Teknologi Informasi
β3 = koefisien regresi Sistem Pengendalian Intern
X1 = variabel Kompetensi Sumber Daya Manusia
X2 = variabel Pemanfaatan Teknologi Informasi
X3 = variabel Sistem Pengendalian Intern
e
= variabel pengganggu α = Konstanta e
= variabel pengganggu menggambarkan nilai variabel dependen dan variabel independen dengan akurat. menggambarkan nilai variabel dependen dan variabel independen dengan akurat. Hasil Uji Analisis Regresi Linear Berganda Berikut
ini
adalah
hasil
pengujian
analisis
regresi
linear
berganda
dengan
menggunakan SPSS untuk pengujian seluruh hipotesis. Tabel 3
Hasil Uji Analisis Regresi Linear Berganda Tabel 3
Hasil Uji Analisis Regresi Linear Berganda Tabel 3
Hasil Uji Analisis Regresi Linear Berganda Tabel 3
Hasil Uji Analisis Regresi Linear Berganda
Coefficientsa
Model
Unstandardized
Coefficients
Standardized
Coefficients
t
Sig. B
Std. Error
Beta
1
(constant)
7,261
3,055
2,377
,020
Kompetensi Sumber Daya (X1)
,287
,096
,289
2,982
,004
Pemanfaatan Teknologi Informasi
(X2)
,263
,088
,279
2,990
,004
Sistem Pengendalian Intern (X3)
,291
,094
,308
3,090
,003
a
Dependent Variable: Kualitas Laporan Keuangan Pemerintah Daerah (Y)
Sumber: Olah data dengan menggunakan SPSS Berdasarkan hasil uji di atas, dapat disimpulkan bahwa seluruh variabel independen
dalam penelitian ini berpengaruh positif terhadap variabel dependen. Diketahui dari masing-
masing nilai Beta variabel dependen yang jika dibulatkan menjadi 1 satuan, maka dapat
meningkatkan variabel Kualitas Laporan Keuangan Pemerintah Daerah sebesar nilai B
masing-masing variabel dependen, dengan asumsi variabel lain tetap. Justika Zebua, Muhammad Rasuli, Vera Oktari
DETERMINAN KUALITAS LAPORAN KEUANGAN PEMERINTAH DAERAH: STUDI PADA OPD PEMERINTAH KOTA
PEKANBARU Justika Zebua, Muhammad Rasuli, Vera Oktari
DETERMINAN KUALITAS LAPORAN KEUANGAN PEMERINTAH DAERAH: STUDI PADA OPD PEMERINTAH KOTA
PEKANBARU Justika Zebua, Muhammad Rasuli, Vera Oktari 178 Hasil Uji Statistik Deskriptif Tabel berikut menampilkan hasil uji statistik deskriptif berdasarkan data yang
diperoleh dari pengisian kuesioner oleh responden. Tabel 2 Tabel 2
Hasil Uji Statistik Deskriptif
N
Min
Max
Mean
Std. Deviation
Kompetensi Sumber Daya Manusia (X1)
83
20
29
24,75
2,053
Pemanfaatan Teknologi Informasi (X2)
83
29
40
33,14
2,159
Sistem Pengendalian Intern (X3)
83
31
42
37,29
2,156
Kualitas Laporan Keuangan Pemerintah Daerah (Y)
83
31
39
33,96
2,039
Valid N (listwise)
83
Sumber: Olah data dengan menggunakan SPSS Hasil Uji Statistik Deskriptif Hasil Uji Statistik Deskriptif Dari data di atas, dapat dilihat Kompetensi Sumber Daya Manusia (X1) dengan nilai
minimum 20, nilai maksimum 29, nilai rata-rata 24,75 dengan standar deviasi 2,053. Pemanfaatan Teknologi Informasi (X2) dengan nilai minimum sebesar 29, nilai maksimum 40
dan nilai rata-rata sebesar 33,14 dengan standar deviasi sebesar 2,159. Sistem Pengendalian
Intern (X3) dengan nilai minimum sebesar 31, nilai maksimum 42 dan nilai rata-rata sebesar
37,29 dengan standar deviasi sebesar 2,156. Kualitas Laporan Keuangan Pemerintah Daerah
(Y) dengan nilai minimum sebesar 31, nilai maksimum 39 dan nilai rata-rata sebesar 33,96
dengan standar deviasi sebesar 2,039. Nilai rata-rata dan nilai standar deviasi setiap variabel
menunjukkan bahwa terdapat penyebaran data yang baik karena nilai rata-ratanya lebih besar
dari pada standar deviasinya. Artinya, data yang telah dikumpulkan dan dianalisis dapat Pemerintah Daerah Berdasarkan uji parsial t pada hipotesis pertama, telah didapat nilai t tabel adalah
1,994, t hitung adalah 2,982 dan signifikansinya 0,004. Ketentuan dari uji parsial t ini adalah
jika t hitung < t tabel maka H01 diterima dan Ha1 ditolak begitu juga sebaliknya. Jika H01
ditolak dan Ha1 diterima, maka nilai signifikansinya harus < 0,05. Dari hasil uji t hipotesis
pertama didapat t hitung > t tabel yaitu 2,982 > 1,994 dengan nilai sig.t 0,004 < 0,05. Dari
keterangan tersebut, ditarik kesimpulan bahwa hasil dari uji t hipotesis pertama adalah
Kompetensi Sumber Daya Manusia berpengaruh signifikan terhadap Kualitas Laporan
Keuangan Pemerintah Daerah. Berdasarkan data yang diperoleh dalam penelitian, diketahui sumber daya manusia
bagian keuangan di tiap OPD Pemerintah Kota Pekanbaru sudah baik. Berdasarkan hasil
analisis dan pengujian yang dilakukan, menunjukkan bahwa semakin kompeten sumber daya
manusia yang dimiliki maka semakin berkualitas laporan keuangan yang dihasilkan. E ISSN 2721-1819 | P ISSN 2721-2416 CURRENT: Jurnal Kajian Akuntansi dan Bisnis Terkini . Vol .1, No.2, Juli 2020, pp. 169-182 CURRENT: Jurnal Kajian Akuntansi dan Bisnis Terkini . Vol .1, No.2, Juli 2020, pp. 169-182 179 Hubungan antara kompetensi sumber daya manusia dan kualitas laporan keuangan dapat
dijelaskan oleh Teori Keagenan. Teori ini menjelaskan bahwa pemerintah daerah sebagai
agent diwajibkan memenuhi kebutuhan rakyat sebagai principal yang diwakilkan oleh DPRD
dalam menghasilkan laporan keuangan yang berkualitas dan akuntabel agar memudahkan
principal dalam mengambil keputusan. Sumber daya yang kompeten akan memudahkan
pemerintah daerah untuk menghasilkan laporan keuangan yang berkualitas. Penelitian ini
mendukung penelitian Indriasih (2014), Karsana dan Suaryana (2017), Tawaqal dan Suparno
(2017), Evicahyani dan Setiawina (2016), serta Oktavia dan Rahayu (2019). Program Studi Akuntansi, Fakultas Ekonomi dan Bisnis, Universitas Riau Daerah Berdasarkan uji parsial t pada hipotesis ketiga, telah didapat nilai t tabel adalah 1,994,
t hitung adalah 3,090 dan signifikansinya 0,003. Ketentuan dari uji parsial t ini adalah jika t
hitung < t tabel maka H03 diterima dan Ha3 ditolak begitu juga sebaliknya, jika H03 ditolak
dan Ha3 diterima, maka nilai signifikansinya harus < 0,05. Dari hasil uji t hipotesis pertama
didapat t hitung > t tabel yaitu 3,090 > 1,994 dengan nilai sig.t 0,003 < 0,05. Dari penjelasan
di atas, dapat disimpulkan bahwa hasil dari uji t hipotesis ketiga adalah Sistem Pengendalian
Intern berpengaruh signifikan terhadap Kualitas Laporan Keuangan Pemerintah Daerah. Berdasarkan data yang diperoleh, diketahui penerapan sistem pengendalian intern di
bagian keuangan tiap OPD Pemerintah Kota Pekanbaru dapat dikatakan baik. Semakin baik
sistem pengendalian intern yang diterapkan maka semakin berkualitas laporan keuangan yang
dihasilkan. Hubungan antara sistem pengendalian intern dan kualitas laporan keuangan dapat
dijelaskan oleh Teori Keagenan. Teori ini menjelaskan bahwa pemerintah daerah sebagai
agent diwajibkan memenuhi kebutuhan rakyat sebagai principal yang diwakilkan oleh DPRD
dalam menghasilkan laporan keuangan yang berkualitas dan akuntabel agar memudahkan
principal
dalam
mengambil
keputusan. Penerapan
sistem
pengendalian
intern
dapat
membantu pemerintah daerah dalam meminimalisir kelemahan dan temuan dalam laporan
keuangan sehingga laporan keuangan yang dihasilkan berkualitas. Penelitian ini sejalan
dengan penelitian Indriasih (2014), Karsana dan Suaryana (2017), Muhammad (2019) serta
Herawati (2014). Pemerintah Daerah Berdasarkan uji parsial t pada hipotesis kedua, telah didapat nilai t tabel adalah 1,994,
t hitung adalah 2,990 dan signifikansinya 0,004. Ketentuan dari uji parsial t ini adalah jika t
hitung < t tabel maka H02 diterima dan Ha2 ditolak begitu juga sebaliknya, Jika H02 ditolak
dan Ha2 diterima, maka nilai signifikansinya harus < 0,05. Dari hasil uji t hipotesis kedua
didapat t hitung > t tabel yaitu 2,990 > 1,994 dengan nilai sig.t 0,004 < 0,05. Dari keterangan
tersebut, ditarik kesimpulan bahwa hasil dari uji t hipotesis kedua adalah Pemanfaatan
Teknologi Informasi berpengaruh signifikan terhadap Kualitas Laporan Keuangan Pemerintah
Daerah. Berdasarkan data yang diperoleh, diketahui pemanfaatan teknologi informasi bagian
keuangan di tiap OPD Pemerintah Kota Pekanbaru dikatakan baik. Berdasarkan hasil analisis
dan pengujian yang dilakukan, menunjukkan bahwa semakin efisien teknologi informasi yang
digunakan maka semakin berkualitas laporan keuangan yang dihasilkan. Hubungan antara
pemanfaatan teknologi informasi dan kualitas laporan keuangan dapat dijelaskan oleh Teori
Keagenan. Teori ini menjelaskan bahwa pemerintah sebagai agent memiliki kewajiban untuk
memenuhi
kebutuhan
rakyat
sebagai
principal
yang
diwakilan
oleh
DPRD
dalam
menghasilkan laporan keuangan yang berkualitas dan akuntabel agar memudahkan principal
dalam mengambil keputusan. Dengan memanfaatkan teknologi informasi secara efektif dan
efisien akan memudahkan pemerintah daerah untuk menghasilkan laporan keuangan yang
berkualitas dan tepat waktu. Penelitian ini sejalan dengan penelitian Tawaqal dan Suparno
(2017), Wardani dan Nugroho (2018), Oktavia dan Rahayu (2019) serta Hasanah, dkk. (2019). Justika Zebua, Muhammad Rasuli, Vera Oktari
DETERMINAN KUALITAS LAPORAN KEUANGAN PEMERINTAH DAERAH: STUDI PADA OPD PEMERINTAH KOTA
PEKANBARU Justika Zebua, Muhammad Rasuli, Vera Oktari Justika Zebua, Muhammad Rasuli, Vera Oktari
DETERMINAN KUALITAS LAPORAN KEUANGAN PEMERINTAH DAERAH: STUDI PADA OPD PEMERINTAH KOTA
PEKANBARU 180 Pengaruh Sistem Pengendalian Intern Terhadap Kualitas Laporan Keuangan Pemerintah Pengaruh Sistem Pengendalian Intern Terhadap Kualitas Laporan Keuangan Pemerintah
Daerah SIMPULAN Hasil dari penelitian ini dapat disimpulkan bahwa seluruh variabel independen, yaitu
Kompetensi
Sumber
Daya
Manusia,
Pemanfaatan
Teknologi
Informasi
dan
Sistem
Pengendalian Intern berpengaruh signifikan terhadap Kualitas Laporan Keuangan Pemerintah
Daerah di OPD Pemerintah Kota Pekanbaru. Keterbatasan dalam penelitian ini yaitu; (a) hanya beberapa responden yang bisa
ditemui
secara
langsung
untuk
dilakukannya
wawancara;
(b)
penelitian
ini
hanya
menggunakan tiga pengaruh variabel independen terhadap variabel dependen; dan (c) terdapat
kuesioner yang tidak direspon oleh responden, dikarenakan lamanya birokrasi penyampaian
kuesioner ke responden yang bersangkutan dari rentang waktu yang ditentukan, sehingga
tingkat pengembalian kuesioner tidak 100%. Saran untuk menutupi keterbatasan penelitian ini untuk peneliti selanjutnya adalah Saran untuk menutupi keterbatasan penelitian ini untuk peneliti selanjutnya adalah E ISSN 2721-1819 | P ISSN 2721-2416 CURRENT: Jurnal Kajian Akuntansi dan Bisnis Terkini . Vol .1, No.2, Juli 2020, pp. 169-182 CURRENT: Jurnal Kajian Akuntansi dan Bisnis Terkini . Vol .1, No.2, Juli 2020, pp. 169-182 181 dengan menambahkan wawancara untuk melengkapi metode penelitian sehingga hasil yang
didapat lebih baik; dapat menambahkan variabel independen maupun intervening/moderating
yang diketahui dapat mempengaruhi kualitas laporan keuangan pemerintah daerah; dan
diharapkan untuk dapat memperoleh data secara keseluruhan dari semua sampel yang telah
ditetapkan, agar hasil yang diperoleh maksimal. Implikasi dari penelitian ini bagi Pemerintah Kota Pekanbaru yaitu, diharapkan hasil
penelitian ini dapat menjadi bahan evaluasi dan pertimbangan untuk OPD di Pemerintah Kota
Pekanbaru dalam proses penyusunan laporan keuangan agar dapat tercapai kualitas laporan
keuangan yang baik, yang mana dapat meminimalisir kelemahan dan temuan BPK dalam
laporan keuangan yang akan diaudit. Serta bagi peneliti selanjutnya, diharapkan penelitian ini
dapat menjadi jurnal acuan yang digunakan untuk meneliti kembali topik yang sama. REFERENSI Evicahyani, S. I., & Setiawina, N. D. (2016). Analisis Faktor-Faktor yang Mempengaruhi
Kualitas Laporan Keuangan Pemerintah Daerah Kabupaten Tabanan. E-Jurnal. Ekonomi dan Bisnis Universitas Udayana. Faristina, R. (2011). Faktor-Faktor yang Mempengaruhi Keandalan dan Timeliness Pelaporan
Keuangan. Skripsi. Universitas Diponegoro. Hasanah, Y. A., Sasongko, N., & Bawono, A. D. B. (2019). Pengaruh Pemahaman Akuntansi,
Penerapan Sistem Akuntansi Keuangan Daerah, Peran Internal Audit, dan Penerapan
Standar Akuntansi Pemerintah terhadap Kualitas Laporan Keuangan Pemerintah
Daerah (Studi pada SKPD di Pemerintah Kabupaten Sukoharjo). The 9th URECOL. Herawati, T. (2014). Pengaruh Sistem Pengendalian Intern terhadap Kualitas Laporan
Keuangan (Survei pada Organisasi Perangkat Daerah Pemda Cianjur). STAR – Study
& Accounting Research, 1-14. Indriasih, D. (2014). The Effect of Government Apparatus Competence and the Effectiveness
of Government Internal Control Toward the Quality of Financial Reporting in Local
Government. Research Journal of Finance and Accounting, 38-47. Karsana, I. W., & I Gusti N. A. S. (2017). Pengaruh Efektivitas Penerapan SAP, Kompetensi
SDM, dan SPI pada Kualitas Laporan Keuangan Pemerintah Kabupaten Bangli. E-
Jurnal Akuntansi. Universitas Udayana. Muhammad, F. A. (2019). Diterapkannya Sistem Akuntansi dan Sistem Pengendalian Intern
untuk Mempengaruhi Kualitas Laporan Keuangan. Syntax Idea. Nurillah, A. (2014). Pengaruh Kompetensi Sumber Daya Manusia, Penerapan Sistem Program Studi Akuntansi, Fakultas Ekonomi dan Bisnis, Universitas Riau Justika Zebua, Muhammad Rasuli, Vera Oktari
DETERMINAN KUALITAS LAPORAN KEUANGAN PEMERINTAH DAERAH: STUDI PADA OPD PEMERINTAH KOTA
PEKANBARU 182 Akuntansi Keuangan Daerah (SAKD), Pemanfaatan Teknologi Informasi, dan Sistem
Pengendalian Intern Terhadap Kualitas Laporan Keuangan Pemerintah Daerah (Studi
Empiris pada SKPD Kota Depok). Skripsi. Universitas Diponegoro. Akuntansi Keuangan Daerah (SAKD), Pemanfaatan Teknologi Informasi, dan Sistem
Pengendalian Intern Terhadap Kualitas Laporan Keuangan Pemerintah Daerah (Studi
Empiris pada SKPD Kota Depok). Skripsi. Universitas Diponegoro. Oktavia, S., & Rahayu, S. (2019). Pengaruh Kompetensi Sumber Daya Manusia, Komitmen
Organisasi dan Sistem Akuntansi Keuangan Daerah terhadap Kualitas Laporan
Keuangan Daerah (Studi Kasus pada Satuan Kinerja Perangkat Daerah Kota Bandung
Tahun 2018), e-Proceeding of Management, 652-659. Oktavia, S., & Rahayu, S. (2019). Pengaruh Kompetensi Sumber Daya Manusia, Komitmen
Organisasi dan Sistem Akuntansi Keuangan Daerah terhadap Kualitas Laporan
Keuangan Daerah (Studi Kasus pada Satuan Kinerja Perangkat Daerah Kota Bandung
Tahun 2018), e-Proceeding of Management, 652-659. Sholohah, S. A., & Sulistyawati. (2018). Kualitas Laporan Keuangan dan Faktor-Faktor yang
Mempengaruhinya (Studi Kasus pada DPKAD Kota Semarang). FOKUS EKONOMI
Jurnal Ilmiah Ekonomi, 280-300. Tawaqal, Irzal, & Suparno. (2017). REFERENSI Pengaruh Penerapan Sistem Informasi Akuntansi, Sistem
Pengendalian Internal, dan Kompetensi Sumber Daya Manusia terhadap Kualitas
Laporan Keuangan Satuan Kerja Perangkat Daerah di Pemerintah Kota Banda Aceh. JIMEKA, 125-135. Wardani, D. K., & Nugroho, S. P. (2018). The Impact of Information Technology on
Financial Statement Quality: A Moderating Role of Internal Control System. ICASI,
10-15. Wawo, A. B., Idrus, M. S., Rahayu, M., & Djumahir (2013). The Influence of Internal and
External Monitoring, Leadership Style and Good Public Governance Implementation
on Financial Reporting Performance (Study on SKPDs/Local Government Task
Forces at South-East Sulawesi Province). J, Basic. Appl. Sci. Res, 402-412. http://pekanbaru.bpk.go.id
https://www.bpk.go.id/
https://www.cakaplah.com E ISSN 2721-1819 | P ISSN 2721-2416
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English
| null |
Some Remarks on the Culture of Eastern Near-Arctic Indians
|
Science
| 1,909
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public-domain
| 2,202
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SCIENCE SCIENCE 150 [N. S. VOL. XXIX. NO. 734 their natural or planted range a height of at
least eight feet, and in diameter of not less
than two inches."
This definition is not,
however,
allowed to exclude unbranched
cactuses, yuccas and palms. The uniform
recapitalization of all specific names is greatly
to be commended, as also the clear type (of
two sizes) and the exact illustrations. Two
good maps of the region covered and a good
index complete this altogether admirable
publication. Carbon Assimilation of Penicillium " in the
Botanical Gazette for Narch, 1908, is a con-
tribution to our knowledge of the chemistry of
the assimilation of some of the simpler com-
pounds by plants. Anlong the results noted is
the fact that "alcohol, acetic acid and the sub-
stances from which the acetic acid radicIe
CH,COO-
is easily derived are assimilated
by Penicellium glaucum." Xention should be made here of Scott and
Rorer's paper "Apple Leaf-spot caused by
Sphaeropsis rnnlorum" in Bulletin 121 of the
Bureau of Plant Industry of the IT. S. Depart-
ment of Agriculture; of W. H. Lawrence's
record of " Some Imported Plant Diseases of
Washington," in Bulletin 83 of the Oregon
Experiment Station, and Cook and Rome's
"Insects and Diseases of the Orange," in
Bulletin 9 of Estacion Central Agronomics
de Cuba. FUNGUS EOTES INa recent number of Ahodom (January,
1908) Dr. W. J. Farlow begins the publication
of " Notes on Fungi," which promise to yield
critical discussions of much value. He shows
that what has been known as Corticium, tre-
rnellinum var. reticulaturn is in the first place
not a Corticium, but a Tremella, and that the
variety is a distlnct species, to be known here-
after as Trernella reticulata. R e shows that
what has been known as Xynchytriurn plurian-
nulaturn (a parasite in species of Xnnicula)
is in reality Urophlyctis pluriannulatus, and
that a uredineous parasite of Rubus neglectus
and R. strigosus, hitherto known as, or con-
fused trith, Plzragmidium gracile is Puccini-
astrum arcticum var. americanum. He is
further of the opinion that the Pucciniastrum
on Potentilla hidentata is P. potentillae. Further notes from this source will be eagerly
looked for by mycologists. Here also may be mentioned Professor
Warshberger's paper '(A Crass-killing Slime
31ould" in the Proceedings of the American
Philosophical Society, Vol. XLV., recording a
case in which the plasmodia of Ph2/sarum
cinerezmz killed the blades of grass over which
they had grown. CHARLESE. BESSEY
THE UNIVERSITYOF NEBRASKA SOME REMARIKS ON TI-IE CULTURE OF EASTERN
NEAR-ARCTIC INDIANS A shamanistic society, very loose in
form, but apparently corresponding to the
Midewin of the Ojibway, occurs. There are
but two degrees, and admission to these is
through dreams. There are no initiatory cere-
monies. So far as could be learned, members
of this society do not attempt to cure disease. Apparently this is done by herb doctors. as Eastmain, and that they did not come out
to any of the posts along that shore. At
Nitchequon, a post situated in the Labrador
interior, fiftyfive days' journey from Rupert's
House by canoe, and still in the hunting terri-
tory of the Northern Cree, the Naskapi are
known to come in winter when driven by
starvation. Otherwise they are confined to the
interior of Labrador proper, held back on the
east and north by the Esquimau, on the west
by the Northern Cree, and on the south by the
Montagnais. As conditions were not favorable for study
of the Naskapi, our attention was turned to
the ethnology of the Northern Cree. The
Northern, or perhaps more properly the
Eastern, Cree range from Nitchequon on the
north, south to the height of land around
James Bay, west to the Albany River and
Agomska Island. There are no Cree between
this post and York, because the Northern
Ojibway have worked northward to the coast
and thus have separated the York Cree from
-the rest. So far as could be learned from the
EIudson's Bay men and Indians, this has
taken place within comparatively recent times,
and it was also stated that the Western Cree
came originally from the vicinity around
James Bay, being induced to go westward by
the Fludson's Bay Company. Both the York
Cree and the Western Cree seem to be con-
sidered by the Eastern Cree to be somewhat
different from the Eastern Cree, although ad-
mittedly the same people. Several dialects of
their language are recognized by the eastern
band, but the changes appear to be perfectly
regular and phonetic, not affecting the gram-
mar in any way. The material culture of these people is now
considerably debased through constant contact
with the Hudson's Bay Company. Clothing
in the old days was made of caribou skin,
tanned without the hair in summer; in winter,
of caribou skin with the hair, or of beaver and
other furs. Garments were often made of
twisted and woven rabbit skins. SOME REMARIKS ON TI-IE CULTURE OF EASTERN
NEAR-ARCTIC INDIANS I n Anmles JIycologici (V., No. 7, 1907)
Professor F. L. Stevens figures and describes
"Some Remarkable Nuclear Structures in
Synchytriu~n." The paper is a record of
facts, and the author does not attempt to base
any conclusions upon what he has yet seen. Other recent fungus papers by the same
author are " An Apple Rot due to Volutella "
and a "List of New York Fungi" in the
March and May numbers of the Journal of
Jfycology (1907), and " The Chrysantheinum
Ray Blight" in the Botanical Gazette (Oc-
tober, 1907). The fungus which causes the
ray blight on the chrysanthemum appears to
be new and is described as Ascochyta chry-
santlzenzi. DGRINGthe past summer, 1908, the writer
undertook an ethnological expedition into the
James Bay region of Canada, for the Depart-
ment of Anthropology of the American Mu-
seum of h'atural History of New Pork. The
original intention was to journey to Moose
Factory and thence to the eastern coast of the
bay, touching at Rupert's House, Eastmain
River and, perhaps, Fort George, at which
posts it was supposed access might possibly
be had to the Naslrapi Indians of Labrador,
who, it was thought, might come down to these
points during the summer, for the purpose of
trade. On arriving at Noose Factory, it was
learned that the Naskapi could not be reached
via the west coast of Labrador, usually k n o w Heinrich Hasselbring's
paper on "The made every spring at some spot previously de-
cided upon for the purpose of reuniting. At
this time, the feasts, councils and meager cere-
monies of these people were held. Nowadays,
the hunters come to the Hudson's Bay posta
every spring to trade their furs for supplies
for the next year, and this coming together
takes the place of the old spring meeting. Chiefs were never elected or chosen, but ac-
quired their office through prestige by tacit
consent on the death of the former incumbent. As the people rarely came together, excepting
at the spring meetings, or in case of war, the
chiePs influence was small in comparison with
that of the shaman. Shamanism, or "con-
juring," as it is called in tlie north, is still
quite extensively carried on for warlike ex-
peditions, hunting, love-making and other
purposes. Conjuring houses are still built and
used. THE AdfERICAAT A8SOCIATION
FOR THB
ADPANCEMENT OF SCIENCE
fJEPY'ION A-MAT'HB$fATZCS
AND
ASTRONOJIY CO~EIJAI~~T~VELT
papers on pure mathe-
few
matics appeared on the program of Scction A
in view of the fact that the American Mathe-
maticit1 Society held its annual meeting in affrlia-
tion with thc asiociation. The address of the
rctiring ricc-president, P~esidcnt E. 0. Lovett,
tlie Rice Institute, IIouston, Texas, was read by
thc secretary of the section. It was entitlcd "The
Problell~ of Several Bodies, Recent Progress in its
Solutio~~,"and an abstract of it has appeared in
a recent number of S c ~ ~ s c c . Little folk-lore was collected, and this was,
in tho main, typically Algonkin, but some ap-
parently resenibles the Esqnimau. A comparison of the writer's notes with
Lucien M. Turner's account of "The Nenenot
or 'Naskopie '" Indians: and conference with
Indians and white men who had been in the
Naslrapi country, seems to show that the cul-
ture 01these people is identical with that of
the old Cree. Considering the absence of agri-
culture, the lack of village life and clan sys-
tems, the loose social and political organiza-
tion, the absence of pottery and the ordinary
forms of fabrics, and the colnparative differ-
ence of artifacts i n general, as here noted-it
may perhaps be well no longer t o consider the
region inhabited by the Kilstern Cree and the
Naslrapi as belonging to the Eastern Woodland
culture area, a region characterized through-
out by its agricultural and village life, its
comparatively highly developed social and
political organization, its pottery, clothing
'Lucien hl. Turner, " Ethnology of the Ungava
District,"
Eleventh Annual Report, Bureau of
Ethnology, 1589-90, pp. 167-350. The following members of the section were
elected as fellow of the association: David R. Allen, Josepli Allen, R. 13. Allen, Harriet W. Bigclow, Oskar Rolza, W. 1%.Bussey, B. E. Carter,
E. F. Chandler, Abraham Cohen, E. H. Coinstock,
IT. A. Converse, S. A. Corcy, J. A. Cragwall,
F. F. Decker, C. C. Engbcrg, F. C. Ferry, F. E. Fomle, Philip Fox, William Gillespic, C. C. Gore,
C. 0. Gunthcr, U. S. Hanna, A. E. IIaynes, Alfred
IIurne, W. J.IIussey, Kurt Laves, A. IT.McDougall,
Jfax Mason, Frank E. Miller, J. S. Miller, n'. F. Osgood, J. M. Page, 3i. T. Pcecl, James Pierpont,
S. C. Recse, W. J. Rusk, P. L. Gxnrel, G. T. Sellcw,
E. D. Skinner, A. G. Smith, D. E. Smith, P. F. Smith, Joel Stebbins, R. SOME REMARIKS ON TI-IE CULTURE OF EASTERN
NEAR-ARCTIC INDIANS Coats with
sleeves, hoods and mittens were worn by both
sexes. The habitations consisted of conical or
dome-shaped lodges, covered with painted
skins, bark or brush. No mats were used for
this or any other purpose, as articles of woven
rabbit-skin seem the only fabrics made. Ow-
ing to climatic conditions, agriculture was un-
known, a few berries furnishing the only vege-
table food. Eunting, and, secondarily, fish-
ing were the great resources of life. As hunt-
ing has not been checked, but rather given an
impetus, by the advent of the Hudson's Bay
Company, all the ancient superstitions regard-
ing animal life may still be found in full
force. Most interesting of these are a series The Eastern Cree claim to have always lived
in the region that they now inhabit, and
~ecognixe several bands or subdivisions, known
according to the locality which they inhabit. The Crees know themselves generally as
Xusk6ko-wug, or "Swamp People."
The
social unit is the patriarchal family and there
geems never to have been any clan organiza-
tion among them. Village life is, and appar-
ently was, unknown; for economic conditions
caused single families to live by themselves,
far apart from any others, and rendezvous was SCIENCE [N. S. VOL. XXIX. NO. 734 [N. S. VOL. XXIX. NO. 734 made from skins tanned without the hair,
fabrics, woven basketry, and the like. Dr. Frank G. Speck, of the Department of Arche-
ology of the University of Pennsylvania, who
spent last summer among the Montagnais of
Lalie St. Johns, arrived independently a t the
sarne conclusion in studying these people. It
is the suggestion of tlie writer, then, that the
culture of the region of Subarctic Eastern
Anierica inhabited by the Cree, Naskapi, and
Montagnais, might better be known hereafter
as the Eastern Subarctic, or Labradorean,
cultural area, as it is apparently so different
from the eastern woodland area with which it
has hitherto been classed. of observances regarding the killing of the
bear. While all the eastern Algonkins have
observances of this order, they seem to have
become much more elaborated among the
Eastern Cree. Pottery was unknown, steatite taking its
place. Semilunar kaives,
here used
as
scrapcrs, other linives and some arrow points
were rubbed ont from slate. I n some parts, at
least, arrow points seen1 to have been cliippcd;
and in others, made of bone and antler. The sectional conimittee of Section A nomi-
nated the following members of the association, SOME REMARIKS ON TI-IE CULTURE OF EASTERN
NEAR-ARCTIC INDIANS The
grooved axe was used. Basketry,
except
simple vessels of birch or pine barlr, was un-
known. Birch bark canoes were used. Syllabics, invented by missionaries, are now
used for cominunication in their own lan-
guage, though the Cree still employ inncmonic
devices of their own invention for the same
purpose. Information was obtained which
seemed to show that in olden times pictorial
writings on birch bark, similar to those found
among the Ojibways, were Imo~vn. The
priniitive forin of art seems to have been
painting, and the lines ernployed were geo-
metric. THE AdfERICAAT A8SOCIATION
FOR THB
ADPANCEMENT OF SCIENCE
fJEPY'ION A-MAT'HB$fATZCS
AND
ASTRONOJIY THE AdfERICAAT A8SOCIATION
FOR THB
ADPANCEMENT OF SCIENCE
fJEPY'ION A-MAT'HB$fATZCS
AND
ASTRONOJIY P. Stcl?l~cns,1,. l3. Stewart, EI. D. Thompson, E. B. Van Vleck,
Osmald Peblen, H. S. White, F. S. Wood3. h
i
l
i i
f S
i
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English
| null |
Interfacial Behaviour in Ferroalloys: The Influence of FeMn Slag Composition
|
Metallurgical and materials transactions. B, Process metallurgy and materials processing science
| 2,022
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cc-by
| 11,213
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ORIGINAL RESEARCH ARTICLE ORIGINAL RESEARCH ARTICLE SERGEY BUBLIK, MERETE TANGSTAD, and KRISTIAN ETIENNE EINARSRUD The present study investigated the interfacial interaction between FeMn alloy and slags of
different compositions and basicity from 0.4 to 1.2 in a sessile drop furnace. Interfacial tension
between the FeMn alloy and the slags was measured, and the results were analyzed to assess the
sensitivity of the applied methodology. The measurement of the interfacial tension was based on
combining the results from experiments, multiphase flow simulations in OpenFOAM,
equilibrium calculations in FactSage, and calculation of slag density and surface tension
based on numerical models. The results demonstrate that the interfacial tension between the
FeMn alloy and slag increases with the slag basicity. It was found that the addition of Al2O3 to
the slag with basicity of 0.8 and 1.2 increases the interfacial tension, while increasing MnO
content from 30.0 to 45.0 wt pct does not have any statistically significant influence on the
interfacial tension. EPMA analysis of slag and FeMn phases showed that slags at lower
basicities and the FeMn alloy form a metal–slag emulsion due to the destabilization of the
interface induced by chemical reactions, partial reduction of SiO2 in the slag and the mass
transfer of Si across the metal–slag interface. https://doi.org/10.1007/s11663-022-02605-3
The Author(s) 2022 Interfacial Behaviour in Ferroalloys: The Influence
of FeMn Slag Composition SERGEY BUBLIK, MERETE TANGSTAD, and KRISTIAN ETIENNE EINARSRUD SERGEY BUBLIK, MERETE TANGSTAD, and KRISTIAN
ETIENNE EINARSRUD are with the Department of Materials
Science and Engineering, Norwegian University of Science and
Technology (NTNU), 7491 Trondheim, Norway. Contact e-mail:
sergey.bublik@ntnu.no
Manuscript submitted December 6, 2021; accepted July 13, 2022. https://doi.org/10.1007/s11663-022-02605-3
The Author(s) 2022 A. Material Preparation Synthetic FeMn alloy and slags have been prepared
from pure powders for studying interfacial tension in the
sessile drop furnace. Compositions of the FeMn alloy
and the slags have been selected based on the compo-
sition of industrial materials and, in addition, the
synthetic slags have been selected to cover the most
relevant slags for FeMn production. The calculated
composition of the synthetic raw materials and the
purity of powders used for material preparation are
specified in Tables IV and V in ‘‘Appendix A’’,
respectively. The composition of FeMn alloy and slag
measured by the X-ray fluorescence (XRF) and the
combustion infrared detection (combustion-IR) tech-
niques is shown in Table I. The corresponding normal-
ized data is given in Table VI in ‘‘Appendix A’’. After the experiments, the FeMn alloy and the slags
were cast in epoxy, sectioned in the centre of the slag
droplet, re-cast into epoxy, polished and coated with
carbon prior to analysis in EOL JXA-8500F Electron
Probe Micro Analyzer (EPMA). The composition of
FeMn and slag phases were measured in several points,
and the average composition of each phase was calcu-
lated based on this. Additionally, the chemical compo-
sition of the synthetic FeMn alloy and the slags before
the experiments was determined by XRF and combus-
tion-IR. The FeMn alloy was analyzed for Mn, Fe, Si
and C, and the slags were analyzed for MnO, CaO,
MgO, SiO2, Al2O3 and FeO. g
pp
The powders were mixed and melted separately in
graphite crucibles in an induction furnace in Ar atmo-
sphere at temperature of 1773 K and holding time of 60
minutes for the FeMn alloy and 5 minutes for master
slags. After the first melting, the FeMn alloy and the
master slags were cooled down in the crucibles and
ground in a ball mill. Thereafter, the master slags were
divided into batches of 20 g and an additional amount
of the oxide powders has been added according to the
composition of the final slags. Both the FeMn alloy and
the master slags were then remelted using the same
operational parameters in the induction furnace. The
amount of the powders required to produce the syn-
thetic materials has been calculated taking into account
the purity of the powders and the composition of the
industrial materials[1] and is presented in Table VII in
‘‘Appendix A’’. I.
INTRODUCTION In FeMn production, the FeMn alloy is highly saturated
with carbon, which results in reduction of oxides such as
MnO, FeO or SiO2 at the interface between FeMn and
slag, and subsequently in the mass transfer of Mn, Fe
and Si to the FeMn alloy. The thermodynamic equilib-
rium between ferroalloy and slag can be described by
several reduction reactions:[13] THE carbothermic reduction of manganese ores for
producing FeMn alloy in submerged arc furnaces is
related to the formation of a significant amount of
molten slag, which is then tapped simultaneously with
FeMn to ladles, where a metal–slag emulsion is formed. Small FeMn droplets are entrained in the slag phase due
to the intensive mixing of FeMn and slag. This decreases
efficiency of the ferroalloy production process because
further removal of metal from slag may cause logistics
difficulties and is heavily time- and energy-consum-
ing.[1,2] Interfacial tension between molten metal and
slag characterizes the metal-slag separation[3] and thus it
is important for ferroalloy production. High interfacial
tension promotes a high degree of separation of metal
and slag, while a low interfacial tension will promote
formation of metal–slag emulsion and small metal
droplets in slag.[4] Several studies have shown that the
interfacial tension is greatly dependent upon the slag
composition due to chemical reactions at the metal–slag
interface and the mass transfer across the interface.[5–12] ðMnOÞ þ ½C ¼ ½Mn þ COðgÞ;
½1 ½1 ðMnOÞ þ ½C ¼ ½Mn þ COðgÞ; ðSiO2Þ þ 2½C ¼ ½Si þ 2COðgÞ;
½2 ½2 ðFeOÞ þ ½C ¼ ½Fe þ COðgÞ;
½3 ½3 ðMnOÞ þ ½Fe ¼ ½Mn þ ðFeOÞ;
½4 ½4 ðSiO2Þ þ 2½Mn ¼ ½Si þ 2ðMnOÞ;
½5 ½5 where [] and () denote chemical constituents in the
FeMn alloy and the slag, respectively. The determination of the metal–slag interfacial ten-
sion is challenging as it requires applying high temper-
atures and recording metal–slag interaction with X-ray
imaging[14] or a digital camera.[6] Generally, these
methods may have significant experimental uncertainty SERGEY BUBLIK, MERETE TANGSTAD, and KRISTIAN
ETIENNE EINARSRUD are with the Department of Materials
Science and Engineering, Norwegian University of Science and
Technology (NTNU), 7491 Trondheim, Norway. Contact e-mail:
sergey.bublik@ntnu.no
Manuscript submitted December 6, 2021; accepted July 13, 2022. METALLURGICAL AND MATERIALS TRANSACTIONS B METALLURGICAL AND MATERIALS TRANSACTIONS B contained in the graphite crucible—this allows to
saturate the required amount of carbon, 7 wt pct
for the FeMn alloy. I.
INTRODUCTION due to sensitivity to impurities present in raw mate-
rials
and
crucibles,
or
the
resolution
of
X-ray
images.[15] Therefore, the use of advanced analysis
and modelling tools can be of great help in under-
standing the interfacial phenomena and in improving
and simplifying the experimental setup for measuring
interfacial tension. A. Material Preparation Note that carbon has not been added as
a raw material for melting of the FeMn alloy as it is B. Experimental Setup A sessile drop furnace (Figure 1) has been applied for
investigating the interfacial interaction between the
FeMn alloy and the slags as shown in Figure 2. The
sessile drop furnace was equipped with a pyrometer and
a C-type thermocouple for measuring temperature, and
with a digital video camera (Allied Vision Prosilica
GT2000, Edmund Optics, Inc., Barrington) with a
telecentric lens (Navitar 1-50993D) for recording images
of molten samples with the resolution of 2048 1088
pixels at one frame per second after reaching melting
temperature of the FeMn alloy and the slags. Every 10
to 15 experiments, the thermocouple was calibrated by
melting pure Fe in Ar and assessing its melting
temperature from recorded images. The experiments
were performed using graphite cups in Ar atmosphere
(6N grade) at temperature of 1673 K and holding time
of 5 minutes in sets, where three parallels were per-
formed for each slag composition. The heating rate was
300 K/min up to 1473 K and approximately 25 K/min
from 1473 K to 1673 K. The graphite cups were cleaned
before the experiments with a paper towel dipped in
ethanol or acetone and then dried using compressed air. A schematic representation of the slag and the FeMn
alloy in the graphite cup before and during the exper-
iments is shown in Figure 3. In the
current
study, the
interfacial interaction
between FeMn alloy and slags of different compositions
was investigated in a sessile drop furnace using the
methodology developed by the authors previously,[16,17]
which was improved in this study to improve repro-
ducibility and reduce uncertainty due to the precipita-
tion of solid carbon during melting of FeMn alloy and
slag. The improved methodology combines analysis of
images from experiments in the sessile drop furnace,
numerical data obtained in multiphase flow simulations
in OpenFOAM v8,[18] the numerical models for calcu-
lation of density and surface tension of slag developed
by Mills et al.[19,20] and results of equilibrium calcula-
tions in FactSage 7.3.[21] In addition, the sensitivity of
the
results
was
assessed
by
comparing
different
experiments. C. Methodology for Determination of Interfacial Tension p
y
y
g
Material
Chemical Composition (Wt Pct)
Mn
Fe
Si
C
MnO
CaO
MgO
SiO2
Al2O3
FeO
Total
Basicity*
FeMn Alloy
77.30
14.50
0.11
7.52
—
—
—
—
—
—
99.43
—
Slags with Al2O3 Addition
Slag A1
—
—
—
—
30.62
14.23
6.77
39.52
10.23
0.89
102.26
0.42
Slag A2
—
—
—
—
29.30
25.50
6.75
29.18
10.17
0.79
101.69
0.82
Slag A3
—
—
—
—
28.91
32.00
6.64
22.14
10.19
0.79
100.67
1.20
Slag B1
—
—
—
—
37.90
12.87
6.72
34.15
10.18
0.77
102.59
0.44
Slag B2
—
—
—
—
36.77
22.50
6.58
25.21
10.08
0.74
101.88
0.82
Slag B3
—
—
—
—
37.53
27.87
6.83
18.33
10.01
0.74
101.31
1.22
Slag C1
—
—
—
—
45.43
10.76
6.61
28.84
10.23
0.73
102.60
0.44
Slag C2
—
—
—
—
44.65
19.04
6.83
20.80
10.19
0.73
102.24
0.83
Slag C3
—
—
—
—
45.39
23.92
6.70
15.04
10.01
0.72
101.78
1.22
Slags Without Al2O3 Addition
Slag D1
—
—
—
—
30.67
14.76
6.10
48.83
0.67
0.89
101.92
0.42
Slag D2
—
—
—
—
29.83
25.30
6.31
38.32
0.77
0.85
101.38
0.81
Slag D3
—
—
—
—
29.27
31.91
6.18
32.23
0.61
0.80
101.00
1.16
Slag E1
—
—
—
—
38.68
12.93
6.09
43.32
0.58
0.77
102.37
0.43
Slag E2
—
—
—
—
37.01
22.69
6.16
34.40
0.69
0.75
101.70
0.82
Slag E3
—
—
—
—
37.24
28.17
6.10
28.36
0.68
0.61
101.16
1.18
Slag F1
—
—
—
—
46.00
10.58
6.23
38.14
0.69
0.71
102.35
0.43
Slag F2
—
—
—
—
44.33
19.64
6.10
30.53
0.56
0.68
101.84
0.83
Slag F3
—
—
—
—
45.33
24.53
6.01
25.02
0.47
0.70
102.06
1.20
Basicity (B) = CaOþMgO
SiO2þAl2O3, calculated on mass basis. Fig. 1—Schematic of the sessile drop furnace. Reprinted from Ref. [22] under the terms of the Creative Commons CC BY license. Fig. 1—Schematic of the sessile drop furnace. Reprinted from Ref. [22] under the terms of the Creative Commons CC BY license. Fig. 2—FeMn alloy and slag in the graphite cup before the
experiments in the sessile drop furnace. Reprinted from Ref. [17]. C. Methodology for Determination of Interfacial Tension The Measured Chemical Composition of the Synthetic FeMn Alloy and Slags
Material
Chemical Composition (Wt Pct)
Mn
Fe
Si
C
MnO
CaO
MgO
SiO2
Al2O3
FeO
Total
Basicity*
FeMn Alloy
77.30
14.50
0.11
7.52
—
—
—
—
—
—
99.43
—
Slags with Al2O3 Addition
Slag A1
—
—
—
—
30.62
14.23
6.77
39.52
10.23
0.89
102.26
0.42
Slag A2
—
—
—
—
29.30
25.50
6.75
29.18
10.17
0.79
101.69
0.82
Slag A3
—
—
—
—
28.91
32.00
6.64
22.14
10.19
0.79
100.67
1.20
Slag B1
—
—
—
—
37.90
12.87
6.72
34.15
10.18
0.77
102.59
0.44
Slag B2
—
—
—
—
36.77
22.50
6.58
25.21
10.08
0.74
101.88
0.82
Slag B3
—
—
—
—
37.53
27.87
6.83
18.33
10.01
0.74
101.31
1.22
Slag C1
—
—
—
—
45.43
10.76
6.61
28.84
10.23
0.73
102.60
0.44
Slag C2
—
—
—
—
44.65
19.04
6.83
20.80
10.19
0.73
102.24
0.83
Slag C3
—
—
—
—
45.39
23.92
6.70
15.04
10.01
0.72
101.78
1.22
Slags Without Al2O3 Addition
Slag D1
—
—
—
—
30.67
14.76
6.10
48.83
0.67
0.89
101.92
0.42
Slag D2
—
—
—
—
29.83
25.30
6.31
38.32
0.77
0.85
101.38
0.81
Slag D3
—
—
—
—
29.27
31.91
6.18
32.23
0.61
0.80
101.00
1.16
Slag E1
—
—
—
—
38.68
12.93
6.09
43.32
0.58
0.77
102.37
0.43
Slag E2
—
—
—
—
37.01
22.69
6.16
34.40
0.69
0.75
101.70
0.82
Slag E3
—
—
—
—
37.24
28.17
6.10
28.36
0.68
0.61
101.16
1.18
Slag F1
—
—
—
—
46.00
10.58
6.23
38.14
0.69
0.71
102.35
0.43
Slag F2
—
—
—
—
44.33
19.64
6.10
30.53
0.56
0.68
101.84
0.83
Slag F3
—
—
—
—
45.33
24.53
6.01
25.02
0.47
0.70
102.06
1.20
Basicity (B) = CaOþMgO
SiO2þAl2O3, calculated on mass basis. Fig. 1—Schematic of the sessile drop furnace. Reprinted from Ref. [22] under the terms of the Creative Commons CC BY license. C. Methodology for Determination of Interfacial Tension The methodology for determination of the interfacial
tension is based on a comparison of geometrical features
obtained from multiphase CFD simulations and the
experiments in the sessile drop furnace[17] as illustrated
in Figure 4. This methodology was developed by the
authors previously[16,17] and has been modified in this
paper to improve reproducibility and reduce uncertainty
due to the precipitation of solid carbon on the surface of
FeMn and slag during the experiments in the sessile
drop furnace. The experimental step is performed to obtain images
of slag and metal interaction in the molten state. Here, a
set of three individual experiments with the slag droplet
placed on top of the FeMn layer in the graphite cup are
conducted for each slag composition. ‘‘Appendix A’’. Note that carbon has not been added as
a raw material for melting of the FeMn alloy as it is METALLURGICAL AND MATERIALS TRANSACTIONS B Table I. C. Methodology for Determination of Interfacial Tension The Measured Chemical Composition of the Synthetic FeMn Alloy and Slags
Material
Chemical Composition (Wt Pct)
Mn
Fe
Si
C
MnO
CaO
MgO
SiO2
Al2O3
FeO
Total
Basicity*
FeMn Alloy
77.30
14.50
0.11
7.52
—
—
—
—
—
—
99.43
—
Slags with Al2O3 Addition
Slag A1
—
—
—
—
30.62
14.23
6.77
39.52
10.23
0.89
102.26
0.42
Slag A2
—
—
—
—
29.30
25.50
6.75
29.18
10.17
0.79
101.69
0.82
Slag A3
—
—
—
—
28.91
32.00
6.64
22.14
10.19
0.79
100.67
1.20
Slag B1
—
—
—
—
37.90
12.87
6.72
34.15
10.18
0.77
102.59
0.44
Slag B2
—
—
—
—
36.77
22.50
6.58
25.21
10.08
0.74
101.88
0.82
Slag B3
—
—
—
—
37.53
27.87
6.83
18.33
10.01
0.74
101.31
1.22
Slag C1
—
—
—
—
45.43
10.76
6.61
28.84
10.23
0.73
102.60
0.44
Slag C2
—
—
—
—
44.65
19.04
6.83
20.80
10.19
0.73
102.24
0.83
Slag C3
—
—
—
—
45.39
23.92
6.70
15.04
10.01
0.72
101.78
1.22
Slags Without Al2O3 Addition
Slag D1
—
—
—
—
30.67
14.76
6.10
48.83
0.67
0.89
101.92
0.42
Slag D2
—
—
—
—
29.83
25.30
6.31
38.32
0.77
0.85
101.38
0.81
Slag D3
—
—
—
—
29.27
31.91
6.18
32.23
0.61
0.80
101.00
1.16
Slag E1
—
—
—
—
38.68
12.93
6.09
43.32
0.58
0.77
102.37
0.43
Slag E2
—
—
—
—
37.01
22.69
6.16
34.40
0.69
0.75
101.70
0.82
Slag E3
—
—
—
—
37.24
28.17
6.10
28.36
0.68
0.61
101.16
1.18
Slag F1
—
—
—
—
46.00
10.58
6.23
38.14
0.69
0.71
102.35
0.43
Slag F2
—
—
—
—
44.33
19.64
6.10
30.53
0.56
0.68
101.84
0.83
Slag F3
—
—
—
—
45.33
24.53
6.01
25.02
0.47
0.70
102.06
1.20
Basicity (B) = CaOþMgO
SiO2þAl2O3, calculated on mass basis. Fig. 1—Schematic of the sessile drop furnace. Reprinted from Ref. [22] under the terms of the Creative Commons CC BY license. Table I. The Measured Chemical Composition of the Synthetic FeMn Alloy and Slags Table I. C. Methodology for Determination of Interfacial Tension METALLURGICAL AND MATERIALS TRANSACTIONS B The composition of the slag and metal changes during
the experiments, and therefore the composition of the
liquid slag and the formed solid monoxide phase must
be taken into account to address the change in compo-
sition. The amount and composition of liquid slag phase
is determined at the adjustment step, which is then used
to estimate surface tension and density of the slag
droplet at the equilibrium using numerical models
developed by Mills et al. [19,20] The adjustment step is
performed as follows: (a) Calculate amount and composition of the liquid slag
and solid phases after interaction with the FeMn
alloy at 1673 K in FactSage in the equilibrium
module using FactPS, FToxid and FTmisc data-
bases. (b) Calculate the surface tension and the density of the
slag droplet and its liquid slag phase using the
numerical models developed by Mills et al. Fig. 2—FeMn alloy and slag in the graphite cup before the
experiments in the sessile drop furnace. Reprinted from Ref. [17]. METALLURGICAL AND MATERIALS TRANSACTIONS B METALLURGICAL AND MATERIALS TRANSACTIONS B Fig. 3—(a) Schematic cross section of the slag droplet on top of the FeMn alloy in the graphite cup before the experiments. (b) The force
balance at the contact point of Ar, slag and FeMn during the experiments. cFeMn is surface tension of FeMn, cslag is surface tension of slag,
cFeMnslag is interfacial tension between FeMn and slag. It should be observed that the slag droplet is small compared to the surface of FeMn,
ensuring that it does not come in contact with the graphite cup. Fig. 3—(a) Schematic cross section of the slag droplet on top of the FeMn alloy in the graphite cup before the experiments. (b) The force
balance at the contact point of Ar, slag and FeMn during the experiments. cFeMn is surface tension of FeMn, cslag is surface tension of slag,
cFeMnslag is interfacial tension between FeMn and slag. It should be observed that the slag droplet is small compared to the surface of FeMn,
ensuring that it does not come in contact with the graphite cup. results. In addition, the surface roughness due to the
formation of solid carbon and the amount of FeMn
layer filling the graphite cup are addressed. C. Methodology for Determination of Interfacial Tension The follow-
ing substeps are conducted at this step: (a) Determine the surface correction coefficient for each
experiment, kR, by direct measurement and averag-
ing of the size of particles at the interfaces. In this
study, kR was up to 0.7 mm, depending on the
surface roughness, which corresponds to 25 pct of
the typical droplet size. yp
p
(b) Calculate total volume of the slag droplet, Vtotal,
using slag density in the molten state and weight of
the slag droplet measured before the experiments: Vtotal ¼ mslag
qslag
;
½6 ½6 where mslag is the weight of the slag droplet. where mslag is the weight of the slag droplet. g
(c) Calculate visible volume of the slag droplet in the
experiments, Vexp
vis , assuming that the slag droplet
forms a spherical cap both above and below the
FeMn–gas interface and subtracting kR from visible
height and radius of the slag droplet: g
(c) Calculate visible volume of the slag droplet in the
experiments, Vexp
vis , assuming that the slag droplet
forms a spherical cap both above and below the
FeMn–gas interface and subtracting kR from visible
height and radius of the slag droplet: Vexp
vis ¼ 1
6 pðhexp
vis kRÞð3ðaexp kRÞ2 þ ðhexp
vis kRÞ2Þ; ½7 where hexp
vis is the visible height of the slag droplet and
aexp is the measured radius of the slag droplet. g
p
(d) Calculate non-visible volume of the slag droplet in
the experiments, Vexp
nonvis: Fig. 4—Schematic
describing
steps
for
the
determination
of
interfacial tension in the experiments based on the geometrical
parameters of the slag droplet. Vexp
nonvis ¼ Vexp
total Vexp
vis :
½8 ½8 (e) Calculate non-visible height of the slag droplet in the
experiments, hexp
nonvis, by expressing it from the
equation: (c) Calculate the average surface tension and the aver-
age density which can be found as the average be-
tween the slag droplet and its liquid phase. Vexp
nonvis ¼ 1
6 phexp
nonvisð3ðaexp kRÞ2 þ ðhexp
nonvisÞ2Þ:
½9 ½9 Then, a geometric analysis of the slag droplet in the
experiments is carried out to characterize the interfacial
interaction between the FeMn alloy and the slags and
measure geometrical parameters of the slag droplet. C. Methodology for Determination of Interfacial Tension The non-visible height in the simulation is then com-
pared to the non-visible height in the experiment, and if
they differ significantly, a new simulation is started. Here, the following substeps are performed: ½11 hadd ¼ Vcup VFeMn
pr2
cup
;
½12 ½12 (a) Perform three parallel simulations using the inter-
facial tension between the FeMn alloy and the slag
droplet of 0.30, 1.15 and 2.00 N/m. (a) Perform three parallel simulations using the inter-
facial tension between the FeMn alloy and the slag
droplet of 0.30, 1.15 and 2.00 N/m. where Vcup is the volume of the cup, rcup is the inner
radius of the cup (4 mm), hcup is the inner height of
the cup (3 mm), VFeMn is the volume of the FeMn
layer, mFeMn is the weight of the FeMn layer, qFeMn is
the density of FeMn in the molten state (5612 kg/
m3[23]), hadd is the distance from the FeMn–gas inter-
face to the edge of the graphite cup, correcting for the
cup not being 100 pct filled. A visual explanation of
all geometrical parameters is shown in Figure 5. (b) Knowing the apex position of the slag droplet on
Y-axis (Yslag apex) and the edge position of the gra-
phite cup on Y-axis (Yedge), visible height of the slag
droplet in simulations can be obtained: hsim
vis ¼ Yslag apex Yedge;
½15 ½15 hsim
vis ðpctÞ ¼
hsim
vis
hsim
droplet
100:
½16 (g) Considering hadd, calculate the non-visible height of
the slag droplet in the experiments (in pct): ½16 hexp
nonvis ðpctÞ ¼ hexp
nonvis hadd
hexp
nonvis þ hexp
vis
100:
½13 (c) Non-visible height is then expressed as: ½13 ½17 hsim
nonvis ðpctÞ ¼ 100 hsim
vis ðpctÞ:
½17 (h) Calculate the average non-visible height for each
experiment: Fig. 6—The slag droplet at the FeMn–Ar interface in the steady
state in the simulations. The semi-transparent blue filled area
represents the walls of the graphite cup with rcup of 4 mm and hcup
of 3 mm (Color figure online). h
exp
nonvis ðpctÞ ¼
P hexp
nonvisðpctÞ
n
;
½14 ½14 where n is the number of observations, considering
the minimum volume expansion of the slag droplet. C. Methodology for Determination of Interfacial Tension These are assessed after 2 minutes of holding time at the
minimum volume expansion of the slag droplet to
minimize the influence of volume fluctuations due to
chemical reactions and gas evolution on experimental Then, a geometric analysis of the slag droplet in the
experiments is carried out to characterize the interfacial
interaction between the FeMn alloy and the slags and
measure geometrical parameters of the slag droplet. Th
d f
2
i
f h ldi
i
h (f) The FeMn layer does not completely fill the graphite
cup, therefore, distance from the FeMn–gas interface
to the edge of the graphite cup has to be taken into
account as well. It can be calculated using volume of
the cup, density of the FeMn alloy in the molten
state and weight of the FeMn layer: (f) The FeMn layer does not completely fill the graphite
cup, therefore, distance from the FeMn–gas interface
to the edge of the graphite cup has to be taken into
account as well. It can be calculated using volume of
the cup, density of the FeMn alloy in the molten
state and weight of the FeMn layer: (f) The FeMn layer does not completely fill the graphite
cup, therefore, distance from the FeMn–gas interface
to the edge of the graphite cup has to be taken into
account as well. It can be calculated using volume of
the cup, density of the FeMn alloy in the molten
state and weight of the FeMn layer: METALLURGICAL AND MATERIALS TRANSACTIONS B Vcup ¼ pr2
cuphcup;
½10
VFeMn ¼ mFeMn
qFeMn
;
½11
hadd ¼ Vcup VFeMn
pr2
cup
;
½12 ½10 alloy and the slag droplet is simulated without walls of
the graphite cup. However, the geometrical features of
the slag droplet have to be calculated considering that a
part of the slag droplet cannot be seen due to the walls. The non-visible height in the simulation is then com-
pared to the non-visible height in the experiment, and if
they differ significantly, a new simulation is started. Here, the following substeps are performed: alloy and the slag droplet is simulated without walls of
the graphite cup. However, the geometrical features of
the slag droplet have to be calculated considering that a
part of the slag droplet cannot be seen due to the walls. C. Methodology for Determination of Interfacial Tension Furthermore, at the modelling step, simulations are
performed using the physical properties and parameters
from the experiments: surface tension (assumed to be a
constant value of 1.50 N/m as was calculated previ-
ously[24] by the authors for the FeMn alloy composition
considered in this study) and the density of the FeMn
alloy in the molten state; the average surface tension and
the average density of slag in the molten state; the
weight of the slag droplet and the FeMn layer. As shown
in Figure 6, the interfacial interaction between the FeMn Fig. 6—The slag droplet at the FeMn–Ar interface in the steady
state in the simulations. The semi-transparent blue filled area
represents the walls of the graphite cup with rcup of 4 mm and hcup
of 3 mm (Color figure online). Fig. 5—Geometrical parameters of the slag droplet (a) and the real position of the interface (b) used in the calculations. Note that the lines
shown in the figures are schematic only and may not represent actual measured geometrical parameters. Fig. 5—Geometrical parameters of the slag droplet (a) and the real position of the interface (b) used in the calculations. Note that the lines
shown in the figures are schematic only and may not represent actual measured geometrical parameters. METALLURGICAL AND MATERIALS TRANSACTIONS B Consequently, the average non-visible height of the
slag droplet in a steady state is calculated (in pct): Consequently, the average non-visible height of the
slag droplet in a steady state is calculated (in pct): reduced to the metal phase by Reaction (2). Note that
Al2O3 remains in the liquid slag phase and FeO
introduced into the slag by impurities in the raw
materials is completely reduced to the metal phase
through Reaction (3). h
sim
nonvis ðpctÞ ¼
P hsim
nonvis ðpctÞ
nt
;
½18 ½18 nt g
( )
The surface tension and density of the slags, as given
in Table III, were calculated considering the composi-
tion of the initial slag (Table I) and the liquid slag phase
calculated in FactSage. The surface tension does not
vary significantly between the initial slag and the liquid
slag phase; the maximum variation of 0.025 N/m (5.5
pct) was observed for slag D1. Higher values of the
surface tension at higher basicities are related to the
composition
change,
thereby
the
content
of
CaO
increases while SiO2 decreases. C. Methodology for Determination of Interfacial Tension CaO has a high surface
tension of 0.625 N/m in its pure form, whereas surface
tension of pure SiO2 (0.260 N/m) is substantially
lower.[20] In addition, slags without Al2O3 addition
have lower surface tension due to the low content of
Al2O3 which has a high surface tension of 0.655 N/m. However, the variation in the density between the initial
slag and the liquid slag phase is higher and reaches 301
kg/m3 or 9.6 pct for slag C3. The density for the initial
slags is almost constant regardless of the slag basicity,
but it increases when MnO and Al2O3 content in the
slags increases as they have a higher density of 5370 and
3965 kg/m3, respectively, in their pure form compared
with CaO (3340 kg/m3), MgO (3580 kg/m3) and SiO2
(2650 kg/m3).[26] For the liquid slag phase, the density
decreases with the slag basicity due to the significant
change in the composition of the liquid phase after
interacting with the FeMn alloy, which leads to a lower
MnO content. where nt is the number of time steps in the steady state
corresponding to the last 0.4 seconds of the simula-
tion. hsim
nonvis is calculated for each time step and the
simulation is stopped when two conditions are met:
simulation
time
is
higher
than
0.8
seconds
and
h
sim
nonvis ðpctÞ is almost constant (2 pct) during the last
0.4 seconds of the simulation, i.e., when the steady
state is reached. i (d) Linearly interpolate h
sim
nonvis ðpctÞ from the current
set of simulations as a function of the interfacial
tension and determine the corresponding value of
the interfacial tension to h
exp
nonvis. non vis
(e) Perform a simulation using the interpolated inter-
facial tension and calculate h
sim
nonvis ðpctÞ as de-
scribed in step (c). sim non vis
(e) Perform a simulation using the interpolated inter-
facial tension and calculate h
sim
nonvis ðpctÞ as de-
scribed in step (c). sim p ( )
(f) Repeat steps from (c) to (e) until h
sim
nonvis ðpctÞ is 1
pct from the experimental value (h
exp
nonvis). p ( )
(f) Repeat steps from (c) to (e) until h
sim
nonvis ðpctÞ is 1
pct from the experimental value (h
exp
nonvis). B. Effect of Slag Composition on Interfacial Tension The interfacial tension between the FeMn alloy and
the slags with and without Al2O3 addition is shown in
Figures 7 and 8, respectively. It can be seen from the
figures that the interfacial tension sharply increases with
the slag basicity for both groups of slags. Moreover, for
slags with Al2O3, the increase in interfacial tension is
more pronounceable at higher basicities. As such, the
average interfacial tension for the slags with Al2O3
addition is 0.21 to 0.40 N/m higher at the basicity of 0.8,
and 0.05 to 0.36 N/m higher for the basicity of 1.2. In
addition, the 80 pct confidence intervals overlap greatly
in each group of basicities due to the low number of
experiments in parallels; however, the measured values
of the interfacial tension fall within the proposed ranges
of the confidence intervals. Increasing MnO content in
slag from 30.0 to 45.0 wt pct does not show statistically
significant differences in the interfacial tension for all
slags. Here, it is important to note that due to the
fluctuations of the slag droplet during reaction with the
FeMn alloy, which can influence the experimental
results, the non-visible height and, accordingly, the
interfacial tension were measured only at local mini-
mum expansion of the slag droplet as shown in
Figure 9. s ¼
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
Pn
i¼1ðyi yÞ2
n 1
s
;
½20 ½20 where yi
y1; y2; . . . ; yn
f
g represents one measured value
of the sample (i.e., interfacial tension in a single
experiment). III.
RESULTS AND DISCUSSION A. Surface Tension and Density of Slags C. Methodology for Determination of Interfacial Tension non vis
The 80 pct confidence interval was calculated for each
set
of
three
experiments
based
on
the
Student’s
t-distribution[25]: CI ¼ y t sffiffiffin
p ;
½19 ½19 where y is the sample mean, t is the critical value
found from the confidence level and degrees of free-
dom of the sample (1.886 for a two-tailed test with a
statistical significance of 0.2 and two degrees of free-
dom), s is the sample standard deviation and n is the
number of experiments in a set. Here, s is expressed
as: A. Surface Tension and Density of Slags The composition of liquid slag phase of the slags after
interaction with the FeMn alloy at 1673 K was
calculated in FactSage and is shown in Table II. As
can be seen from this table, the composition of the liquid
slag changes after reacting with FeMn, which can be
partially attributed to the reduction of MnO to Mn by
Reaction (1) and mainly due to the formation of solid
MnO–MgO–CaO phase resulting in a lower MnO and
MgO content in the liquid slag when the basicity
increases. In addition, there is no solid slag phase at
low slag basicity of 0.4 and SiO2 in the slag is partially METALLURGICAL AND MATERIALS TRANSACTIONS B Table II. The Chemical Composition of the Liquid Slag Phase After Interaction with the FeMn Alloy Calculated in FactSage
Liquid Phase Corresponding to
Chemical Composition (Wt Pct)
MnO
CaO
MgO
SiO2
Al2O3
Slags with Al2O3 Addition
Slag A1
34.76
14.05
6.68
34.41
10.10
Slag A2
26.60
26.22
6.95
29.72
10.46
Slag A3
21.15
36.27
4.92
25.57
12.11
Slag B1
38.15
12.96
6.77
31.87
10.25
Slag B2
31.42
24.09
6.68
27.02
10.79
Slag B3
22.82
35.91
3.92
24.26
13.09
Slag C1
42.75
11.11
6.82
28.75
10.56
Slag C2
33.28
23.20
5.52
25.53
12.46
Slag C3
24.77
34.36
3.33
22.70
14.83
Slags Without Al2O3 Addition
Slag D1
42.66
13.56
5.60
37.55
0.62
Slag D2
31.69
25.55
6.37
35.61
0.78
Slag D3
26.18
33.26
6.51
33.39
0.65
Slag E1
44.43
12.76
6.01
36.22
0.58
Slag E2
35.92
23.33
6.34
33.70
0.71
Slag E3
29.87
31.51
6.14
31.72
0.76
Slag F1
48.34
10.65
6.28
34.03
0.69
Slag F2
40.96
20.64
6.41
31.41
0.59
Slag F3
31.82
30.69
5.47
31.42
0.59 Table II. The Chemical Composition of the Liquid Slag Phase After Interaction with the FeMn Alloy Calculated in FactSage Table II. The Chemical Composition of the Liquid Slag Phase After Interaction with the FeMn Alloy Table III. A. Surface Tension and Density of Slags The Calculated Surface Tension and Density of the Initial Slag and the Liquid Slag Phase
Slag
Surface Tension (N/m)
Density (kg/m3)
Initial Slag
Liquid Phase
Average
Initial Slag
Liquid Phase
Average
Slags with Al2O3 Addition
A1
0.460
0.472
0.466
3138
3223
3181
A2
0.502
0.501
0.502
3170
3126
3148
A3
0.529
0.521
0.525
3187
3076
3132
B1
0.474
0.485
0.479
3270
3313
3292
B2
0.510
0.506
0.508
3292
3209
3251
B3
0.536
0.524
0.530
3318
3108
3213
C1
0.486
0.486
0.486
3414
3376
3395
C2
0.519
0.508
0.514
3426
3247
3337
C3
0.541
0.527
0.534
3446
3145
3296
Slags Without Al2O3 Addition
D1
0.429
0.454
0.442
3025
3266
3146
D2
0.470
0.476
0.473
3083
3115
3099
D3
0.493
0.491
0.492
3103
3050
3077
E1
0.441
0.458
0.450
3169
3298
3234
E2
0.477
0.478
0.478
3201
3185
3193
E3
0.499
0.493
0.496
3229
3110
3170
F1
0.453
0.462
0.458
3311
3375
3343
F2
0.484
0.482
0.483
3327
3270
3299
F3
0.505
0.492
0.499
3360
3139
3250 Table III. The Calculated Surface Tension and Density of the Initial Slag and the Liquid Slag Phase Previous studies[5,8,27] investigated the mechanisms of
the interfacial interaction between molten Fe and
CaO–SiO2–Al2O3 slag, where it was suggested that the
interfacial tension between slag and metal changes due
to reduction or oxidation reactions in molten Fe
containing Al (Eq. [21]) or decomposition and dissolu-
tion of SiO2 into molten Fe without Al (Eq. [22]). The
interfacial tension rapidly decreases due to the proposed reactions, and then gradually increases before reaching a
constant value at the equilibrium between slag and
metal. As shown in Figure 9, the similar behaviour can
be observed in the current study, when the visible
surface area of slag B1 and B2 sharply changes in the
beginning of the holding period (0 to 1.5 minutes) and
then remains practically constant. This may indicate
that the dynamic interfacial oxygen potential and the METALLURGICAL AND MATERIALS TRANSACTIONS B Fig. 7—The interfacial tension between the FeMn alloy and the slag with the addition of Al2O3 at different slag basicities and MnO content at
1673 K. The top part of the bars corresponds to the average interfacial tension between the FeMn alloy and the slag. The grey lines on top of
bars represent the 80 pct confidence interval, and the circle markers represent the experimental measurements. Fig. A. Surface Tension and Density of Slags 7—The interfacial tension between the FeMn alloy and the slag with the addition of Al2O3 at different slag basicities and MnO content at
1673 K. The top part of the bars corresponds to the average interfacial tension between the FeMn alloy and the slag. The grey lines on top of
bars represent the 80 pct confidence interval, and the circle markers represent the experimental measurements. Fig. 8—The interfacial tension between the FeMn alloy and the slag without the addition of Al2O3 at different slag basicities and MnO content
at 1673 K. The top part of the bars corresponds to the average interfacial tension between the FeMn alloy and the slag. The grey lines on top of
bars represent the 80 pct confidence interval, and the circle markers represent the experimental measurements. Fig. 8—The interfacial tension between the FeMn alloy and the slag without the addition of Al2O3 at different slag basicities and MnO content
at 1673 K. The top part of the bars corresponds to the average interfacial tension between the FeMn alloy and the slag. The grey lines on top of
bars represent the 80 pct confidence interval, and the circle markers represent the experimental measurements. mass transfer rate across the interface decrease as
reactions between slag and metal proceed as previously
was found by Gu et al.[28] content and therefore oxygen activity in molten Fe,
which causes an increase of the interfacial tension.[6,7]
As such, the addition of CaO and Al2O3 to the slag
slightly increases the interfacial tension, while the addi-
tion of MnO and FeO substantially decreases it. How-
ever, it was pointed out that the interfacial tension,
depending on MnO content, decreases exponentially,
reaching a steady value at high MnO content (after
adding 20 mol pct MnO). As it is shown in the present
results, the interfacial tension does not change with the
MnO content, meaning that a steady value has been
reached, after which the interfacial tension does not
change after the addition of MnO. The Al2O3 addition
and the slag basicity also show a similar effect on the
interfacial tension, similar to what was reported in the
literature. 4½Al þ 3ðSiO2Þ ! 2ðAl2O3Þ þ 3½Si;
½21 ½21 ðSiO2Þ ! A. Surface Tension and Density of Slags ½Si þ 2½O:
½22 ½22 Nakashima and Mori[11] studied interfacial interaction
between
molten
Fe
and
CaO–SiO2–Al2O3,
and
CaO–MgO–Al2O3 slags, where it was concluded that
the interfacial tension can be greatly modified due to
the change of slag composition. Several research-
ers[10,29–31] have shown that a decrease of SiO2 in slag
with
increasing
CaO/SiO2
ratio
decreases
oxygen Nakashima and Mori[11] studied interfacial interaction
between
molten
Fe
and
CaO–SiO2–Al2O3,
and
CaO–MgO–Al2O3 slags, where it was concluded that
the interfacial tension can be greatly modified due to
the change of slag composition. Several research-
ers[10,29–31] have shown that a decrease of SiO2 in slag
with
increasing
CaO/SiO2
ratio
decreases
oxygen METALLURGICAL AND MATERIALS TRANSACTIONS B Interfacial interaction of the slag droplet with the
FeMn alloy in the graphite cup depending on different
slag composition is shown in Figures 10, 11 and 12. The
non-visible height of the slag droplet with MnO content
of 30 wt pct decreases with the basicity (Figure 10),
which means that the droplet tends to stay at the interface and leads to better separation of slag and
metal. Furthermore, the angle between the tangent of
the droplet and the top of the crucible hscr increases
from 73.8 to 90.4 deg, indicating a reduced contact area
between slag and metal owing to increased interfacial
tension between these phases. Fig. 9—Temporal change in visible surface area for slags B1–B3 during holding at 1673 K. The grey cross markers correspond to the local
minimum expansion of the slag droplet in the experiments. Note that interfacial tension was measured only after 2 minutes of holding time as
indicated with the vertical dashed line. Fig. 9—Temporal change in visible surface area for slags B1–B3 during holding at 1673 K. The grey cross markers correspond to the local
minimum expansion of the slag droplet in the experiments. Note that interfacial tension was measured only after 2 minutes of holding time as
indicated with the vertical dashed line. Fig. 10—Slag droplets with various basicity and constant MnO content of 30 wt pct during interaction with the FeMn alloy at 1673 K: (a) slag
A1, (b) slag A2, and (c) slag A3. The droplet contour and the real position of the interface are shown by the solid and the dashed line,
respectively. Fig. A. Surface Tension and Density of Slags 10—Slag droplets with various basicity and constant MnO content of 30 wt pct during interaction with the FeMn alloy at 1673 K: (a) slag
A1, (b) slag A2, and (c) slag A3. The droplet contour and the real position of the interface are shown by the solid and the dashed line,
respectively. METALLURGICAL AND MATERIALS TRANSACTIONS B METALLURGICAL AND MATERIALS TRANSACTIONS B METALLURGICAL AND MATERIALS TRANSACTIONS B Fig. 11—Slag droplets with constant basicity of 0.8 and various MnO content during interaction with the FeMn alloy at 1673 K: (a) slag A2, (b)
slag B2, and (c) slag C2. The droplet contour and the real position of the interface are shown by the solid and the dashed line, respectively. Fig. 11—Slag droplets with constant basicity of 0.8 and various MnO content during interaction with the FeMn alloy at 1673 K: (a) slag A2, (b)
slag B2, and (c) slag C2. The droplet contour and the real position of the interface are shown by the solid and the dashed line, respectively. Fig. 11—Slag droplets with constant basicity of 0.8 and various MnO content during interaction with the FeMn alloy at 1673 K: (a) slag A2, (b)
slag B2, and (c) slag C2. The droplet contour and the real position of the interface are shown by the solid and the dashed line, respectively. Fig. 11—Slag droplets with constant basicity of 0.8 and various MnO content during interaction with the FeMn alloy at 1673 K: (a) slag A2, (b)
slag B2, and (c) slag C2. The droplet contour and the real position of the interface are shown by the solid and the dashed line, respectively. Fig. 12—Slag droplets with various basicity and constant MnO content of 37.5 wt pct during interaction with the FeMn alloy at 1673 K. Images
(a) and (c) correspond to slags B1 and B2 (with Al2O3 addition); images (b) and (d) show slags E1 and E2 (without Al2O3 addition). The droplet
contour and the real position of the interface are shown by the solid and the dashed line, respectively. Fig. 11—Slag droplets with constant basicity of 0.8 and various MnO content during interaction with the FeMn alloy at 1673 K: (a) slag A2, (b)
slag B2, and (c) slag C2. The droplet contour and the real position of the interface are shown by the solid and the dashed line, respectively. Fig. A. Surface Tension and Density of Slags 12—Slag droplets with various basicity and constant MnO content of 37.5 wt pct during interaction with the FeMn alloy at 1673 K. Images
(a) and (c) correspond to slags B1 and B2 (with Al2O3 addition); images (b) and (d) show slags E1 and E2 (without Al2O3 addition). The droplet
contour and the real position of the interface are shown by the solid and the dashed line, respectively. Fig. 12—Slag droplets with various basicity and constant MnO content of 37.5 wt pct during interaction with the FeMn alloy at 1673 K. Images
(a) and (c) correspond to slags B1 and B2 (with Al2O3 addition); images (b) and (d) show slags E1 and E2 (without Al2O3 addition). The droplet
contour and the real position of the interface are shown by the solid and the dashed line, respectively. METALLURGICAL AND MATERIALS TRANSACTIONS B is disturbed and the interfacial area increases due to the
chemicalreactionsbetweentheslagandmetal.Thechemical
composition of slagand metal phases(Tables VIII and IX in
‘‘AppendixB’’)confirmsthatinterfacial tensiondecreasesat
low slag basicity due to chemical reactions between slag and
FeMn alloy and mass transfer of Si to the metal phase. As
such, Si can be found in the metal phases after interaction
with the low basicity slags at high temperatures, despite the
fact that Si was not originally added to the FeMn alloy,
which indicates that SiO2 in the slag is reduced through
Reaction (2). In addition, FeMn droplets were observed on
top of the slag phase (Figures 13(a) and 14(a)), specifying
that the slag of low basicity and the FeMn alloy form a
metal–slag emulsion during the reactions, resulting in their
poor separation. As illustrated in Figure 11, the variation in MnO
content in the slag from 30.0 to 45.0 wt pct at basicity of
0.8 shows that both the non-visible height of the slag
droplet and the angle between slag and the top of the
crucible are nearly constant which results in comparable
values of the interfacial tension for all MnO contents. A. Surface Tension and Density of Slags Similarly, the addition of Al2O3 to the low basicity slag
(Figures 12(a) and (b)) does not show any influence on
the non-visible height and the angle hscr, while the
effect of alumina addition is more obvious at a higher
slag basicity of 0.8 (Figures 12(c) and (d)) as the angle
hscr decreases and the non-visible height increases
which leads to lower values of the interfacial tension
and therefore worse separation of slag and metal. Chung and Cramb[12] suggested that the emulsifica-
tion phenomena are closely related to the interaction of
fluids at the interface and that the driving force for fluid
flow across the interface depends on reactions in the
system under isothermal conditions due to the concen-
tration gradient at the interface. In case of the concen-
tration gradient and the fluid flow driven by the C. EPMA Results A general view of the FeMn alloy and slags with and
without Al2O3 addition after the experiments in the sessile
drop furnace is shown in Figures 13 and 14, respectively. As
observed for the slags with low basicities (0.4 and 0.8), the
slagpenetratesthe FeMnphase, indicatingthat theinterface Fig. 13—BSE images of the FeMn alloy and slags with Al2O3 addition after the experiments: (a) slag B1 (basicity = 0.4), (b) slag B2 (basicity
= 0.8), and (c) slag B3 (basicity = 1.2). Fig. 13—BSE images of the FeMn alloy and slags with Al2O3 addition after the experiments: (a) slag B1 (bas
= 0.8), and (c) slag B3 (basicity = 1.2). Fig. 13—BSE images of the FeMn alloy and slags with Al2O3 addition after the experiments: (a) slag B1 (basicity = 0.4), (b) slag B2 (basicity
= 0.8), and (c) slag B3 (basicity = 1.2). Fig. 14—BSE images of the FeMn alloy and slags without Al2O3 addition after the experiments: (a) slag E1 (basicity = 0.4), (b) slag E2
(basicity = 0.8), and (c) slag E3 (basicity = 1.2). Fig. 14—BSE images of the FeMn alloy and slags without Al2O3 addition after the experiments: (a) slag E1 (basicity = 0.4), (b) slag E2
(basicity = 0.8), and (c) slag E3 (basicity = 1.2). FeMn alloy and slags without Al2O3 addition after the experiments: (a) slag E1 (basicity = 0.4), (b) slag E2
E3 (basicity = 1.2). METALLURGICAL AND MATERIALS TRANSACTIONS B interfacial reaction, the interface eventually becomes
unstable and the metal–slag interfacial area increases
(the Kelvin–Helmholtz interfacial instability). Based on
the Kelvin–Helmholtz model, Gopal[3] discussed the
mechanism of small droplet formation and emulsifica-
tion of slag and metal during reactions between them. If
the mass transfer rate of elements or compounds across
the interface is low, it results in a slight destabilization of
the interface (Figure 15(a)), corresponding to the high
slag basicity of 1.2 in this study. At higher mass transfer
rates, the destabilization of the interface is more severe
as shown in Figures 15(b) and (c), which results in the formation of the metal–slag emulsion and droplets of
one phase into another as it was observed for the low
slag basicities from 0.4 to 0.8. Solid carbon particles in the form of graphite whiskers
have been observed on top of FeMn layer and along the
walls of the graphite cup in all experiments as illustrated in
Figure 16. C. EPMA Results The formation of the solid carbon particles
may be attributed to different temperatures used for the
materials preparation and the experiments.[24] Bao
et al.[32] also confirmed this finding and showed that solid
carbon particles can form extensively when non-carbon
saturated FeMn alloy reacts with slag and a graphite
substrate, especially if CO is present in the system. In
addition, carbon is consumed from the FeMn alloy due to
the partial reduction of MnO, SiO2 and FeO by Reactions
(1), (2) and (3), therefore this creates an additional driving
force for the FeMn alloy to dissolve carbon from the
graphite cup, which causes the molten FeMn alloy to
penetrate the walls of the graphite cup. Fig. 15—Emulsification of slag and metal and formation of small
droplets due to the Kelvin–Helmholtz instability. Images (a) through
(c) represent disturbances of the interface between slag and metal at
different slag basicities. CONFLICT OF INTEREST On behalf of all authors, the corresponding author
states that there is no conflict of interest. Since the study was limited to using only graphite
crucibles for the materials preparation and graphite cups
for the melting experiments in the sessile drop furnace,
solid carbon particles were observed on the surface of
the slag and the FeMn alloy in the experiments, which
may introduce additional uncertainty to the experimen-
tal results. However, the surface roughness correction
coefficient has been used in this study to address this
issue. IV.
CONCLUSIONS In the present study, the interaction between FeMn
alloy and slags of different compositions has been
studied by comparing experimental results from a sessile
drop furnace and modelling results from multiphase
flow simulations in OpenFOAM. p
The current results show that the interfacial tension
between FeMn alloy and slag increases when the slag
basicity changes from 0.4 to 1.2. It was also found that
the addition of Al2O3 to the slag with basicity of 0.8 and Fig. 15—Emulsification of slag and metal and formation of small
droplets due to the Kelvin–Helmholtz instability. Images (a) through
(c) represent disturbances of the interface between slag and metal at
different slag basicities. Fig. 16—FeMn alloy inclusions in the graphite cup and graphite whiskers formed along the walls of the graphite cup. . 16—FeMn alloy inclusions in the graphite cup and graphite whiskers formed along the walls of the graphite cup. METALLURGICAL AND MATERIALS TRANSACTIONS B ACKNOWLEDGMENTS 1.2 increases the interfacial tension, while increasing
MnO content from 30.0 to 45.0 wt pct does not have any
statistically
significant
influence
on
the
interfacial
tension. 1.2 increases the interfacial tension, while increasing
MnO content from 30.0 to 45.0 wt pct does not have any
statistically
significant
influence
on
the
interfacial
tension. Open access funding provided by NTNU Norwegian
University of Science and Technology (incl St. Olavs
Hospital - Trondheim University Hospital). This publi-
cation has been funded by the Research Council of Nor-
way (KPN Project, 267621). The authors gratefully
acknowledge the financial support from the Research
Council of Norway and the Norwegian Ferroalloy Pro-
ducers Research Association (FFF). EPMA analysis demonstrates an important phe-
nomenon which contributes to the understanding of
interfacial tension drop at low slag basicity. SiO2 in the
slag is reduced by the saturated carbon from the FeMn
alloy at the interface and afterwards Si distributes to the
metal phase. The mass transfer of Si across the interface
creates the interface instability due to the growing
interfacial area between slag and FeMn, which leads to
the formation of a metal–slag emulsion and small
droplets and, thus, to an increase in losses of the FeMn
alloy with the slag. OPEN ACCESS This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use,
sharing, adaptation, distribution and reproduction in any
medium or format, as long as you give appropriate credit
to the original author(s) and the source, provide a link to
the Creative Commons licence, and indicate if changes
were made. The images or other third party material in
this article are included in the article’s Creative Com-
mons licence, unless indicated otherwise in a credit line to
the material. If material is not included in the article’s
Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permit-
ted use, you will need to obtain permission directly from
the copyright holder. To view a copy of this licence, visit
http://creativecommons.org/licenses/by/4.0/. Further experiments, using a broader range of MnO
content in the slag, could provide more data on the
effect of MnO influence on the interfacial tension
between slag and metal, which might be crucial for the
ferroalloy industry. In addition, experiments in the
sessile drop furnace with cups made of different mate-
rials can be a further development of the current
methodology. APPENDIX A Normalized Values of the Measured Chemical Composition of the Synthetic FeMn Alloy and Slags
Material
Chemical Composition (Wt Pct)
Mn
Fe
Si
C
MnO
CaO
MgO
SiO2
Al2O3
FeO
Total
Basicity
FeMn Alloy
77.75
14.58
0.11
7.56
—
—
—
—
—
—
100.00
—
Slags with Al2O3 Addition
Slag A1
—
—
—
—
29.94
13.92
6.62
38.65
10.00
0.87
100.00
0.42
Slag A2
—
—
—
—
28.81
25.08
6.64
28.70
10.00
0.78
100.00
0.82
Slag A3
—
—
—
—
28.72
31.79
6.60
21.99
10.12
0.78
100.00
1.20
Slag B1
—
—
—
—
36.94
12.55
6.55
33.29
9.92
0.75
100.00
0.44
Slag B2
—
—
—
—
36.09
22.08
6.46
24.74
9.89
0.73
100.00
0.82
Slag B3
—
—
—
—
37.04
27.51
6.74
18.09
9.88
0.73
100.00
1.22
Slag C1
—
—
—
—
44.28
10.49
6.44
28.11
9.97
0.71
100.00
0.44
Slag C2
—
—
—
—
43.67
18.62
6.68
20.34
9.97
0.71
100.00
0.83
Slag C3
—
—
—
—
44.60
23.50
6.58
14.78
9.83
0.71
100.00
1.22
Slags Without Al2O3 Addition
Slag D1
—
—
—
—
30.09
14.48
5.99
47.91
0.66
0.87
100.00
0.42
Slag D2
—
—
—
—
29.42
24.96
6.22
37.80
0.76
0.84
100.00
0.81
Slag D3
—
—
—
—
28.98
31.59
6.12
31.91
0.60
0.79
100.00
1.16
Slag E1
—
—
—
—
37.78
12.63
5.95
42.32
0.57
0.75
100.00
0.43
Slag E2
—
—
—
—
36.39
22.31
6.06
33.82
0.68
0.74
100.00
0.82
Slag E3
—
—
—
—
36.81
27.85
6.03
28.03
0.67
0.60
100.00
1.18
Slag F1
—
—
—
—
44.94
10.34
6.09
37.26
0.67
0.69
100.00
0.43
Slag F2
—
—
—
—
43.53
19.29
5.99
29.98
0.55
0.67
100.00
0.83
Slag F3
—
—
—
—
44.42
24.03
5.89
24.51
0.46
0.69
100.00
1.20 Normalized Values of the Measured Chemical Composition of the Synthetic FeMn Alloy and Slags Table VI. Normalized Values of the Measured Chemical Composition of the Syntheti ues of the Measured Chemical Composition of the Synthetic FeMn Alloy and Slags Table VII. APPENDIX A Table IV. The Calculated Chemical Composition of the Synthetic FeMn Alloy and Slags
Material
Chemical Composition (Wt Pct)
Mn
Fe
C
MnO
CaO
MgO
SiO2
Al2O3
Basicity
FeMn Alloy
78.00
15.00
7.00
—
—
—
—
—
—
Slags with Al2O3 Addition
Master Slag #1
—
—
—
38.00
23.00
6.00
23.00
10.00
0.9
Slag A1
—
—
—
30.00
14.00
6.00
40.00
10.00
0.4
Slag A2
—
—
—
30.00
25.11
6.00
28.89
10.00
0.8
Slag A3
—
—
—
30.00
32.18
6.00
21.82
10.00
1.2
Slag B1
—
—
—
37.50
11.86
6.00
34.64
10.00
0.4
Slag B2
—
—
—
37.50
21.78
6.00
24.72
10.00
0.8
Slag B3
—
—
—
37.50
28.09
6.00
18.41
10.00
1.2
Slag C1
—
—
—
45.00
9.71
6.00
29.29
10.00
0.4
Slag C2
—
—
—
45.00
18.44
6.00
20.56
10.00
0.8
Slag C3
—
—
—
45.00
24.00
6.00
15.00
10.00
1.2
Slags Without Al2O3 Addition
Master Slag #2
—
—
—
42.52
25.74
6.00
25.74
—
1.2
Slag D1
—
—
—
30.00
14.00
6.00
50.00
—
0.4
Slag D2
—
—
—
30.00
25.11
6.00
38.89
—
0.8
Slag D3
—
—
—
30.00
32.18
6.00
31.82
—
1.2
Slag E1
—
—
—
37.50
11.86
6.00
44.64
—
0.4
Slag E2
—
—
—
37.50
21.78
6.00
34.72
—
0.8
Slag E3
—
—
—
37.50
28.09
6.00
28.41
—
1.2
Slag F1
—
—
—
45.00
9.71
6.00
39.29
—
0.4
Slag F2
—
—
—
45.00
18.44
6.00
30.56
—
0.8
Slag F3
—
—
—
45.00
24.00
6.00
25.00
—
1.2 The Calculated Chemical Composition of the Synthetic FeMn Alloy and Slags METALLURGICAL AND MATERIALS TRANSACTIONS B Table V. The Purity of the Powders Used for the Preparation of the Synthetic Raw Materials
Purity of Corresponding Powders (Pct)
Mn
Fe
MnO
CaO
MgO
SiO2
Al2O3
99.30
99.00
99.00
95.00
99.00
99.50
99.00 Table V. The Purity of the Powders Used for the Preparation of the Synthetic Raw Materials Table VI. APPENDIX A Weight of the Powders Used for the Preparation of the Synthetic Raw Materials
Material
Weight of Powders (g)
Mn
Fe
MnO
CaO
MgO
SiO2
Al2O3
Total
FeMn
211.16
40.73
—
—
—
—
—
251.89
Slags with Al2O3 Addition
Master Slag #1
—
—
95.00
57.50
15.00
57.50
25.00
250.00
Powders Addition to Master Slag #1, g
Slag A1
—
—
2.28
—
0.78
8.59
1.30
12.95
Slag A2
—
—
—
1.85
0.32
2.73
0.54
5.44
Slag A3
—
—
—
3.74
0.32
0.93
0.54
5.53
Slag B1
—
—
7.02
—
1.14
8.88
1.90
18.94
Slag B2
—
—
0.32
—
0.07
0.63
0.11
1.13
Slag B3
—
—
1.79
2.55
0.30
—
0.50
5.14
Slag C1
—
—
13.85
—
1.66
9.31
2.76
27.58
Slag C2
—
—
3.66
—
0.30
0.53
0.50
4.99
Slag C3
—
—
6.26
2.91
0.65
—
1.08
10.90
Slags Without Al2O3 Addition
Master Slag #2
—
—
106
64
15
64
—
250.00
Powders Addition to Master Slag #2, g
Slag D1
—
—
3.47
0.58
1.02
13.85
—
18.92
Slag D2
—
—
0.91
2.65
0.51
6.46
—
10.53
Slag D3
—
—
0.91
4.76
0.51
4.44
—
10.62
Slag E1
—
—
8.77
0.58
1.42
14.86
—
25.63 Table VII. Weight of the Powders Used for the Preparation of the Synthetic Raw Materials METALLURGICAL AND MATERIALS TRANSACTIONS B Table VII. continued Material
Weight of Powders (g)
Mn
Fe
MnO
CaO
MgO
SiO2
Al2O3
Total
Slag E2
—
—
1.28
0.58
0.22
3.63
—
5.71
Slag E3
—
—
0.91
1.86
0.16
1.85
—
4.78
Slag F1
—
—
16.41
0.58
2.00
16.30
—
35.29
Slag F2
—
—
5.01
0.58
0.48
3.95
—
10.02
Slag F3
—
—
2.07
0.58
0.09
0.77
—
3.51 See Tables IV, V, VI and VII. See Tables IV, V, VI and VII. Table VIII. APPENDIX A Average Chemical Composition (in Wt Pct) of Slag Phases after the Experiments in the Sessile Drop Furnace
Slag
Matrix*
Dendrites*
MnO
CaO
MgO
SiO2
Al2O3
FeO
Total
Bulk
B1
100
—
28.85
16.23
8.01
33.35
13.35
0.09
99.89
B2
100
—
27.30
25.78
7.36
27.01
12.72
0.06
100.21
B3
82
18
30.20
30.36
6.43
19.77
13.11
0.08
99.95
E1
70/30**
—
40.86
13.58
8.44
36.54
0.46
0.10
99.98
E2
87/13**
—
34.79
22.51
7.15
34.36
0.54
0.10
99.45
E3
87
13
33.65
28.13
7.97
29.53
0.05
0.09
99.43
Slag Inclusions in the Metal Phase
B1
100
—
32.41
14.88
7.59
32.87
12.21
0.15
100.11
B2
100
—
27.94
25.38
7.24
27.02
13.02
0.40
101.00
B3
—
—
—
—
—
—
—
—
—
E1
70/30**
—
37.94
13.84
9.95
37.41
0.64
0.18
99.96
E2
87/13**
—
35.26
22.52
6.91
33.17
0.77
0.20
98.82
E3
—
—
—
—
—
—
—
—
—
Percentage from the total surface area of the slag phase. Matrix consists of two distinctive phases: bright and grey. al Composition (in Wt Pct) of Slag Phases after the Experiments in the Sessile Drop Furnace Table VIII. Average Chemical Composition (in Wt Pct) of Slag Phases after the Experiments i Percentage from the total surface area of the slag phase. Matrix consists of two distinctive phases: bright and grey. Table IX. Average Chemical Composition (in Wt Pct) of the FeMn Alloy After the Experiments in the Sessile Drop Furnace
Slag in experiment
Phase
Mn
Fe
Si
C
Total
Bulk
B1
Bright
75.90
19.23
0.57
5.07
100.78
Grey
79.23
13.61
0.00
6.12
98.96
Dark
79.46
13.69
2.78
5.18
101.11
B2
Bright
75.61
19.57
0.12
4.69
99.98
Dark
79.39
13.53
0.00
5.87
98.79
B3
Bright
73.79
22.66
0.10
5.19
101.75
Grey
75.74
19.15
0.00
6.19
101.08
Dark
78.78
14.30
0.00
6.96
100.04
E1
Bright
67.95
26.10
3.95
4.59
102.58
Grey
72.79
21.00
1.28
5.73
100.80
Dark
79.08
14.66
0.00
5.80
99.55
E2
Grey
79.96
13.76
0.01
6.49
100.23
E3
Bright
72.51
27.54
0.32
4.11
104.48
Grey
77.66
17.54
0.00
5.62
100.82
Dark
80.19
13.63
0.00
6.08
99.89
Near the Interface
B1
Bright
72.36
22.12
2.32
4.43
101.22
Dark
80.25
13.35
0.01
6.22
99.83 omposition (in Wt Pct) of the FeMn Alloy After the Experiments in the Sessile Drop Furnace Table IX. APPENDIX A Average Chemical Composition (in Wt Pct) of the FeMn Alloy After the Experiments in t METALLURGICAL AND MATERIALS TRANSACTIONS B METALLURGICAL AND MATERIALS TRANSACTIONS B Table IX. continued Slag in experiment
Phase
Mn
Fe
Si
C
Total
B2
Bright
75.57
23.56
0.13
2.98
102.24
Grey
79.59
15.31
0.00
5.16
100.06
Dark
79.74
13.81
0.00
5.75
99.30
B3
Bright
74.52
23.03
0.03
4.64
102.22
Grey
76.77
17.88
0.00
6.22
100.87
Dark
79.40
13.85
0.00
6.83
100.08
E1
Bright
73.13
20.09
4.50
4.87
102.58
Dark
81.68
11.76
0.03
5.92
99.39
E2
Bright
77.25
17.81
0.01
6.00
101.07
Grey
79.71
14.15
0.00
6.63
100.50
E3
Bright
74.56
23.87
0.19
3.80
102.42
Grey
76.59
19.20
0.02
5.76
101.56
Dark
79.89
13.41
0.00
6.42
99.72
FeMn Inclusions in the Graphite Cup
B1
Bright
77.17
14.82
0.02
8.08
100.08
B2
Bright
75.93
14.81
0.00
7.67
98.42
B3
Bright
76.42
14.18
0.00
8.99
99.60
E1
Bright
77.32
13.83
0.00
7.32
98.48
E2
Bright
76.79
14.89
0.00
8.10
99.79
E3
Bright
73.84
14.60
0.03
9.48
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Medicina veterinária e a saúde única: ação socioeducativa aplicada a
idosos no contexto da COVID-19 Ana Laura Martins Ferreira1, Raquelline Figueiredo Braz2, Mariana Comassio Chueiri1, Deborah Araujo
Policarpo1, Andrezza Brigato Siqueira1, Denise Ramos Pacheco1, Roberta Torres de Melo3 Originais recebidos em
11 de junho de 2020
Aceito para publicação em
1 ° de dezembro de 2020 Resumo: A saúde única integra as esferas da saúde humana, ambiental e animal, por meio de
políticas públicas focadas na prevenção e controle de enfermidades infecciosas. O médico
veterinário é um profissional atuante na área da saúde pública pelo contato direto com as três
esferas, e uma das principais ações desse profissional inclui a promoção de medidas comunitárias
educativas para disseminar informações em relação à prevenção de doenças zoonóticas. Diante
da atual pandemia, desenvolvemos o projeto: “Efeito borboleta aplicado a asilos de Uberlândia-
MG no combate à COVID-19” com o objetivo de divulgar informações sobre a doença de maneira
lúdica, e atender as demandas das instituições envolvidas. Foram produzidos e distribuídos dez
materiais didáticos como vídeos e jogos interativos com orientações em relação à COVID-19 em
16 abrigos de idosos, junto à arrecadação de produtos de limpeza e higiene pessoal, máscaras e
gêneros alimentícios, para enfatizar as medidas preventivas, provenientes de doações de 23
empresas. Funcionários das instituições desenvolveram as atividades propostas com os idosos,
ressaltando a importância do distanciamento social e da higiene pessoal no momento atual. A ação
executada permitiu a ampliação do conhecimento a respeito da pandemia, em virtude do uso de
metodologias aplicadas às dificuldades e necessidades dos idosos, aliada à motivação emocional
utilizada na proposta, o que favoreceu o processo ensino-aprendizagem. A presente ação social
nas instituições auxiliou na conscientização e esclarecimento de dúvidas em relação à COVID-19
ao principal grupo de risco, e reforçou o contexto de atuação do médico veterinário na saúde
única. 1
Acadêmica do curso de
Medicina Veterinária na
Universidade Federal de
Uberlândia, Minas Gerais,
Brasil. 1
Acadêmica do curso de
Medicina Veterinária na
Universidade Federal de
Uberlândia, Minas Gerais,
Brasil. Mestranda do Programa de
Pós-Graduação em Ciências
Veterinárias (PPGCV) na
Universidade Federal de
Uberlândia, Minas Gerais, Brasil Mestranda do Programa de
Pós-Graduação em Ciências
Veterinárias (PPGCV) na
Universidade Federal de
Uberlândia, Minas Gerais, Brasil Palavras-chave: Educação; Epidemia; Grupo vulnerável; Promoção de saúde Revista Brasileira de Extensão Universitária v. 11, n. 3, p. 429-438, set.–dez. 2020 e-ISSN 2358-0399 Content shared under Creative Commons Attribution 4.0 Licence CC-BY Docente de Saúde Pública do
curso de Medicina Veterinária e
do PPGCV na Universidade
Federal de Uberlândia, Minas
Gerais, Brasil Introdução O conceito de saúde única foi elencado pela primeira vez por Rudolf Virchow (1821-1902), médico e político
alemão do século XIX, que defendia a necessidade de haver um equilíbrio entre a saúde humana, animal e
ambiental (Brown, 2003). Apesar disso, a ideia só ganhou espaço a partir de 2008, momento em que as
organizações mundiais da Saúde (OMS), da Saúde Animal (OIE) e das Nações Unidas para alimentação e
agricultura (FAO) instituíram os objetivos da saúde única na promoção de políticas públicas voltadas à
prevenção e controle de enfermidades infecciosas (King et al., 2008). Junto ao conceito de saúde única, Virchow também trouxe a denominação de zoonoses, compreendida pelas
enfermidades infecciosas transmissíveis entre humanos e animais. Hoje está bem estabelecido que 60% das
doenças infecciosas que acometem humanos são de origem animal e a globalização, aliada às mudanças
climáticas e aos hábitos peculiares de diferentes sociedades, tem levado à emergência de zoonoses, como a
COVID-19 (Chen et al., 2020). A formação dos médicos veterinários é aliada a conteúdos que envolvem não só a área biomédica animal, mas
também aspectos sociais voltados à atenção primária, ligada à prevenção de enfermidades zoonóticas e à
promoção da saúde humana. Essa atuação ganhou ainda mais espaço com a inclusão do profissional aos
Núcleos de Apoio à Saúde da Família (NASF), passando a atuar junto aos demais profissionais de saúde na
avaliação de fatores de risco ligados à interação homem-animal, na prevenção e controle de zoonoses, em
ações socioeducativas na comunidade, e na vigilância em saúde (Ministério da Saúde, Portaria n. 2488, de 21
de outubro de 2011). A recente pandemia da COVID-19, iniciada na província de Wuhan, China, tornou ainda mais clara a influência
das interações homem-animal silvestre na emergência de doenças zoonóticas. Trata-se de uma enfermidade
cujo agente causador é um coronavírus (SARS-CoV-2). As pesquisas até então publicadas sugerem que o vírus
se mantém de forma comensal em algumas espécies de morcegos e se adaptou aos pangolins, espécie que
apresenta amplo contato com humanos nos mercados chineses (Zhang et al., 2020). A América Latina recebeu o status de epicentro da pandemia a partir de maio de 2020, sendo o Brasil o país
mais atingido, com mais de 1,600 milhão infectados e mais de 64 mil mortos, e até o momento o município
de Uberlândia contabiliza mais de 4.324 casos confirmados, dos quais 486 (11,2%) são idosos (Ministério da
Saúde, 2020a). Veterinary medicine and the one health system: socio-educational action
applied to elderly in the context of COVID-19 Docente de Saúde Pública do
curso de Medicina Veterinária e
do PPGCV na Universidade
Federal de Uberlândia, Minas
Gerais, Brasil Abstract: The one health system integrates the human, environmental, and animal health spheres
through public policies focused on preventing and controlling infectious diseases. The veterinary
doctor is a professional actor in public health through direct contact with the three spheres. One
of these professional's main actions includes promoting community educational measures to
disseminate information regarding the prevention of zoonotic diseases. In the face of the current
pandemic, we developed the project: "Butterfly effect applied to asylums of Uberlândia, Minas
Gerais State, Brazil, in the combat to COVID-19" to spread information about the disease playfully
and to attend to the demands of the involved institutions. Ten educational materials were
produced and distributed, such as videos and interactive games with guidelines regarding COVID-
19, in 16 shelters for the elderly, along with the collection of cleaning and personal hygiene
products, masks, and food products from donations from 23 companies, to emphasize preventive
measures. Institutional staff developed the proposed activities with the elderly, emphasizing the
importance of social distance and personal hygiene at this time. The action performed allowed the
amplification of knowledge about the pandemic due to the use of methodologies applied to the
difficulties and needs of the elderly, allied to the emotional motivation used in the proposal, which
favored the teaching-learning process. This social action in the institutions helped to raise
awareness and clarify doubts concerning VOCID-19 to the leading risk group and reinforced the
context of the veterinarian's action in the one health. (autora para correspondência)
roberta.tmelo@ufu.br Keywords: Education; Epidemic; Vulnerable group; Health promotion DOI: https://doi.org/10.36661/2358-0399.2020v11i3.11560 DOI: https://doi.org/10.36661/2358-0399.2020v11i3.11560 430 | Ferreira, A. L. M. et al. 430 Introdução A doença se configura pelo seu status de maior risco em pacientes idosos, principalmente
aqueles com comorbidades. As dificuldades inerentes a esse grupo de risco impossibilitam uma melhor
compreensão da situação vivenciada, em virtude da ocorrência comum de alterações cognitivas, que
inviabilizam a compreensão e adaptação às orientações de controle, mesmo aquelas mais simples (Alzheimer
Europe, 2020). As instituições de longa permanência para idosos apresentam carências não só relacionadas aos cuidados com
esse grupo de risco, mas também deficiências estruturais e de recursos humanos, que permitiriam uma melhor
forma de controle de doenças como a COVID-19. A sinergia desses fatores é refletida no primeiro grande
estudo da COVID-19 em um lar de idosos, em que quase dois terços dos residentes foram infectados por um
período de três semanas, com uma taxa de mortalidade de 33% (McMichael et al., 2020). Apesar de ser uma
questão alarmante, ainda é muito baixo o reconhecimento dado aos desafios únicos a serem enfrentados
nessas instituições. A Organização Mundial da Saúde definiu que procedimentos de isolamento e formas de educação em saúde
adaptadas aos residentes de lares de idosos devem ser excepcionalmente elaboradas, considerando o
comprometimento cognitivo significativo de parte deles. O número de pessoas dessa classe social que se Ação socioeducativa no contexto da COVID-19 431 sentem solitárias e deprimidas já é muito alto em condições normais, com 30% dos residentes de asilos com
depressão em sua fase aguda. Com a inclusão das medidas de isolamento e distanciamento social esse valor
pode alcançar de 50-55% (Read et al., 2020). sentem solitárias e deprimidas já é muito alto em condições normais, com 30% dos residentes de asilos com
depressão em sua fase aguda. Com a inclusão das medidas de isolamento e distanciamento social esse valor
pode alcançar de 50-55% (Read et al., 2020). A comunicação aberta, adequada, compreensiva, repetida e gradual a respeito da importância da doença é
fundamental para favorecer a sensibilização ao conceito de prevenção contra a COVID-19. Além disso,
precauções adicionais que abordem conselhos práticos de forma lúdica sobre as restrições ao contato social e
medidas intensivas de higiene pessoal foram recomendadas, uma vez que podem auxiliar não só no combate
à pandemia, mas também na redução dos impactos negativos na saúde mental decorrentes das restrições de
convívio com familiares (World Health Organization [WHO], 2020). Introdução Diante desse contexto, a atuação do profissional médico veterinário se torna fundamental no quesito
orientação e conscientização da comunidade geral, principalmente aos idosos. Isso subsidiou o objetivo do
presente trabalho de desenvolver e aplicar materiais educativos com conteúdo informativo lúdico voltado às
necessidades do principal grupo de risco envolvido na COVID-19, e reforçar a informação por meio da entrega
de doações de produtos relacionados às medidas de combate ao SARS-CoV-2. Procedimentos metodológicos As atividades extensionistas do projeto “Efeito Borboleta aplicado a asilos de Uberlândia no combate à COVID-
19” foram executadas pelos alunos coordenadores do Grupo de Estudos sobre Zoonoses (GEZ), da Faculdade
de Medicina Veterinária (FAMEV), da Universidade Federal de Uberlândia (UFU). A proposta foi desenvolvida
no período de março a junho de 2020. Para obter domínio teórico sobre o novo coronavírus (SARS-CoV-2), os alunos envolvidos participaram de uma
palestra apresentada pela orientadora do grupo, na qual foram apresentadas informações a respeito da
definição, etiologia, histórico, epidemiologia, sintomas, diagnóstico, tratamento e controle da doença. Determinação das propostas didáticas, instituições e doações Material didático físico Todo material físico foi produzido de acordo com a quantidade específica de residentes de cada asilo. No total
foram elaborados quatro jogos, dois materiais informativos e uma simulação de coronavírus produzido em
biscuit. Os jogos interativos incluem o ‘Jogo da Memória: conhecendo os sintomas da COVID-19’, o jogo ‘Como
Controlar o Coronavírus’, a ‘Cruzadinha da COVID-19’ e o ‘Bingo da COVID-19’. Os materiais informativos foram
a planilha de contatos das Unidades Básicas de Saúde de Uberlândia-MG e o folder ‘Coronavírus e a COVID-
19: Você sabia?’. O ‘Jogo da Memória: conhecendo os sintomas da COVID-19’ foi proposto para abordar desde os sintomas
leves até os mais graves ligados à doença, a fim de promover a familiarização e reconhecimento desses
sintomas caso necessário. A proposta do jogo interativo ‘Como Controlar o Coronavírus’ foi proporcionar
informações sobre as medidas de distanciamento e isolamento social por meio de estratégias de associação. O intuito da ‘Cruzadinha da COVID-19’ foi utilizar de forma criativa palavras-chave relacionadas à prevenção
da doença. Abordagens ligadas à manutenção da saúde mental dos idosos foram utilizadas no ‘Bingo da
COVID-19’ por meio de frases de conforto. Já o folder ‘Coronavírus e a COVID-19: Você sabia?’ propôs uma maneira simples e resumida para diferenciar
os tipos de coronavírus e ressaltar a inexistência da transmissão do SARS-CoV-2 de animais para humanos. Material didático digital Todo material digital produzido foi disponibilizado às instituições via e-mail e em CD gravado para cada abrigo
de idosos. Foram elaborados dois vídeos informativos, um jogo digital e uma história em quadrinhos. Os vídeos
foram denominados ‘Que vírus é esse?’ e ‘Queridos idosos’, o jogo interativo de ‘Verdades e Mitos da Prevenção
da COVID-19’ e a história em quadrinhos de ‘Diálogo entre o Coronavírus x Mundo’. O objetivo do vídeo ‘Que vírus é esse?’ foi esclarecer as características gerais do vírus de maneira simples e
ilustrativa. Já no vídeo ‘Queridos idosos’ a ideia foi esquematizar medidas de controle aliadas a mensagens de
carinho e conforto. O jogo ‘Verdades e Mitos da Prevenção da COVID-19’ enfatizou o entendimento sobre a
doença por meio de perguntas sobre as formas de transmissão e prevenção. A proposta da história em
quadrinhos ‘Coronavírus x Mundo’, por sua vez, foi informar sobre a origem e as ‘predileções’ do SARS-CoV-2
(ou seja, situações que o favorecem), de maneira lúdica, com linguagem informal. Determinação das propostas didáticas, instituições e doações Para as ações educativas a respeito da atual pandemia da COVID-19 foram desenvolvidas dez propostas
didáticas que abordaram aspectos relacionados às características, origem, relação com animais, sintomas,
transmissão e, principalmente, às formas de combate ao novo coronavírus. Adicionalmente, foi disponibilizada
uma planilha com telefones e endereços de todos os postos de saúde da cidade de Uberlândia para facilitar o
acesso dos asilos às informações, à medida que surgirem atualizações sobre a COVID-19. A elaboração dos
jogos incluiu estratégias lúdicas com o objetivo de informar e conscientizar os idosos sobre o coronavírus, de
maneira adaptada às necessidades desse grupo. De forma complementar, os materiais abordaram aspectos
ligados à manutenção da saúde mental por meio do uso de mensagens positivas e estimuladoras, em meio à
pandemia. A escolha das instituições beneficiadas foi baseada em uma pesquisa em 35 asilos da cidade de Uberlândia,
MG. Desses, 16 foram selecionados pelo aspecto filantrópico, pela rápida resposta e retorno formal à realização
de todas as atividades do projeto, em virtude da dificuldade de maiores contatos em época de isolamento
social. Os abrigos contemplam um total de 364 idosos, que apresentam diferentes níveis de capacidade
cognitiva e morbidades crônicas. Com o objetivo de intensificar de forma ainda mais prática a proposta, foi inserida uma ação de cunho social,
ligado à arrecadação de produtos de higiene pessoal e limpeza, assim como máscaras descartáveis e de dupla
camada de algodão 100%, e gêneros alimentícios para todos os residentes das instituições (disponível em Ferreira, A. L. M. et al. 432 Material Suplementar 1). As doações foram provenientes de um total de 23 empresas Material Suplementar 1). As doações foram provenientes de um total de 23 empresas. Aplicação prática A necessidade de respeitar as medidas de distanciamento social fez com que a ideia inicial do projeto fosse
adaptada à aplicação dos jogos pelos próprios funcionários e assistentes sociais de cada abrigo. Por isso, em
todas as propostas foram desenvolvidas regras específicas de aplicação explicadas aos funcionários nos
próprios jogos, via videoconferência, contato telefônico e/ou e-mail. Todas as entregas foram realizadas pessoalmente, respeitando as medidas de distanciamento social. Os
materiais didáticos foram apresentados aos idosos em nove dias distintos, de acordo com o cronograma de
atividades local, que seguiu a ordem estabelecida na Tabela 1, para garantir a aquisição gradativa do
conhecimento. Todas as aplicações práticas foram acompanhadas via videoconferência, gravações e/ou fotos. Ação socioeducativa no contexto da COVID-19 433 Tabela 1. Ordem estabelecida de aplicação das atividades propostas aos idosos para o processo ensino-
aprendizagem gradual sobre a COVID-19. Ordem
Atividade
Justificativa
1º dia
Vídeo ‘Que vírus é esse’ e contato com o vírus
de biscuit
Conhecimento inicial sobre o agente. 2º dia
Folder ‘Coronavírus e a COVID-19: Você sabia?’
Esclarecimento sobre origem e a relação
com animais. 3º dia
Jogo digital ‘Verdades e Mitos da COVID-19’
Abordagem principal sobre os aspectos
ligados à transmissão da doença. 4º dia
‘Jogo da memória: conhecendo os sintomas da
COVID-19’
Compreensão dos diferentes sinais
clínicos e dos níveis de gravidade. 5º dia
‘Cruzadinha da COVID-19’
Aprofundamento e fixação acerca das
medidas de controle e prevenção da
COVID-19. 6º dia
Jogo ‘Como Controlar o Coronavírus’
7º dia
História em quadrinhos digital ‘Coronavírus x
Mundo’
8º dia
‘Bingo da COVID-19’
Apoio às questões relativas à saúde
mental. 9º dia
Vídeo ‘Queridos Idosos’ Resultados e Discussão O trabalho foi realizado a partir de dez atividades educativas, a fim de levar informações completas sobre a
COVID-19 aos idosos residentes de 16 casas de repouso da cidade de Uberlândia, Minas Gerais. As atividades
foram realizadas do mês de abril ao mês de junho de 2020. Ao todo, foram 364 residentes beneficiados com
os materiais didáticos, além de toda a equipe de funcionários em contato direto com os idosos. De maneira
complementar, foram arrecadados e doados, a partir de empresas e de pessoas físicas, produtos de limpeza,
de higiene pessoal, máscaras e gêneros alimentícios, com o intuito de enfatizar as ações práticas voltadas à
prevenção dessa doença. Quanto às atividades educativas, os jogos interativos propostos foram ‘Verdades e mitos da prevenção da
COVID-19’ (Figura 1a), ‘Bingo da COVID-19’, ‘Cruzadinha da COVID-19’, ‘Como Controlar o Coronavírus’ e ‘Jogo
da Memória: conhecendo os sintomas da COVID-19’, todos acompanhados com as devidas instruções para
auxiliar o mediador no momento da aplicação. O material informativo produzido consistiu dos vídeos ‘Que
vírus é esse?’ - acompanhado do vírus de biscuit para facilitar a compreensão - e ‘Queridos idosos’ (Figura 1b),
além do folder: ‘Coronavírus e a COVID-19: Você sabia?’ e da história em quadrinhos ‘Diálogo entre o
Coronavírus x Mundo’. Os materiais distribuídos foram aplicados pelas equipes de assistência social e terapia
ocupacional de cada asilo (Figura 2), uma vez que as medidas de isolamento e distanciamento social 4 | Ferreira, A. L. M. et al. 434 impossibilitaram nossa participação direta. Figura 1. Materiais educativos desenvolvidos nos formatos físico e digital – ‘Verdades e mitos da prevenção da
COVID-1.9’ (A) e vídeo ‘Queridos idosos’ (B). Figura 1. Materiais educativos desenvolvidos nos formatos físico e digital – ‘Verdades e mitos da prevenção da
COVID-1.9’ (A) e vídeo ‘Queridos idosos’ (B). Figura 2. Momento de aplicação dos materiais didáticos: ‘Jogo da Memória: conhecendo os sintomas da
COVID-19’ (A) e jogo ‘Jogo Como Controlar o Coronavírus’ (B), nos diferentes asilos beneficiados. Figura 2. Momento de aplicação dos materiais didáticos: ‘Jogo da Memória: conhecendo os sintomas da
COVID-19’ (A) e jogo ‘Jogo Como Controlar o Coronavírus’ (B), nos diferentes asilos beneficiados. Antes da realização das entregas dos materiais didáticos e das doações aos asilos, nos foi relatado que grande
parte dos idosos apresentava elevada resistência em seguir as recomendações de combate à doença, em
relação ao contato físico e isolamento social, principalmente. Resultados e Discussão No Brasil, culturalmente, há maior proximidade
física entre a população, de modo que a transmissão da doença é favorecida, e os meios de comunicação que
retratam as medidas de prevenção e controle são de difícil compreensão, especialmente entre as pessoas de
idade avançada. A partir dos resultados e relatos feitos posteriormente pela equipe de enfermeiros, terapeutas ocupacionais e
assistentes sociais de cada casa de repouso, os materiais didáticos permitiram uma maior conscientização e
adesão por parte dos idosos em relação às medidas de controle e prevenção da COVID-19. De acordo com
Damasceno et al. (2018), ações educativas são fundamentais no envolvimento dos idosos quanto ao cuidado
com sua saúde. Além disso, proporcionam maior interação entre os profissionais, familiares e idosos, o que
possibilita a troca de informações e favorece hábitos mais saudáveis, importantes no controle de diversas
doenças e complicações. Logo, a conscientização através de medidas educativas lúdicas sobre a COVID-19 é
importante para essa faixa-etária, de modo a envolver ativamente os idosos no cuidado com a saúde física e
mental nas casas de repouso, além do impacto positivo a toda comunidade de convivência com estes idosos. O vídeo ‘Que Vírus é Esse?’ representou uma forma de interação digital para divulgação do conhecimento e
consequente promoção da saúde. O material potencializou o alcance da informação por meio do Ação socioeducativa no contexto da COVID-19 435 esclarecimento de dúvidas sobre características e funcionalidades do vírus causador da doença. Essa
compreensão deixa claros os fundamentos das formas de prevenção e facilita a sua aceitação e adaptação
pelos idosos. O ‘vírus de biscuit’ complementou de forma palpável essas características e induziu reflexões e
discussões que favorecem a fixação do conteúdo. O folder ‘Coronavírus e a COVID-19: Você Sabia?’ consistiu em informações sobre a origem da doença e as
diferenças do SARS-CoV-2 com os demais coronavírus que afetam animais. O material informativo contemplou
imagens de animais com a descrição simples dos outros coronavírus, focando no aprendizado visual. A ideia
central foi deixar claro que o contato com animais não favorece a transmissão da COVID-19. Até o momento,
sabe-se que o SARS-CoV-2 provavelmente apresentou como hospedeiro primário os morcegos, que abrigam
uma variedade de subtipos de coronavírus, de maneira comensal. Por meio de transferência adaptativa, esse
vírus sofreu processos de recombinação, tendo os pangolins como hospedeiros intermediários e humanos
como hospedeiros finais. Resultados e Discussão De maneira concomitante, vários estudos têm tentado mostrar o risco de transmissão
da COVID-19 a partir de animais, como furões, gatos, cães, porcos, galinhas e patos, para humanos (Shi et al.,
2020; Zhang et al., 2020), mas nenhum comprova seu risco efetivo, e são questionados quanto ao desenho
experimental (Hora, 2020). O material digital ‘Verdades e Mitos da Prevenção da COVID-19’ permitiu momentos de descontração, ao
mesmo tempo em que testava o conhecimento dos envolvidos. Consistiu em 11 perguntas que o jogador
deveria responder quanto à veracidade, sendo vencedor aquele que acertasse mais de sete questões. A
abordagem focou nas formas de transmissão da COVID-19. De acordo com o Ministério da Saúde (2020b), a
principal forma de disseminar a doença se dá por contato direto entre as pessoas, por meio de aperto de mãos,
gotículas de saliva, espirro, tosse, secreções nasais e objetos ou superfícies contaminadas, como mesas,
maçanetas, entre outros itens compartilhados, seguido do contato do vírus com as mucosas ocular, nasal e
bucal do paciente. Outro material físico foi o ‘Jogo da Memória: conhecendo os sintomas da COVID-19’, que abordou aspectos
ligados aos sintomas da COVID-19, subdivididos em três categorias, conforme a gravidade. A atividade
apresentou 16 pares de figuras correspondentes ao nome do sintoma, acompanhados das instruções do jogo
e de um informativo que discrimina os sintomas no humano conforme a intensidade. Os sintomas foram
divididos em respiratórios, digestivos e sistêmicos, e incluíram: dor de cabeça, dor nas articulações, tosse seca,
febre, dificuldade de respirar, pneumonia, falência renal, diarreia, perda do olfato, insuficiência respiratória, dor
abdominal, náusea, coriza, dor muscular, dor de garganta e vômito. Sabe-se que a COVID-19 está associada
especialmente a alterações respiratórias, e que a maioria das pessoas infectadas apresenta sintomas leves e se
recupera. Em alguns casos os sintomas nem sequer são apresentados. Porém, a doença pode levar também a
sintomas mais graves, como a pneumonia, e, dependendo do grau de debilidade, à morte, mais relevante em
pacientes idosos (Huang et al., 2020). A ‘Cruzadinha da COVID-19’ possibilitou o aprendizado individual por meio de palavras cruzadas com
informações sobre o controle e a prevenção da doença, utilizando uma linguagem acessível. Sua composição
incluía 14 palavras-chave dispostas vertical e horizontalmente, cruzando-se umas com as outras, além de dicas
enumeradas sobre cada palavra a ser descoberta, estimulando o aprendizado. Resultados e Discussão Paralelamente, o vídeo ‘Queridos Idosos’, apresentou de maneira digital uma homenagem aos
idosos, que valorizou a importância referencial do grupo à sociedade e reforçou a ideia de exemplos de vida
aos seus familiares. Se, por um lado, há necessidade de inclusão de cuidados voltados às restrições e medidas
de isolamento social, por outro, é também essencial o atendimento centrado na pessoa idosa e no cuidado à
manutenção da qualidade de vida, reforçando a importância dessa classe social, mediada pela interação
carinhosa com os residentes, apoiados na atenção psicossocial (Wang et al., 2020). Quanto aos resultados quantitativos observados, dos 16 asilos beneficiados pelo projeto, obtivemos o retorno
de 14 das instituições quanto aos casos de COVID-19. Felizmente, até o encerramento das ações de extensão
(junho de 2020), não houve nenhum caso positivo da doença nos idosos desses locais. Além disso, os idosos
demonstraram maior sensibilização à adoção dos cuidados contra o coronavírus. Tais fatos indicam o impacto
positivo do projeto no controle e prevenção da doença ao público-alvo, e corrobora com o que foi observado
por Damasceno et al. (2018), em estudo sobre a Extensão Universitária como estratégia para a Educação em
Saúde com um grupo de idosos. Os autores observaram que a inserção de ações universitárias junto aos idosos
permitiram o desenvolvimento de uma abordagem holística e aprendizagem significativa, alcançando a
promoção da saúde, recuperação e, principalmente, a prevenção de doenças, atendendo às necessidades reais
do público-alvo assistido. Para reforçar as ações, foram realizadas doações às instituições envolvidas, por meio de parcerias com
indivíduos e empresas de diversos segmentos, como clínicas veterinárias, consultórios de psicologia, serviços
de alimentação, fábricas de produtos de limpeza e higiene pessoal, drogarias e farmácias de manipulação
veterinária. Os insumos adquiridos incluíram gêneros alimentícios, itens de limpeza e higiene pessoal, além de
máscaras, tanto para os idosos quanto para seus colaboradores. Os materiais arrecadados contribuíram para a
aplicação de forma prática do conteúdo abordado, principalmente o referente ao controle e prevenção da
doença. De acordo com a World Health Organization (WHO, 2020), a recepção de doações gera entusiasmo
nas pessoas envolvidas, e se estabelece como importante estratégia de superação ou como um fator de
sucesso no enfrentamento de agravos, como a COVID-19. A abordagem positiva sobre as doações se alinha às
estratégias de acesso facilitado às formas de prevenção da pandemia. Resultados e Discussão De maneira semelhante, o jogo ‘Como Controlar o Coronavírus’ permitiu a disseminação de informações
ligadas ao combate à pandemia de maneira coletiva e interativa. A atividade era composta por um cartão de
definições, um cartão de respostas, um quadro produzido em cartolina, o qual possuía quatro áreas delimitadas
com as expressões ‘Isolamento social’, ‘Distanciamento social’, ‘Quarentena’ e ‘Bloqueio total’, além de 12
imagens, as quais o jogador teria que distribuir conforme os diferentes conceitos. O vencedor seria o
jogador/grupo que mais acertasse as figuras colocadas nas áreas adequadas. Ferreira, A. L. M. et al. 436 Seguindo a mesma ideia de controle da COVID-19, foi desenvolvido ainda um diálogo digital denominado
‘Coronavírus x Mundo’, no qual o coronavírus conversa com o mundo, de maneira lúdica, sobre seu surgimento
e sobre os cuidados que o mundo deve tomar que impedem sua propagação. O material foi elaborado
seguindo a ideia de história em quadrinhos, unindo texto e imagens ao narrar o diálogo, de fácil entendimento
ao leitor (disponível em Material Suplementar 2). A maior quantidade de materiais produzidos focou nas medidas de controle e prevenção da doença pela
necessidade imperativa de levar esse tipo de informação e sensibilizar ao máximo os idosos a essas novas
regras. Devido à rápida disseminação da COVID-19 e ao alto poder de contágio do vírus, além da ausência de
vacinas e antivirais, as autoridades de saúde têm buscado implementar medidas tradicionais de Saúde Pública
para conter a doença (Wilder-Smith & Freedman, 2020). Essas medidas consistem na redução da
movimentação e do contato entre as pessoas, dada a maneira de transmissão do vírus, além das formas básicas
de higiene, como lavar as mãos e higienizar adequadamente as superfícies. A conscientização sobre essas
medidas, aliada à distribuição de produtos aplicáveis a essas necessidades e à atual realidade, representou
nossa principal estratégia para efetivar a promoção da saúde dos envolvidos. O ‘Bingo da COVID-19’, por sua vez, utilizou de palavras de encorajamento ao público-alvo do projeto. O jogo
consistiu em uma adaptação do bingo tradicional, no qual, em vez de bolas enumeradas, havia 50
palavras/frases motivadoras dispostas aleatoriamente em 20 cartelas compostas de cinco linhas e cinco
colunas. A ideia central do jogo era favorecer a saúde mental, que se encontra mais debilitada nesse momento
de pandemia. Resultados e Discussão Dessa maneira, o impacto dessa ação
tende a gerar uma motivação na comunidade geral no combate à COVID-19, no que se refere ao cuidado Ação socioeducativa no contexto da COVID-19 437 próprio e dos funcionários com os idosos. Algumas dificuldades foram encontradas na realização do projeto, sendo principalmente relacionadas aos
recursos necessários para a confecção das atividades propostas, que foram supridas por meio de doações
provenientes de cinco empresas do ramo de papelaria. As restrições relacionadas à execução à distância das
atividades, por sua vez, culminaram em atrasos na produção dos materiais e no estabelecimento de contatos
entre os asilos, as empresas parceiras e a comunidade. Além disso, as restrições de contato físico e as medidas
de isolamento social limitaram as etapas de entrega e realização das atividades práticas junto aos idosos por
parte do grupo. No entanto, tais adversidades foram contornadas por meio de ajustes no cronograma de
entregas e contatos estabelecidos, para a sincronia das atividades. De maneira complementar, foram incluídas
instruções específicas a cada material, aliado a uma comunicação reafirmada periodicamente junto às casas de
repouso, a fim de esclarecer aos funcionários o detalhamento sobre as atividades a serem realizadas com os
idosos. De maneira geral, as atividades permitiram reflexões sobre prevenção da COVID-19 e promoção de saúde,
tanto nos idosos, quanto na equipe de seu convívio. O material didático contribuiu no incentivo aos idosos a
seguirem as recomendações das autoridades de saúde, ao transmitir, de maneira lúdica e informal, a
importância dessas ações, bem como a maneira de executá-las. Agradecimentos Às empresas e pessoas envolvidas nas doações. Considerações finais A extensão universitária favoreceu a execução de ações na comunidade de forma simultânea à construção do
conhecimento, unindo tanto o saber acadêmico quanto a linguagem lúdica, para intensificar os vínculos e
permitir o empoderamento tanto dos estudantes, quanto dos profissionais e idosos de cada instituição
participante. Foi perceptível o sentimento de gratidão de todas as partes envolvidas no projeto. Além disso,
foi visível o impacto positivo para o controle e a prevenção da doença, uma vez que houve estímulo a um
maior engajamento por parte da comunidade em relação ao cuidado com os idosos e valorização desses
indivíduos, especialmente neste período de pandemia. Contribuição de cada autor A.L.M.F. coordenou a arrecadação das doações, participou das entregas e contribuiu na introdução; R.F.B. estabeleceu os contatos, participou das entregas, contribuiu na discussão e adequou o inglês e as normas;
M.C.C. coordenou os materiais físicos, participou das entregas e contribuiu nos resultados; D.A.P. elaborou
jogos digitais, contribuiu na arrecadação de doações, na divulgação e nas considerações finais; A.B.S. e D.R.P. elaboraram materiais físicos e digitais e contribuíram nos procedimentos metodológicos. R.T.M coordenou e
orientou todas as etapas. g
Ferreira, A. L. M., Braz, R. F., Chueiri, M. C., Policarpo, D. A., Siqueira, A. B., Pacheco, D. R., Melo, R. T. (2020). Medicina veterinária e
a saúde única: Ação socioeducativa aplicada a idosos no contexto da COVID-19. Revista Brasileira de Extensão Universitária, 11(3),
429-438. https://periodicos.uffs.edu.br/index.php/ RBEU/article/view/11560/pdf Referências Alzheimer Europe. (2020). Alzheimer Europe recommendations on promoting the wellbeing of people with dementia and
carers during the COVID-19 pandemic. Luxemburg: Alzheimer Europe. Recuperado de https://www.alzheimer-
europe.org/Policy/Our-opinion-on/Wellbeing-of-people-with-dementia-during-COVID-19-pandemic Ferreira, A. L. M. et al. 438 Brown, C. (2003). Virchow revisited: Emerging zoonoses. American Society for Microbiology, 69(10), 493-497. Chen, Y., Liu, Q., & Guo, D. (2020). Emerging coronaviruses: Genome structure, replication, and pathogenesis. Journal
of Medical Virology, 92(4), 418-423. Damasceno, A. J. S., Aragão, C. P., Mesquita, F. M. S., Vasconcelos, V. P., de Sousa, L. S., de Sousa, L. V., & Moreira, A. C. A. (2018). A Extensão Universitária como estratégia para a Educação em Saúde com um grupo de idosos. Revista Kairós:
Gerontologia, 21(4), 317-333. Hora, A. S. (2020). Coronavirus: A veterinary perspective. Nature, 580(7803), 321-321. Huang, X., Wei, F., Hu, L., Wen, L., & Chen, K. (2020). Epidemiology and clinical characteristics of COVID-19. Archives of
Iranian Medicine, 23(4), 268-271. King, L. J., Anderson, L. R., Blackmore, C. G., Blackwell, M. J., Lautner, E. A., Marcus, L. C., … & Mahr, R. K. (2008). Executive summary of the AVMA One Health Initiative Task Force report. Journal of the American Veterinary Medical
Association, 233(2), 259-261. McMichael, T. M., Currie, D. W., Clark, S., Pogosjans, S., Kay, M., Schwartz, N. G., … & Duchin, J. S. (2020). Epidemiology
of Covid-19 in a Long-Term Care Facility in King Country, Washington. The New England Journal of Medicine, 382(21),
2005-2011. Ministério da Saúde. (2020a). Guia de Vigilância Epidemiológica do COVID-19. Brasília: Ministério da Saúde do Brasil. Recuperado de https://covid.saude.gov.br/ Ministério da Saúde. (2020b). Coronavírus (COVID-19). Brasília: Ministério da Saúde do Brasil. Recuperado de
https://coronavirus.saude.gov.br/ Read, S., Comas-Herrera, A., & Grundy, E. (2020). Social isolation and memory decline in later-life. The Journals of
Gerontology: Series B, 75(2), 367-376. Shi, J., Wen, Z., Zhong, G., Yang, H., Wang, C., Huang, B., ... & Zhao, Y. (2020). Susceptibility of ferrets, cats, dogs, and
other domesticated animals to SARS–coronavirus 2. Science, 368(6494), 1016-1020. Wang, H., Li, T., Barbarino, P., Gauthier, S., Brodaty, H., Molinuevo, J. L., ... & Weidner, W. (2020). Dementia care during
COVID-19. The Lancet, 395(10231), 1190-1191. Wilder-Smith, A., & Freedman, D. O. (2020). Isolation, quarantine, social distancing and community containment: pivotal
role for old-style public health measures in the novel coronavirus (2019-nCoV) outbreak. Journal of Travel Medicine,
27(2), 1-4. World Health Organization. (2020). Referências Infection Prevention and Control guidance for Long-Term Care Facilities in the context
of COVID-19. [ S. l. ]: WHO. Recuperado de https://apps.who.int/iris/bitstream/handle/10665/331508/WHO-2019-
nCoV-IPC_long_term_care-2020.1-eng.pdf?sequence=1&isAllowed=y World Health Organization. (2020). Infection Prevention and Control guidance for Long-Term Care Facilities in the context
of COVID-19. [ S. l. ]: WHO. Recuperado de https://apps.who.int/iris/bitstream/handle/10665/331508/WHO-2019-
nCoV-IPC_long_term_care-2020.1-eng.pdf?sequence=1&isAllowed=y Zhang, T., Wu, Q., & Zhang, Z. (2020). Probable pangolin origin of SARS-CoV-2 associated with the COVID-19 outbreak. Current Biology, 30(8), 1346-1351. ***
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Avaliação do tipo de substrato e do período de armazenamento para a germinação de sementes de Cordia glabrata (Mart.) DC.
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Acta Scientiarum. Biological Sciences
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Avaliação do tipo de substrato e do período de armazenamento
Avaliação do tipo de substrato e do período de armazenamento
Avaliação do tipo de substrato e do período de armazenamento
Avaliação do tipo de substrato e do período de armazenamento
para a germinação de sementes de
para a germinação de sementes de
para a germinação de sementes de
para a germinação de sementes de Cordia glabrata
Cordia glabrata
Cordia glabrata
Cordia glabrata (Mart.) DC.
(Mart.) DC.
(Mart.) DC.
(Mart.) DC. Ademir Kleber Morbeck de Oliveira1*, Felipe Fernandes Alves2 e Juliana Gadum 1Programa de Pós-graduação em Meio Ambiente e Desenvolvimento Regional, Universidade para o Desenvolvimento do
Estado e da Região do Pantanal, Cx. Postal 2153, 79031-320, Campo Grande, Mato Grosso do Sul, Brasil. 2Departamento de
Ciências Biológicas, Universidade para o Desenvolvimento do Estado e da Região do Pantanal, Campo Grande, Mato Grosso
do Sul, Brasil. 3Departamento de Agronomia, Universidade para o Desenvolvimento do Estado e da Região do Pantanal,
Campo Grande, Mato Grosso do Sul, Brasil. *Autor para correspondência. E-mail: akmorbeck@hotmail.com RESUMO. Informações sobre a viabilidade e substratos adequados para a germinação de
sementes de espécies arbóreas nativas são escassas. Cordia glabrata (louro-preto), espécie
pioneira e de ampla distribuição, está incluída entre essas espécies. Suas características
permitem a utilização em ornamentação, na prática apícola e na medicina popular. Levando
em conta seu potencial de utilização, este trabalho teve por objetivo avaliar as taxas de
germinação, em diferentes substratos, de sementes recém-colhidas e armazenadas até 120
dias em laboratório, as quais foram coletadas no Pantanal do Rio Negro, Estado do Mato
Grosso do Sul. Foram realizados testes com sementes recém-colhidas e após 30, 60, 90 e
120 dias de armazenamento, em três tipos de substrato (papel-filtro, solo arenoso e solo
arenoso + matéria orgânica). Os resultados revelaram que C. glabrata possui pequena
porcentagem de água (1,55%) nas sementes, que decai com o período de armazenamento. Seu período de viabilidade é curto (90 dias) e os melhores resultados foram obtidos com
sementes recém-colhidas germinadas em papel-filtro (34%), com índice de velocidade de
germinação de 10. Palavras-chave: sementes florestais, teste de germinação, conservação da semente, viabilidade, louro-
preto. ABSTRACT. Evaluation of substrate type and storage period for the germination
of Cordia glabrata (Mart.) DC seeds. Information about the feasibility and adequate
substrates for the germination of seeds from native arboreous species are scarce. Cordia
glabrata (louro preto), a pioneering species with wide distribution, is included among these
species. Its characteristics allow its use in ornamentation, apiculture and popular medicine. Considering its potential of utilization, this work had as objective to evaluate the
germination rates, in different substrates, of recently reaped seeds stored for up to 120 days
in laboratory, which were collected in Pantanal of the Rio Negro, Mato Grosso do Sul
State. Avaliação do tipo de substrato e do período de armazenamento
Avaliação do tipo de substrato e do período de armazenamento
Avaliação do tipo de substrato e do período de armazenamento
Avaliação do tipo de substrato e do período de armazenamento
para a germinação de sementes de
para a germinação de sementes de
para a germinação de sementes de
para a germinação de sementes de Cordia glabrata
Cordia glabrata
Cordia glabrata
Cordia glabrata (Mart.) DC.
(Mart.) DC.
(Mart.) DC.
(Mart.) DC. Tests were done with recently reaped seeds and after 30, 60, 90 and 120 days of
storage, in three kinds of substrate (paper filter, sandy soil and sandy soil + organic matter). The results revealed that C. glabrata has a small percentage of water (1.55%) in the seeds,
which decreases with the storage period. Its feasibility period is short (90 days) and the best
results were obtained with recently reaped seeds germinated in paper filter (34%), with a
germination speed rate of 10. Key words: forest seeds, germination test, seed conservation, feasibility, louro preto. Acta Scientiarum. Biological Sciences
ISSN: 1679-9283
eduem@uem.br
Universidade Estadual de Maringá
Brasil
Morbeck de Oliveira, Ademir Kleber; Fernandes Alves, Felipe; Gadum, Juliana
Avaliação do tipo de substrato e do período de armazenamento para a germinação de sementes de
Cordia glabrata (Mart.) DC
Acta Scientiarum. Biological Sciences, vol. 31, núm. 3, 2009, pp. 301-305
Universidade Estadual de Maringá
.png, Brasil Acta Scientiarum. Biological Sciences
ISSN: 1679-9283
eduem@uem.br
Universidade Estadual de Maringá
Brasil
Morbeck de Oliveira, Ademir Kleber; Fernandes Alves, Felipe; Gadum, Juliana
Avaliação do tipo de substrato e do período de armazenamento para a germinação de sementes de
Cordia glabrata (Mart.) DC
Acta Scientiarum. Biological Sciences, vol. 31, núm. 3, 2009, pp. 301-305
Universidade Estadual de Maringá
.png, Brasil Acta Scientiarum. Biological Sciences
ISSN: 1679-9283
eduem@uem.br
Universidade Estadual de Maringá
Brasil Acta Scientiarum. Biological Sciences
ISSN: 1679-9283
eduem@uem.br
Universidade Estadual de Maringá
Brasil Morbeck de Oliveira, Ademir Kleber; Fernandes Alves, Felipe; Gadum, Juliana
Avaliação do tipo de substrato e do período de armazenamento para a germinação de sementes de
Cordia glabrata (Mart.) DC
Acta Scientiarum. Biological Sciences, vol. 31, núm. 3, 2009, pp. 301-305
Universidade Estadual de Maringá
.png, Brasil Disponível em: http://www.redalyc.org/articulo.oa?id=187115796010 Como citar este artigo
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Projeto acadêmico sem fins lucrativos desenvolvido no âmbito da iniciativa Acesso Aberto DOI: 10.4025/actascibiolsci.v31i3.1013 Acta Scientiarum. Biological Sciences Key words: forest seeds, germination test, seed conservation, feasibility, louro preto. Material e métodos
Material e métodos
Material e métodos
Material e métodos Machado et al. (2002) destacaram a importância
das espécies nativas pelo seu amplo aproveitamento
econômico, ornamental e medicinal, entre outros,
existindo a preocupação por parte dos pesquisadores
em realizar estudos que forneçam informações sobre
as características dessas sementes, pois de acordo
com Ferreira et al. (2008), cada vez mais a cobertura
vegetal é alterada pela ação antrópica, ameaçando a
integridade dos ecossistemas. O armazenamento de
sementes, de modo geral, é fundamental, uma vez
que tem a função básica de preservar a qualidade
delas, sendo esta preservação possível, pois, quando
realizada de modo adequado, diminui a velocidade
de deterioração (MELO et al., 1998). Isto permite a
utilização das sementes por mais tempo e garante a
manutenção do germoplasma de espécies ameaçadas
por atividades antrópicas ou com frutificação
irregular (SOUZA et al., 1980; BONNER, 1990). A área de coleta foi o Instituto de Pesquisa do
Pantanal – Ippan, com área de 2.618 ha (19°29’12,2”
a 19°30’49,8” Sul e 55°35’28,5” a 55°42’37,9” Oeste),
Pantanal do Rio Negro, município de Aquidauana,
Estado de Mato Grosso do Sul. A coleta dos frutos
foi realizada com o auxílio de tesoura de poda alta,
no mês de novembro/2006, em quatro árvores,
considerando-se o porte, o vigor e a sanidade. Depois de colhidas, as unidades de dispersão,
compostas por parte do fruto que envolve a semente,
foram deixadas à sombra por 12h, para secagem. A
seguir, foram retirados os pedúnculos e pétalas secas
por meio de esfregaço (LORENZI, 1992), sendo
consideradas sementes para a execução do trabalho
(DONADIO et al., 2004). As sementes foram
acondicionadas
em
sacos
de
papel
Kraft,
transportados para o Laboratório de Fisiologia
Vegetal, Campus de Ciências Biológicas, Agrárias e
da Saúde – Uniderp, Campo Grande, Estado de
Mato Grosso do Sul, e armazenadas em condições
de laboratório, com temperatura média de ±28,5°C. Testes de germinação
foram efetuados com
sementes recém-colhidas (sem armazenamento) e
após 30, 60, 90 e 120 dias de armazenamento (DA),
em três diferentes substratos: papel-filtro, solo
arenoso e solo arenoso com maior proporção de
matéria orgânica. Os substratos arenoso e arenoso
com matéria orgânica foram coletados na região do
Ippan e analisados no Laboratório de Fertilidade do
Solo - Uniderp. Oliveira et al. Oliveira et al. que servem de alimento para pássaros e mamíferos
(LORENZI, 1992). A planta também é utilizada na
medicina popular (GUARIM NETO, 2006), na
forma de chás para o tratamento sintomático de
‘dores no corpo’ e de reumatismo. objetivo avaliar a porcentagem de água nas sementes
e as taxas de germinação de C. glabrata, espécie nativa
do Pantanal do Rio Negro, Estado do Mato Grosso
do Sul, Brasil. Introdução
Introdução
Introdução
Introdução uma espécie nativa pioneira de ampla distribuição,
que pode colonizar desde margens de estradas até
matas, Cerrados e capões (LORENZI, 1992; POTT;
POTT, 1994). Pertence à família Boraginaceae e é
uma planta de altura média (8,0 a 15,0 m), madeira
macia ao corte e utilizada para confecções de móveis. Suas características permitem a utilização em
ornamentação, pois seu porte, beleza e forma da
copa são adequados ao paisagismo, além da prática
apícola. É uma espécie utilizada nos estágios iniciais
de recuperação de áreas impactadas e produz frutos No Brasil, dada a sua riqueza em espécies
florestais, tem-se observado carência de informações
sobre a germinação de sementes de espécies nativas,
essenciais para sua utilização, seja economicamente
ou com finalidade conservacionista (OLIVEIRA
et al., 2005). Entre as diferentes espécies arbóreas
encontra-se
Cordia
glabrata
(Mart.)
A. DC.,
comumente conhecida como louro-preto, claraíba,
louro-de-mato-grosso, peteribi ou louro-branco, Maringá, v. 31, n. 3, p. 301-305, 2009 Acta Scientiarum. Biological Sciences Acta Scientiarum. Biological Sciences 302 Acta Scientiarum. Biological Sciences Maringá, v. 31, n. 3, p. 301-305, 2009 Germinação de sementes de Cordia glabrata 303 queda no teor de água em suas sementes até 150 dias
de armazenamento em duas condições (laboratório e
refrigerador), enquanto Caesalpinia peltophoroides
Benth. apresentou redução no teor, quando
armazenada a 20oC, e aumento, se armazenada a 5oC
(SOUZA et al., 2005). com solução aquosa contendo fungicida Rovral a
0,1%. Para o segundo tratamento, com 200 sementes
(a cada período de tempo), divididas em quatro
repetições de 50 sementes, foram utilizadas caixas
plásticas
transparentes
contendo
solo
arenoso
(87,6%) com pH ácido (5,5) e matéria orgânica
(6,4 g dm-3), esterilizado a 105oC, por 48h. O
terceiro tratamento, com 200 sementes (a cada
período de tempo), foi dividido em quatro
repetições de 50 sementes utilizando-se caixas
gerbox transparentes contendo solo arenoso (88%)
com maior proporção de matéria orgânica (33,1 g
dm-3) e pH ácido (5,7), esterilizado a 105oC, por
48h. O monitoramento da germinação foi realizado
a cada 24h, sendo consideradas germinadas as
sementes que apresentaram raiz primária superior a
2 mm de comprimento. A taxa média de germinação
e o índice de velocidade de germinação (IVG) foram
calculados somando-se o número de sementes
germinadas a cada dia, dividido pelo respectivo
número de dias transcorridos a partir de semeadura
(NAKAGAWA, 1999). Os dados foram analisados
por meio do programa estatístico Estat, em nível de
5% de probabilidade, seguido, em caso de
significância, pelo teste de Tukey, em nível de 5%
(p < 0,05). Quando germinadas em substrato papel-filtro, as
sementes recém-colhidas apresentaram a maior
média
de
germinação
(34%)
e
perderam
gradativamente
seu
poder
germinativo,
não
ultrapassando 120 dias de viabilidade nos substratos
estudados (Tabela 2). Lorenzi (1992) destaca que C. glabrata possui taxa de germinação geralmente baixa,
além de apresentar viabilidade de armazenamento
também curta. Já Donadio et al. (2004) constataram
porcentagens de germinação de Cordia trichotoma
(louro-pardo) superiores a 74%. Utilizando como
substrato areia e papel-filtro, os mesmos autores
afirmam que o melhor substrato é o papel-filtro,
resultando em 83% de germinação, à temperatura de
25oC e alternada de 20-30oC. Tabela 2. Porcentagem média de germinação em sementes de
Cordia glabrata recém-colhidas e com 30, 60, 90 e 120 dias de
armazenamento em três substratos (papel-filtro, arenoso e
arenoso + matéria orgânica - MO), a 26oC. Resultados e discussão
Resultados e discussão
Resultados e discussão
Resultados e discussão A porcentagem de água nas sementes foi pequena
(1,55%), com queda significativa após cada período
de
armazenamento
(Tabela
1);
os
valores
encontrados foram menores que os citados por
Bewley e Black (1994), cuja maioria das sementes
apresentou de 5 a 20% de água. O substrato responsável pela maior porcentagem de
germinação até 60 dias de armazenamento (DA) foi o
papel-filtro, com diferenças significativas para solo
arenoso e solo arenoso com matéria orgânica (Tabela
2). A cada período de armazenamento, constatou-se
queda significativa nas porcentagens das sementes
germinadas. O segundo melhor substrato foi solo
arenoso, com bons resultados com sementes recém-
colhidas e após 60 DA. O substrato arenoso com
matéria orgânica foi o que apresentou resultados
menos interessantes. Em ambos os substratos, ocorreu
queda significativa nas médias de germinação (Tabela
2), após cada período de armazenamento. Estes
resultados estão provavelmente relacionados à alta
porosidade dos solos arenosos, que não são eficientes
na retenção de água, a qual é drenada rapidamente
podendo secar a superfície do substrato e atrasar ou
dificultar o processo de embebição da semente
(CARVALHO; NAKAGAWA, 2000). Donadio et al. (2004) encontraram 16,2% de
umidade nas sementes de Cordia trichotoma (Vell.)
Arrab. ex Steud., valores significativamente maiores
que os encontrados em C. glabrata (Tabela 1), um
possível indicativo de que a baixa taxa de germinação
observada (Tabela 2) pode estar relacionada ao
pequeno teor de água nas sementes. Tabela 1. Porcentagem de umidade obtida em sementes de
Cordia glabrata recém-colhidas e com 30, 60, 90 e 120 dias de
armazenamento em condições de laboratório. Local
Recém-colhidas
30 dias 60 dias 90 dias 120 dias
Laboratório
1,55 a
1,25 b
0,84 c
0,66 d
0,47 e
Médias seguidas de letra minúscula na linha não diferem estatisticamente entre si, pelo
teste de Tukey, em nível de 5% de probabilidade. Local
Recém-colhidas
30 dias 60 dias 90 dias 120 dias
Laboratório
1,55 a
1,25 b
0,84 c
0,66 d
0,47 e
Médias seguidas de letra minúscula na linha não diferem estatisticamente entre si, pelo
teste de Tukey, em nível de 5% de probabilidade. As diferentes espécies vegetais apresentam
variações
em
seu
comportamento
quanto
à
porcentagem de água contida, podendo alterar
conforme a umidade relativa do ar. Espécies como
Tabebuia serratifolia (Vahl.) Nich. Germinação de sementes de Cordia glabrata Substrato
Recém-colhidas
30 dias 60 dias 90 dias 120 dias
Papel-filtro
34 aA
28 aB
18 aC
8 aD
0 aE
Arenoso
20 bA
12 bB
8 bC
6 abC
0 aD
Arenoso + MO
16 cA
10 bB
4 cC
4 bC
0 aD
Médias seguidas na mesma letra minúscula na coluna e maiúscula na linha não diferem
estatisticamente entre si, pelo teste de Tukey, em nível de 5% de probabilidade. Material e métodos
Material e métodos
Material e métodos
Material e métodos Estudos com germinação de sementes são
geralmente
realizados
visando
ampliar
os
conhecimentos fisiológicos e metodológicos, a fim
de verificar as respostas da germinação aos fatores
ambientais, às causas de dormência e aos métodos de
superação, além de adequar os substratos para
aumentar as taxas de germinação. Neste contexto,
Wendling e Gatto (2002) e Floriano (2004) afirmam
que o substrato é importante para os testes de
germinação, pois a capacidade de retenção de água e
oxigênio, os patógenos, a estrutura física, entre
outros, podem variar de um substrato para outro,
interferindo na porcentagem de germinação das
sementes. O substrato deve permanecer úmido,
suprindo as necessidades de água da semente, porém
sem
excesso,
visto
que
pode
diminuir
a
disponibilidade de oxigênio para a semente,
reduzindo os processos metabólicos, além de facilitar
a proliferação de fungos (POPINIGIS, 1985;
FIGLIOLIA et al., 1993; PEREZ et al., 1999). Pelas diferenças encontradas no tamanho das
sementes, foi realizada a biometria das mesmas;
foram utilizadas as que apresentaram diâmetro
médio de 0,60 cm ± 0,10 e comprimento médio de
1,2 cm ± 0,2, tomados com paquímetro manual. O
teor de água das sementes foi quantificado
utilizando-se quatro repetições de 50 sementes para
cada período de tempo, submetidas a 105°C durante
24h (BRASIL, 1992). Como existem poucas recomendações para
espécies florestais, diversos substratos têm sido
testados (PACHECO et al., 2006), porém ainda há
poucas informações para grande parte das espécies
arbóreas nativas. Isto torna necessário avaliar
diferentes substratos, visando a informações que
propiciem a germinação rápida e uniforme das
sementes, importantes para a produção de mudas,
principalmente para espécies com reduzidas taxas de
germinação, como é o caso de C. glabrata
(LORENZI, 1992). Assim, este trabalho teve por Para cada teste de germinação, foram utilizadas
200 sementes (previamente imersas em hipoclorito
de sódio por 5 min. e lavadas em água corrente por 2
min.) por tratamento, colocadas em germinador, sob
luz branca, fotoperíodo de 12h e a 26oC. O primeiro
tratamento foi realizado com 200 sementes (a cada
período de tempo), divididas em quatro repetições
de 50, colocadas em caixas plásticas transparentes,
forradas com duas folhas de papel-filtro umedecidas Maringá, v. 31, n. 3, p. 301-305, 2009 Acta Scientiarum. Biological Sciences Germinação de sementes de Cordia glabrata Oliveira et al. apresentaram alta umidade no momento em que
foram colhidas. apresentaram alta umidade no momento em que
foram colhidas. As velocidades de germinação relatadas por
Donadio et al. (2004), para Cordia trichotoma em dois
substratos (areia e papel-filtro), não diferiram
significativamente (1,09 a 1,10). Porém, ambos os
valores são inferiores àqueles observados para C. glabrata com sementes recém-colhidas e após 30 e 60
DA (exceto o substrato arenoso com matéria
orgânica, aos 60 DA), germinando nos três
substratos. Tal fato indica que C. trichotoma germina
mais lentamente, no entanto com maiores taxas de
germinação para sementes recém-colhidas. Seneme
et al. (2006), estudando Allophylus edulis, não
observaram diferenças significativas na velocidade de
germinação em areia e papel-filtro, após 20 dias de
experimento, a 25 ou 30oC. Várias espécies
apresentam queda na velocidade de germinação de
acordo com o tempo de armazenagem, tais como
Tabebuia serratifolia (SOUZA et al., 2005) com
sementes armazenadas por 150 dias em laboratório
(27 ± 3oC e 62 ± 2% de umidade relativa) e
refrigerador (7oC e 54% de umidade relativa),
indicando que a diminuição no vigor das sementes
após determinado tempo de armazenamento é uma
característica da maioria das espécies. Avaliando
a
germinação
de
sementes
de
Peltophorum dubium (Spreng) Taubert em substrato
papel-filtro e arenoso, entre outros, Perez et al. (1999) não encontraram diferenças significativas nos
resultados obtidos. Similarmente, em Allophylus
edulis (St.-Hil.) Radlk. Seneme et al. (2006) não
observaram diferenças em sementes germinadas em
substrato arenoso e papel-filtro, após 20 dias de
armazenamento. Por outro lado, Pacheco et al. (2006) constataram diferenças significativas entre as
taxas de germinação de Myracrodruon urundeuva Fr. All., quando germinada em substrato arenoso e em
papel, somente em temperaturas superiores a 30oC e
alternadas, indicando que o papel-filtro é mais eficaz
quando ocorre aumento de temperatura ou quando são
utilizadas temperaturas alternadas. Temperaturas de 25
e 27oC não apresentaram diferenças significativas na
germinação entre os substratos utilizados. A redução na porcentagem de germinação, em
condições laboratoriais, é observada em espécies
florestais como Schinus terebinthifolius (MEDEIROS;
ZANON, 1998) e Peltophorum dubium Raddi
(PEREZ et al., 1999). Já Schleder et al. (2003),
trabalhando com Tabebuia impetiginosa (Mart.) Standl
e Oliveira et al. (2006), com Tabebuia aurea (Silva
Manso) Benth. & Hook. f. ex. S. Moore,
encontraram taxas superiores a 55% na germinação,
quando as sementes foram armazenadas por até 90
dias, a 24oC. Resultados e discussão
Resultados e discussão
Resultados e discussão
Resultados e discussão não apresentaram Em relação ao tempo de armazenamento, as
sementes de Cordia glabrata não podem ser
classificadas como ortodoxas, visto que o baixo teor
de água na semente não mantém sua viabilidade. Também
não
são
recalcitrantes,
pois
não Maringá, v. 31, n. 3, p. 301-305, 2009 Acta Scientiarum. Biological Sciences 304 Conclusão
Conclusão
Conclusão
Conclusão A porcentagem de umidade nas sementes é
pequena (1,55%) e decai com o tempo de
armazenamento, atingindo 0,47% após 120 dias. A
germinação das sementes foi melhor no papel-filtro,
porém com baixas médias (34%), ocorrendo quedas
significativas nas taxas de germinação após cada
período
de
armazenamento. Os
substratos
autóctones (solo arenoso e solo arenoso como
matéria orgânica) não foram adequados para a
germinação na temperatura testada. A velocidade de
germinação foi maior no papel-filtro, com sementes
recém-colhidas (10), com quedas significativas na
velocidade após cada período de armazenamento. A
viabilidade das sementes, nas condições testadas, não
ultrapassou 120 dias. A velocidade de germinação indica que sementes
recém-colhidas germinadas em substrato papel-filtro
apresentaram maior vigor (10), com redução
significativa
em
função
do
tempo
de
armazenamento. O uso de papel-filtro, como
substrato, permitiu a obtenção das melhores médias
amostrais até 90 DA, quando o substrato arenoso
passou a ter resultados semelhantes (Tabela 3). Os
substratos solo arenoso e solo arenoso com matéria
orgânica apresentaram resultados similares, com
exceção de 60 DA, quando o primeiro revelou maior
vigor (Tabela 3). Ambos os substratos apresentaram
diferenças significativas, em comparação ao papel-
filtro, que foi o mais eficaz. Agradecimentos
Agradecimentos
Agradecimentos
Agradecimentos À Fundação Manoel de Barros (FMB), pelo
financiamento do projeto GIP e ao CNPq, pela
bolsa de iniciação científica concedida. Tabela 3. Velocidade de germinação em sementes de Cordia
glabrata recém-colhidas, com 30, 60, 90 e 120 dias de
armazenamento em três substratos (papel-filtro, arenoso, arenoso
+ matéria orgânica-MO), em BOD, a 26oC. License information: This is an open-access article distributed under the terms of the
Creative Commons Attribution License, which permits unrestricted use, distribution,
and reproduction in any medium, provided the original work is properly cited. Germinação de sementes de Cordia glabrata Germinação de sementes de Cordia glabrata 305 degradadas. In: BAUER, F. C.; VARGAS JUNIOR, F. M. (Ed.). Produção e gestão agroindustrial. Campo
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Referências
Referências
Referências BEWLEY, J. D.; BLACK, M. Seeds: physiology of development
and germination. New York: Plenum Press, 1994. Substrato
Recém-colhidas
30 dias 60 dias 90 dias 120 dias
Papel-filtro
10 aA
7,2 aB 5,2 aC 2,2 aD
0 aE
Arenoso
4,8 bA
3,8 bA 2,4 bB 1,6 abB
0 aC
Arenoso + MO
4,8 bA
3,0 bB 1,0 cC 1,0 bC
0 aD
Médias seguidas na mesma letra minúscula na coluna e maiúscula na linha não diferem
estatisticamente entre si, pelo teste de Tukey, em nível de 5% de probabilidade. BONNER, F. T. Storage of seeds: potential and
limitations for germplasm conservation. Forest Ecology
and Management, v. 35, n. 1/2, p. 35-41, 1990. Maringá, v. 31, n. 3, p. 301-305, 2009 Maringá, v. 31, n. 3, p. 301-305, 2009 Acta Scientiarum. Biological Sciences Received on March 5, 2008. Accepted on November 3, 2008. Germinação de sementes de Cordia glabrata 3, p. 301-305, 2009 Acta Scientiarum. Biological Sciences
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Classifying Pacific islands
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Geoscience letters
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cc-by
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Abstract An earth-science-based classification of islands within the Pacific Basin resulted from the preparation of a database
describing the location, area, and type of 1779 islands, where island type is determined as a function of the prevail-
ing lithology and maximum elevation of each island, with an island defined as a discrete landmass composed of a
contiguous land area ≥1 ha (0.01 km2) above mean high-water level. Reefs lacking islands and short-lived (<20 years)
transient islands are not included. The principal aim of the classification is to assess the spatial diversity of the geologic
and geomorphic attributes of Pacific islands. It is intended to be valid at a regional scale and based on two attributes:
five types of lithology (volcanic, limestone, composite, continental, surficial) and a distinction between high and low
islands. These attributes yielded eight island types: volcanic high and low islands; limestone high and low islands;
composite high and low islands; reef (including all unconsolidated) islands; and continental islands. Most common are
reef islands (36 %) and volcanic high islands (31 %), whereas the least common are composite low islands (1 %). Conti-
nental islands, 18 of the 1779 islands examined, are not included in maps showing the distribution of island attributes
and types. Rationale for the spatial distributions of the various island attributes is drawn from the available literature
and canvassed in the text. With exception of the few continental islands, the distribution of island types is broadly
interpretable from the proximity of island-forming processes. It is anticipated the classification will become the basis
for more focused investigation of spatial variability of the climate and ocean setting as well as the biological attributes
of Pacific islands. It may also be used in spatial assessments of second-order phenomena associated with the islands,
such as their vulnerability to various disasters, coastal erosion, or ocean pollution as well as human populations, built
infrastructure and natural resources. Compared to most inhabited parts of the earth’s sur-
face, the Pacific Basin bounded by continental margins is
different in land areas that are comparatively small and
isolated from one another and from the Basin rim; land
totals around 0.34 % of the area of the Pacific Basin. The
number of people occupying Pacific islands is approxi-
mately 10.8 million, some 0.15 % of the earth’s popula-
tion. REVIEW Open Access © 2016 Nunn et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. Abstract These two attributes—comparatively small land
area and small population size—have inevitably led to
the marginalization of the Pacific islands region in many
global datasets. Marginalization invariably encourages
generalization and it is clear that Pacific islands and their
inhabitants have not been well served by such generaliza-
tions in the past and would not be served well by them
in the future, particularly as the pace of both natural and
societal change accelerates (Crocombe 2001; Connell
2010, 2011; Nunn 2013).h Classifying Pacific islands Patrick D. Nunn1* , Lalit Kumar2, Ian Eliot3,4 and Roger F. McLean5 Patrick D. Nunn1* Nunn et al. Geosci. Lett. (2016) 3:7
DOI 10.1186/s40562-016-0041-8 Nunn et al. Geosci. Lett. (2016) 3:7
DOI 10.1186/s40562-016-0041-8 Introduction
h As the management of human systems has become
more globalized, so the need to effectively capture envi-
ronmental diversity at global and regional scales has
increased. Spanning almost one-third of the earth’s sur-
face, the Pacific presents a daunting challenge to anyone
seeking to generalize about it, whether this refers to its
geology, its ocean, its islands, or the peoples who inhabit
them. In the past, perceptions of uniformity or emptiness
within the Pacific encouraged generalization (Köppen
1936; Ward 1989) and even today, when the diversity of
its natural environment is almost universally acknowl-
edged, there remains a need for informed generaliza-
tion for particular purposes (e.g., Ellison 2009; Dickinson
2001; Mueller-Dombois and Fosberg 1998; Neall and Tre-
wick 2008; Richardson and Richardson 1986). This situation is redressed here by providing an earth-
science-based classification for islands in the Pacific Basin *Correspondence: pnunn@usc.edu.au
1 University of the Sunshine Coast, Maroochydore, QLD, Australia
Full list of author information is available at the end of the article *Correspondence: pnunn@usc.edu.au
1 University of the Sunshine Coast, Maroochydore, QLD, Australia
Full list of author information is available at the end of the article *Correspondence: pnunn@usc.edu.au
1 University of the Sunshine Coast, Maroochydore, QLD, Australia
Full list of author information is available at the end of the article Page 2 of 19 Nunn et al. Geosci. Lett. (2016) 3:7 New Zealand, as well as the Pacific coast of Antarctica. The defined region includes the independent countries
of Cook Islands, Federated States of Micronesia, Fiji,
Kiribati, Marshall Islands, Nauru, Niue, Palau, Papua
New Guinea, Samoa, Solomon Islands, Tokelau, Tonga,
Tuvalu, Vanuatu; the territories of American Samoa,
French Polynesia, New Caledonia, Wallis, and Futuna;
the commonwealth of the Northern Marianas Islands;
the State of Hawaii; the US-administered islands in the
central Pacific; the Galapagos islands (Ecuador); and the
Chile-administered islands in the southeast Pacific. A
summary of the islands included in the database is given
in Table 1. that is simple and readily applicable to a variety of pur-
poses, while also capturing essential elements of island
diversity. The principal aim of this classification is to cap-
ture the spatial diversity of the geologic and geomorphic
attributes of islands in the Pacific region. This is then
expected to become the basis for more focused descrip-
tions of natural (biogeographic, ecological, pedologic) sys-
tems as well as second-order phenomena associated with
Pacific islands, such as their exposure to climate change
and natural disasters, as well as their vulnerability to
coastal erosion or ocean pollution, for example. This paper first explains the basis for classification,
describes data sources and identifies the island types, and
presents a first-order analysis of the database before dis-
cussing ways in which it might be applied. Islands that are clearly part of continents and/or are
adjacent to the rim are not included; for ease of applica-
tion, nations have not been subdivided into islands and
non-islands except in rare instances. Islands in Indonesia,
Japan, and the Philippines are excluded, largely because
most have a peri-continental location and origin, whereas
the islands of Papua New Guinea (including the large
island of New Guinea) are included because they are
mostly of oceanic origin; note that because it skews the Basis for classificationh The region of interest for this classification is arbitrary
yet usual in most discussions of islands within the Pacific
Basin (Fig. 1). The outer boundary is the continental
Pacific rim, comprising the west coasts of the Americas,
the east coasts of Asia (including Japan), Australia and Fig. 1 The study region showing the locations of the island countries and groups listed in Table 1. Plate boundaries (in red) and hotspots active
within the past 43 Ma (orange circles) are also shown. Active convergent plate boundaries are shown by lines with triangles pointing in the down-
thrust direction; all other plate boundaries are undistinguished, although most are transform except for most of the East Pacific Rise where diver-
gence occurs. Hotspot locations are from King and Adam (2014) Fig. 1 The study region showing the locations of the island countries and groups listed in Table 1. Plate boundaries (in red) and hotspots active
within the past 43 Ma (orange circles) are also shown. Active convergent plate boundaries are shown by lines with triangles pointing in the down-
thrust direction; all other plate boundaries are undistinguished, although most are transform except for most of the East Pacific Rise where diver-
gence occurs. Hotspot locations are from King and Adam (2014) Fig. 1 The study region showing the locations of the island countries and groups listed in Table 1. Plate boundaries (in red) and hotspots active
within the past 43 Ma (orange circles) are also shown. Active convergent plate boundaries are shown by lines with triangles pointing in the down-
thrust direction; all other plate boundaries are undistinguished, although most are transform except for most of the East Pacific Rise where diver-
gence occurs. Hotspot locations are from King and Adam (2014) Page 3 of 19 Page 3 of 19 Nunn et al. Geosci. Lett. Basis for classificationh (2016) 3:7 Table 1 Summary of key data in island database
Note that the island of New Guinea is excluded from the data analysis in this table because of its great size which skews the analysis
a This group is comprised mostly of the Galapagos islands, politically part of Ecuador
b Average island areas for these atoll countries are overestimates as they are based on polygons that subsume multiple islands (see text)
c Note that the large island of New Guinea is excluded from this analysis because it is shared between Papua New Guinea and Indonesia
Country/group of islands
Number
of islands
Total area
of islands (km2)
Average island
area (km2)
Average island
maximum elevation (m)
American Samoa
7
222
32
396
Chilean islands (eastern Pacific)
2
215
108
269
Cook Islands
15
297
20
73
East Pacific outliersa
24
8236
343
509
Federated States of Micronesia
127
799
6
45
Fiji
211
20,857
99
134
French Polynesia
125
3939
31
154
Guam
1
588
588
400
Hawaii
16
19,121
1195
869
Kiribati
33
995
30b
6
Marshall Islands
34
286
8b
3
Nauru
1
23
23
71
New Caledonia
29
21,613
745
121
Niue
1
298
298
60
Northern Mariana islands
16
537
34
444
Palau
33
495
15
58
Papua New Guineac
437
67,754
155
135
Pitcairn islands
4
54
13
97
Samoa
7
3046
435
504
Solomon Islands
415
29,675
72
88
Tokelau
3
16
5b
5
Tonga
124
847
7
56
Tuvalu
10
44
4b
4
US-administered islands (central Pacific)
8
37
5
5
Vanuatu
81
13,526
167
330
Wallis and Futuna
14
190
14
94
Total
1778
1,93,712
171
190 Table 1 Summary of key data in island database data analysis, the island of New Guinea is excluded from
the analysis in Table 1. The continental-outlier islands
of New Caledonia (southwest Pacific) are also included
because they are located within the Pacific Basin and sur-
rounded by oceanic crust. the shores of larger islands, that are close to the lower size
limit. Reefs lacking islands are not considered as islands,
although they are in some island lists [e.g., Motteler
(2006)]. Transient islands, such as those that may appear
or disappear during storms, are not included unless they
had existed continuously for more than 20 years at the
time of data collection in 2013. An island database and data sourcesi A database of islands in the Pacific Basin (n = 1779),
summarized in Table 1 and available as Additional File
1, refers to all islands ≥1 ha (0.01 km2) in area in the
study region. For each island, data on lithology and eleva-
tion were acquired, allowing island type to be identified. Reef islands scattered along an elongate reef are counted
as one island rather than several islands because such
islands are often transient, changing shape and size over
decadal periods, sometimes connected to others (at low
tide or by causeways), sometimes severed by subtidal
channels (after storms). All that was sought from cross-checking was that the
latitude and longitude recorded in Google Earth were
approximately the same as that recorded previously. This
was an important check on whether the correct (named)
island had been identified in Google Earth. Where minor
differences were found in the coordinates for a particular
island, it was assumed that those in Google Earth were
superior. Often a disparity was found to arise because
coordinates from Google Earth were centered within a
particular island, whereas coordinates from other (ear-
lier) sources for an island were often those of a particu-
lar place of interest (like a coastal settlement) within that
island not its geographical center. Diagnostic data including location and shape for each
island were obtained from the world vector shorelines
(WVS) digital data file which contains shorelines along
the ocean-land interface (Soluri and Woodson 1990). At
a scale of 1:250,000, the WVS is a US Defense Mapping
Agency product that supports geographical information
systems at regional and global scales. Since the WVS was
created at very broad scales from satellite imagery, there
will inevitably be slight distortions in the positional accu-
racy and shapes of the islands, although for the spatial
scale and resolution of this work, this data source was
deemed acceptable. g
g
The latitude and longitude information thus recorded
were then converted into a GIS shapefile using the World
Geodetic System 1984 (WGS84) coordinate system. This
file was then overlaid on a WVS polygon file to allow
island names to be assigned to each of the polygons. Since the WVS map layer is created at a very broad scale
and coarse-scale satellite imagery is used, there were
inevitably minor inaccuracies in positions and shapes
of islands. Basis for classificationh Likewise islands that for-
merly existed, perhaps at times of lower sea level in the
past, are not included. The data used in this paper repre-
sent a snapshot of the situation in the Pacific around the
start of the 21st century.hi A fundamental question for any discussion about the
common characteristics of islands is “what is an island?”. For this classification, which treats only Pacific islands,
the answer to that question is easier than for global sur-
veys (Fairbridge 1968; Grigor’yev 1971; Kaplin 1981;
Nunn 1994) because the area of interest (the Pacific
Basin) is geographically bounded. All discrete land-
masses within this area that comprise above mean high-
water level contiguous areas of land ≥1 ha (0.01 km2) are
considered islands. This includes some as large as New
Guinea (~786,000 km2) to numerous others, mostly off The purpose of any classification of natural phenom-
ena (such as islands) is the key to its structure. Given that
our classification seeks to capture the diversity of physi-
cal and natural attributes of islands, the most appropriate
classification is one based at its highest level on lithology Nunn et al. Geosci. Lett. (2016) 3:7 Page 4 of 19 Page 4 of 19 Page 4 of 19 Many reef islands that form atolls, for example, are long
sinuous entities and sometimes a degree of subjective
judgement is needed to identify their approximate center. Sometimes, it is unclear whether two islands form a sin-
gle continuous body or not; indeed, it is possible that
they do at low tide (when people may be accustomed to
walk from one to the other) but not at higher tide levels. and elevation. The choice of these variables reflects the
dominant controls on a broad range of characteristics
of Pacific islands, including their erodability and resist-
ance, their drainage (surficial and subterranean), and
their landscapes (and major landscape processes). It was
decided not to include a measure of climate (because this
is implicit in elevation) nor any measure of exposure to
particular natural hazards (like volcanic eruptions, earth-
quakes, tsunamis, tropical cyclones) given that this is
highly variable both spatially and temporally within the
Pacific Basin and therefore more suited to a second-order
layer of classification. Island locations In the island database, latitude and longitude were
obtained from Google Earth for almost all islands, the
cursor being placed over the center of an island and
coordinates recorded. The advantage of this approach is
its consistency and the resulting impossibility of islands
overlapping in location. One disadvantage is that a single
pair of coordinates does not say anything about the size
or shape of a particular island or its proximity to near-
est neighbors. This is less of a problem with islands (of
any size) that are approximately circular in form but does
pose challenges for those that are less uniformly shaped. Basis for classificationh While having no reason to doubt the accuracy of the
latitudes and longitudes reported in Google Earth for the
vast majority of islands, each location was cross-checked
for correctness against another source, usually a list of
islands generated for a particular country or island group. In some cases, usually for smaller, more isolated island
groups, the only source of earlier locational information
found was a map, from which coordinates were read. For
some smaller (typically more isolated) islands, no source
of latitude and longitude other than Google Earth was
readily available. An island database and data sourcesi With a world base map provided by ESRI as
a background, individual WVS polygons were moved
where necessary to align with this base map. Where the
WVS polygons were not a good match with the actual
island shape or where polygons were missing, especially
for smaller islands, new polygons were digitized from the
background base map at a scale of 1:20,000. These poly-
gons were more accurate than those obtained from the
WVS. In the following subsections, sources of key data (island
locations, names, elevations, areas, lithologies) for these
two databases are discussed separately. Island namesh The main reference for island names was Motteler (2006). This text is widely regarded as the standard listing of
islands in particular Pacific island groups, although it is Nunn et al. Geosci. Lett. (2016) 3:7 Page 5 of 19 incomplete. In some island groups, the names of even
sizeable islands are not listed, while others may be spelled
in ways that are clearly not those preferred by the mod-
ern nation of which the particular island is part. In some
island groups, the names of even the smallest islands—
often uninhabited rocks of less than 0.01 ha in area—are
given, while in others they are omitted. Many reefs (not
reef islands) are also included in the Motteler list but were
omitted from the database for this paper. The prevalence of
European names (and earlier European names) for islands
rather than current (and previous) local/vernacular names
is a weakness of the Motteler list (2006); one source that
was especially useful in rectifying this was Langdon (1978)
who gives two gazetteers of equivalent and obsolete names
for many islands. Despite such concerns, by and large,
the Motteler text represents the most complete source of
information about Pacific island names and is accompa-
nied by a series of informative maps. the Pacific regional sources used, several were espe-
cially helpful for providing context (Gillespie and Clague
2009; Menard 1986; Neall and Trewick 2008; Nunn 1994,
1998b, 1999; Vacher and Quinn 1997; Wiens 1962),
while at a subregional level, a wider range of publications
proved useful (including Anthony 2004; Bonatti et al. 1977; Bonvallot et al. 1993; Brocher 1985; Coleman 1970;
Dana 1875; Davis 1920; Derrick 1957; Dow 1977; Dun-
can and McDougall 1976; Greene and Wong 1988; Jost
1998; Keating and Bolton 1992; Macdonald et al. 1983;
McBirney et al. 1969; Scholl and Vallier 1985; Tracey
et al. 1964; Wood 1967).h There are also numerous sources of sub-national
(island group) information about lithology available, usu-
ally in the form of academic publications (found in librar-
ies but identified using Google Scholar) that describe the
geology of a particular island or group of islands. While
invariably focused on research questions requiring more
than descriptions of island lithology, these are often
included as background details and were extracted to
include in the database. Google Earth and Google maps often provide island
names, although these may be misplaced, especially
with the smaller islands, or misspelled. Island namesh It was found that
Google Earth often confuses islands of the same name
(or similar names) and as a result in some instances may
place the names considerable distances from the islands
to which they correctly refer. The use of photos (loaded
by users without consistent quality control) in Google
Earth is helpful but potentially a source of serious error
both not only because (island) names are often wrong
but also because the photos may be wrongly located, par-
ticularly in more isolated areas, less frequently visited by
tourists. Other sources of locational information were
obtained from various atlases, maps, and encyclopedias
as well as a variety of internet sources, although these are
not always reliable. Solely online sources of information about island lithol-
ogy were used principally to confirm details obtained
from library sources (see above). Online sources included
photographs in Google Earth which, unless misplaced
(see above), were sometimes able to provide good infor-
mation about lithology. The most useful photographs
were those off/from the island’s coast which sometimes
show the composition of cliffs or the form of the island
itself, both of which aid in confirming its dominant
lithology. While both volcanic and limestone islands
have cliffs, those of the latter generally tend to be flatter-
topped and straighter-sided. The form of an emerged
limestone island is generally marked by flat tabular sur-
faces (former reef surfaces) in stark contrast to the form
of volcanic islands where flat surfaces are rarer and the
topography is often dominated by peaks and valleys. Island lithologies Lithology is considered a primary basis for classification
because it is fundamental to numerous physical-natural
attributes of islands as well as their recent development
through which it links to island-forming processes (Her-
zberg 2011; Walsh 1982). It is a major cause of diversity
in the Pacific yet there are dangers in being too specific,
given that few islands are effectively of just one lithology
and that different lithologies sometimes have comparable
attributes. For this reason and in order to make this clas-
sification as easy to use as possible, we distinguish five
types of lithology—volcanic (igneous), limestone (cal-
careous and non-volcanic sedimentary), composite (less
than 80 % volcanic and less than 80 % limestone), reef
(unconsolidated sediment) islands, and continental (non-
oceanic) islands. Supplementary online sources used were encyclope-
dias, generally Wikipedia, that not only gives accounts
and photographs of particular islands, but also links to
more formal and more reliable reference materials. The
authors’ own knowledge was used to verify lithologies of
particular islands. Island elevations Elevation is also considered an important first-order clas-
sifier because it reflects aspects of lithology as well as
recent island development, capturing both island build-
ing (including tectonics) and denudation. It is also a cause
of significant diversity in the Pacific and it is suggested
that a simple twofold distinction between high and low
islands, separated by a 30-m elevation above mean sea
level, best captures this. The use of 30 m as the divider Archival (library) sources of information about Pacific
island lithology are too numerous to review here. Of Page 6 of 19 Nunn et al. Geosci. Lett. (2016) 3:7 in the Pacific based on the lithology and elevation of
each island. The classification involves eight categories,
described below. between high and low is arbitrary but in our experience
best separates lower (less resistant, greater surface low-
ering) islands from higher (more resistant, less denuded)
ones. Similar classificatory procedures have been used
to separate younger (upstanding) volcanic islands from
older (more denuded) ones (Menard 1983; Ramalho et al. 2013). Volcanic high islands are those composed of at least
80 % igneous rock types that rise to a maximum eleva-
tion of at least 30 m above mean sea level. In the Pacific,
these island types are the commonest in those places
where there is active (usually submarine) volcanism
within 500 km. Such places may be in the volcanic island
arcs that develop above convergent plate boundaries or
around mid-plate hotspots where magma reaches the
ocean surface and builds chains of volcanic islands. For the purposes of this project, the only information
about elevation required for classification is whether or
not the maximum elevation of a particular island is less
than 30 m (above mean sea level) or not. This informa-
tion is a crucial component of island type, and is obtain-
able using Google Earth but, after using this a few times,
it became clear that errors in some instances were poten-
tially large (there were apparent differences between the
elevation at Point A in Google Earth and that on a top-
ographic map of the island) and that it was not always
straightforward to identify the highest point on a par-
ticular island with this tool. Island areas Areas for islands were calculated directly from the poly-
gon shapefile layer in a GIS as described in “Island loca-
tions” section. While published records of island areas
exist, these commonly differ with reference to the same
island. In addition, it is likely that some island areas stated
as referring to single entities incorporate smaller offshore
islands, so are unreliable. With these constraints in mind,
published information about island area from various
sources was used to confirm the automatically calcu-
lated areas for most islands in the database. We accept
that areas of islands calculated from polygons generated
from coarse-scale satellite imagery would have larger
errors than those calculated from fine-scale topographi-
cal maps, yet for the broad-scale work undertaken in this
research this level of inaccuracy was deemed acceptable. The areas calculated from the polygon shapefile were
generally within the range of figures obtained for individ-
ual islands from different published sources. The calcula-
tion of all areas using the polygon shapefile ensured that
whatever errors were present were consistent. Limestone low islands are those which are composed
of at least 80 % calcareous rock types and rise to a maxi-
mum elevation of less than 30 m above mean sea level. In the Pacific, these island types are common where high
limestone islands are found (see above), the distinction
often being largely attributable to the degree of net uplift
that particular islands have experienced. Low limestone
islands are also found in many places (not tectonically
active) where reef islands are found (see below) and it is
sometimes difficult to distinguish the two types. The low
limestone islands tend to be significantly older and typi-
cally formed (in part at least) from reefs that were living
around 6 m above present sea level during the Last Inter-
glacial period, around 125,000 years ago and sometimes
during earlier Quaternary interglacial periods. Island elevations Thus, for the purposes of
determining island maximum elevation, other (library,
online, expert knowledge) sources of information (espe-
cially Dahl 1980; Karolle 1993; Lobban and Schefter 1997;
Mueller-Dombois and Fosberg 1998; Rapaport 2013)
were used with Google Earth providing corroboration
only. The authors’ own knowledge was used to verify
maximum elevations of particular islands. Volcanic low islands are those which are composed of
at least 80 % igneous rock types and rise to a maximum
elevation of less than 30 m above mean sea level. In the
Pacific, these island types are common not only where
higher volcanic islands are found but also tend to occur
farther away from contemporary sites of active volcan-
ism. Along hotspot island chains, the highest islands are
usually close to the hotspot itself while farther away, the
volcanic islands (largely through subsidence) are gener-
ally lower. Elsewhere, once removed from the area in
which volcanic islands form, such islands also subside
and become reduced in elevation by subaerial erosion
(Scott and Rotondo 1983). Limestone high islands are those which are composed
of at least 80 % calcareous rock types and rise to a maxi-
mum elevation of at least 30 m above mean sea level. In
the Pacific, these island types are commonest in those
places where (tectonic) uplift has been occurring for
several 100,000 years, most commonly as a result of one
(oceanic) plate being pushed up over another as is usual
along convergent plate boundaries. High limestone
islands commonly develop along such forearcs. Else-
where collision/compression of lithospheric plates may
cause uplift of islands in places where subduction is not
taking place. Results: outcomes of classification Several primary attributes for each of 1779 islands in
the Pacific Basin, namely island location, area and type,
where type is determined by the dominant lithology and
maximum elevation, were compiled in a database. These
attributes were then used to produce maps of the Pacific
at a broad scale to establish the spatial diversity of the
geologic and geomorphic attributes of the islands. The
maps were generated by representing each island in the
database as a single point (Figs. 2, 3, 4, 5 and 6). i Islands are distributed unevenly throughout the Pacific
Basin (Fig. 2), the most numerous being in the southwest
quadrant and fewest in the northeast quadrant. They are
clustered, mostly in linear patterns, yet there are vast
tracts of landless ocean, especially in the eastern Pacific
(approximately 140–100°W) and in high-latitude areas. Areas were determined for all islands in the database
(Fig. 3). The map in Fig. 3 shows the distribution of
islands by size, the key shows the proportion of islands
in each size category. The distribution is dominated by
small islands, with 67 % <10 km2 and 44 % <1 km2 in area. Large islands, those with an area >100 km2, comprise
approximately 6 % of the 1779 islands in the database.i Composite high islands are those which are composed
of both less than 80 % volcanic and less than 80 % calcar-
eous (limestone) rock types and rise to a maximum eleva-
tion of at least 30 m above mean sea level. In the Pacific,
these island types are commonest in places where long-
term subsidence (allowing thick reefs to develop around a
volcanic island) has been interrupted by long-term uplift
(causing those reefs to emerge) resulting in the forma-
tion of makatea islands (Nunn 1994). Other more diverse
composite high islands occur around convergent plate
boundaries, typically formed over long time periods by
processes associated with alternating volcanism (along a
volcanic island arc) and uplift (along a forearc). Five lithological types are recognized for Pacific islands:
composite, continental, limestone, reef, and volcanic. Their spatial distribution is shown in Fig. 4 with propor-
tions given in the key. Results: outcomes of classification Volcanic (39 %) and reef (36 %)
lithologies are the most common; limestone (17 %) and
composite (7 %) follow, islands with continental litholo-
gies are few (1 %).i All islands in the database are classified as either high
(maximum elevation of 30 m or higher) or low (maxi-
mum elevation <30 m), although five elevation catego-
ries have been identified for the purposes of discussion
and are shown in Fig. 5. 45 % of the islands are lower
than 30 m, with 27 % less than 5 m. In contrast, 26 % are
higher than 100 m. Composite low islands are those which are composed
of both less than 80 % volcanic and less than 80 % cal-
careous rock types and less than 80 % unconsolidated
sediments (forming reef islands) and rise to a maximum
elevation of less than 30 m above mean sea level. In the
Pacific, these island types are common where compos-
ite high islands are found, the difference often being that
island-forming processes (volcanism and/or uplift) were
less active and therefore produced a lower island. Also
it is clear that such processes often produced compos-
ite high islands that are comparatively large surrounded
by smaller islands, representing peripheral parts of the
structure, that are lower. Island type is the combination of lithology and eleva-
tion and is regarded as the highest-level basis for mean-
ingful classification of Pacific islands. The distribution of
islands by type is shown in Fig. 6 with the key showing
the proportions of each. Perhaps unsurprisingly, the most
common types are reef islands (36 %) and volcanic high
islands (31 %) and the least common are composite low
islands (1 %). Continental islands, 18 of the 1779 islands
examined, are not included in the classification. Continental islands are those which are composed of
at least 80 % continental (not of oceanic origin) rocks. Given that these island types are so few within the study
area and that almost all are above 30 m maximum ele-
vation, this island type is not subdivided on the basis of
elevation. Such islands are found exclusively in the New
Caledonia group. Island types Using the data described in “An island database and data
sources” section, we produce a classification of islands Nunn et al. Geosci. Lett. (2016) 3:7 Page 7 of 19 Page 7 of 19 Reef islands are those which are composed of at least
80 % unconsolidated sediments (derived from adjacent
offshore/underwater areas) that have accumulated on a
shallow flat (shoal), commonly biogenic (reefal) in ori-
gin. While sometimes confused with lower types of low
limestone island (see above), reef islands tend to rise no
more than 3 m above mean sea level and are character-
ized by shorelines that change position faster than those
of other island types. Often reef islands are long and sin-
uous, accumulated on sub-circular (atoll) reefs that have
grown up from the submerged flanks of a drowned vol-
canic island. Reef islands also develop on other types of
reef (Stoddart et al. 1978). Of all the island types in this
classification, reef islands are the most transient, many of
those that exist today having formed only after sea level
in the Pacific began falling from its Holocene maximum
around 4000 years ago. Reef islands are widespread in the
low-latitude Pacific, concentrated along atoll reefs in its
equatorial central part. Results: outcomes of classification Discussion A rationale for the separate spatial distributions of island
types and their attributes (Figs. 2, 3, 4, 5 and 6) is consid-
ered below with the distributions interpreted in the con-
text of available information. Additionally, preliminary Nunn et al. Geosci. Lett. (2016) 3:7 Page 8 of 19 Fig. 2 Island locations within the Pacific Basin showing their relationship with principal island-forming island locations (plate boundaries and
hotspots from Fig. 1) Fig. 2 Island locations within the Pacific Basin showing their relationship with principal island-forming island locations (plate boundaries and
hotspots from Fig. 1) (to the west) or the Cook Islands (to the east). Other
islands countries/groups with comparatively few num-
bers (see Table 1) may also be artifacts of history as well
as geographical isolation. statistics describing the number of islands, their total and
average area and average maximum elevation have been
determined for each country or group of islands included
in the database (see Table 1).h Those countries with the greatest number of islands—
Papua New Guinea, Solomon Islands and Fiji—are coun-
tries with comparatively large populations, most of whom
subsist from the land on which they reside. That these
islands are also quite large (>31 km2) and high (>88 m)
exemplifies the points that the larger an island, the more
possibilities it generally presents for human livelihoods,
and the higher the island, the more likely it is to receive
sufficient rainfall for a diversity of human needs. Island locationsh The pattern of islands in the Pacific Basin can be broadly
interpreted by the prevalence of (the most effective)
island-forming processes in particular areas. The excep-
tions, which are not a central focus of this paper, are the
continental islands in New Caledonia (SW Pacific) that
represent emergent parts of a sliver of continental litho-
sphere that acquired its present oceanic location only
following the opening of the Tasman Sea about 84 Ma
(Lafoy et al. 2005). All other Pacific islands originated as
ocean-floor volcanoes, which means that the locations of
most of them today can be linked to places where ocean-
floor volcanism continues or where it ceased within the
past few million years (Nunn 1999).h fi
At the other end of the scale, generalization is less
straightforward. There are several single island countries/
groups, most born from distinct political histories rein-
forced in some cases by relative geographical isolation. Examples include Nauru, which owes its independence
to a history of phosphate extraction and indeed its phos-
phorites to its geographical isolation (the only landmass
for roosting birds within a large area), and Niue, inde-
pendent largely because of similar isolation which pre-
vented it from becoming an obvious part of either Tonga There are three principal geophysical situations in the
Pacific Basin in which ocean-floor volcanism occurs
(Frisch et al. 2011; Duncan and Richards 1991; Husson
and Conrad 2012; Nunn 1994). The first is at a divergent Nunn et al. Geosci. Lett. (2016) 3:7 Page 9 of 19 Fig. 3 Areas of islands within the Pacific Basin with plate boundaries (not hotspots) also shown ig. 3 Areas of islands within the Pacific Basin with plate boundaries (not hotspots) also shown Fig. 3 It is apparent that there are very few islands close to
divergent plate boundaries in the Pacific, largely because
both the crests and (steep-sided) flanks of most mid-
ocean (or back-arc basin) ridges are in deep ocean. Following island formation at the crests, islands are
generally carried into deeper water and often may not
reach the surface until they cross a lithospheric swell, for
instance, which may be thousands of kilometers from the
plate boundary at which they originated. For this reason,
divergent plate boundary islands are comparatively few
and usually emergent only when other processes have
contributed to their upgrowth. Island locationsh Examples from the Pacific
Basin were once thought to include Easter Island, which
is close to the mid-ocean ridge (MOR) named the East
Pacific Rise, but no evidence of MOR basalts is found
there (Ray et al. 2012). Better examples come from the
back-arc basins in the western Pacific where small MORs
have produced islands like Niuafo’ou (Tonga) and Mota
Lava (Vanuatu) (Sorbadere et al. 2013; Tian et al. 2011). plate boundary (often a mid-ocean ridge) and involves
magma rising up from the asthenosphere at places
where the overlying lithosphere is fractured and diverg-
ing. The second is at a convergent plate boundary, above
the place where a downthrust lithospheric plate is melt-
ing as a result of subduction. The third is in intraplate
(mid-plate) situations where a (mantle) plume has pene-
trated through the lithosphere to create a hotspot, which
is comparatively stationary relative to the movement
of the lithospheric plate. In all three situations, island
building is attributable to both extrusive volcanism and,
often more importantly, to intrusion. While allowing for
islands to have moved considerable distances since their
time of origin, the simplest explanation for the locations
of Pacific islands is, depending in which of the three geo-
physical situations they formed, their proximity to con-
vergent plate boundaries or intraplate hotspots. Figure 2
shows the distribution of divergent and convergent plate
boundaries and intraplate hotspots (active since 43 Ma)
in the Pacific relative to the distribution of islands. Com-
pared to other ocean basins, divergent plate bounda-
ries (mid-ocean ridges or MOR) are less numerous in
the Pacific, while convergent plate boundaries are more
numerous and concentrated in the western Pacific. plate boundary (often a mid-ocean ridge) and involves
magma rising up from the asthenosphere at places
where the overlying lithosphere is fractured and diverg-
ing. The second is at a convergent plate boundary, above
the place where a downthrust lithospheric plate is melt-
ing as a result of subduction. The third is in intraplate
(mid-plate) situations where a (mantle) plume has pene-
trated through the lithosphere to create a hotspot, which
is comparatively stationary relative to the movement
of the lithospheric plate. In all three situations, island
building is attributable to both extrusive volcanism and,
often more importantly, to intrusion. Island locationsh While allowing for
islands to have moved considerable distances since their
time of origin, the simplest explanation for the locations
of Pacific islands is, depending in which of the three geo-
physical situations they formed, their proximity to con-
vergent plate boundaries or intraplate hotspots. Figure 2
shows the distribution of divergent and convergent plate
boundaries and intraplate hotspots (active since 43 Ma)
in the Pacific relative to the distribution of islands. Com-
pared to other ocean basins, divergent plate bounda-
ries (mid-ocean ridges or MOR) are less numerous in
the Pacific, while convergent plate boundaries are more
numerous and concentrated in the western Pacific. Far more numerous in the Pacific (but not in other
ocean basins) are islands formed at convergent plate
boundaries. Such islands are invariably organized in lin-
ear or arcuate groups and have often not moved far from
the places at which they formed. Such islands include Nunn et al. Geosci. Lett. (2016) 3:7 Page 10 of 19 Fig. 4 Lithology of islands within the Pacific Basin with plate boundaries (not hotspots) also shown ig. 4 Lithology of islands within the Pacific Basin with plate boundaries (not hotspots) also shown Fig. 4 chain, which is believed to have been forming for at least
the past 81 million years as a consequence of the Pacific
Plate passing over a hotspot currently located just south-
west of Hawai’i island (the Big island) at its southwest end
(Ballmer et al. 2011). Most hotspot chains in the Pacific
are younger. Those that have formed most islands include
the Caroline, Marquesas, Macdonald, Samoa, and Soci-
ety hotspot chains, all of which have been active within
the past 1.2 million years (Clouard and Bonneville 2001;
Wessel and Kroenke 2008). At such hotspots, a line of
volcanic islands is produced by the movement of the lith-
ospheric plate over a hotspot. Island locationsh Generally the further away,
an island is from the hotspot, the more it has subsided
so the older and smaller/lower it is, which explains why
many of the oldest islands in the Hawaii-Emperor chain
became atolls and finally (undersea) guyots (Nunn 1994).h (volcanic) islands that form above the zones of subsurface
magma accumulation, emerged (limestone) islands that
form adjacent to the places (forearcs) where one plate
is pushed beneath another, and (composite) islands that
represent amalgams of the preceding types that are often
found in remnant locations at a distance from contem-
porary subduction zones (Nunn 1994). In the southwest
Pacific, the three types are geographically separated in
Solomon Islands and Vanuatu (Greene and Wong 1988;
Petterson et al. 1999). More common are en echelon lines
of limestone (forearc) and volcanic islands running paral-
lel to an adjacent trench axis, such as are found in Tonga
and the Marianas islands (Nunn 1998b; Oakley et al. 2009). In their simplest expression, usually where sub-
duction began only comparatively recently, a single line
of young volcanic islands is found; examples are found
in the Aleutian and Kurile groups in the northernmost
Pacific as well as parts of Papua New Guinea (Prueher
and Rea 2001; Silver et al. 2009). i
There are a small number of often isolated islands in
the Pacific whose origin does not readily fall into one of
the categories discussed above. These include islands that
rise from locally thickened oceanic crust, possibly ter-
ranes, such as those of Ontong Java (Solomon Islands)
and Manihiki (Cook Islands) (Taylor 2006), and those
that originated at complex plate boundaries, including
plate triple junctions, such as Simbo and others in the Around 43 Ma there was a major change in the direc-
tion in which the rigid Pacific plate was moving as a result
of which the oldest hotspot island chains on this plate all
exhibit a marked kink at this point. The best-documented
example is the Hawaiian-Emperor island-seamount Nunn et al. Geosci. Lett. (2016) 3:7 Page 11 of 19 Fig. 5 Maximum elevation of islands within the Pacific Basin with plate boundaries (not hotspots) also shown Fig. Island locationsh 5 Maximum elevation of islands within the Pacific Basin with plate boundaries (not hotspots) also shown westwards what would have been a much smaller island-
free area of ocean before this time.h New Georgia group (Solomon Islands) (Crook and Taylor
1994) as well as the Galapagos islands (Smith et al. 2013). It is also important to comment on the areas within the
Pacific Basin from which islands are absent. Islands tend
to be generally absent from higher latitudes (>30°N and
>30°S) as well as from most areas around the East Pacific
Rise. The absence of islands at higher latitudes in the
Pacific can partly be explained by the cooling of ocean-
surface waters that prevent submerged islands continu-
ing to have surface expression through the upgrowth
of (atoll) reefs (Nunn 1994). This certainly explains dis-
continuities in linear island chains such as the Hawaiian
group but is also a function of the relative inability of
island-forming processes to create and sustain emergent
islands (rather than seamounts and guyots) in such areas. For example, the southernmost hotspot shown in Fig. 2,
the Louisville hotspot, has a comparatively low level of
volcanic activity and has not generated significant lith-
ospheric swells (Sleep 1990) which may be a prerequisite
to island emergence in such situations. The absence of islands around the region’s major diver-
gent plate boundary, the East Pacific Rise, is explainable
by its steep undersea flanks and the likelihood that any
islands formed in such places would soon get carried into
deeper water. Island areash The dominance of smaller (≤10 km2 in area) islands in
Fig. 3 can be explained in two ways. First, since the Last
Glacial Maximum (LGM), when Pacific sea level was per-
haps 120 m lower than today (Oba and Irino 2012), island
coasts have been progressively drowned resulting in their
fragmentation into smaller islands. This is suggested by
the common situation where innumerable smaller islands
are found close to the coast of larger ones, to which they
were once joined. Instinctively this may be considered to
have added to the number of islands in the Pacific since
the LGM but, unless the number to have been com-
pletely submerged by subsequent sea-level rise is known,
this cannot be demonstrated. The second reason is that
once sea level stabilized in the Pacific, beginning around The absence of islands in the northeast quadrant
of the Pacific may be a result of the change in Pacific
Plate motion that occurred 43 Ma, which has stretched Nunn et al. Geosci. Lett. (2016) 3:7 Page 12 of 19 Fig. 6 Types of islands within the Pacific Basin with plate boundaries (not hotspots) also shown ig. 6 Types of islands within the Pacific Basin with plate boundaries (not hotspots) also shown location along the boundary between the Pacific and
Indo-Australian Plates (Nunn 1998b). Elsewhere, large
islands have been created recently by effusive volcan-
ism, the comparative youth of this explaining why they
remain large. Good examples are found close to hotspots
near Easter Island, in the Galapagos, Hawaii and parts of
French Polynesia. Although the shield volcano on which
the island of Savai’i in Samoa is built originated at a hot-
spot >300 km east, its large size (1694 km2) is explainable
by younger volcanism associated with rifting along the
adjacent Tonga Trench (Keating 1992). 6000 years ago (Grossman et al. 1998), broad reef plat-
forms around the fringes of many islands began devel-
oping and came to provide substrates on which islands
made of surficial materials might then form. Island areash Although
strings of such islands (motu) are not counted separately
in the database (see above), they nevertheless contrib-
ute a substantial number, typically to the population of
islands ≤10 km2 in area.h The comparative paucity of larger islands can not only
be explained by their tendency to be fragmented by sea-
level rise (see above) but also by the comparative diffi-
culty of building and maintaining large islands in oceanic
settings. Most large islands in the Pacific Basin are anom-
alous in origin, typically when several distinct island-
building processes have come into play at perhaps the
same time. By far the largest island in the database, New
Guinea (138,958 km2) is an extreme example but none-
theless registers the effects of multiple island-building
processes operating alone or simultaneously in various
parts at various times above part of the Australian (con-
tinental) craton (Davies 2012). The origin of Viti Levu
(10,388 km2), the largest island in the Fiji archipelago, is
similar, having been created along a succession of island
arcs from perhaps 45 Ma until 3 Ma owing to its singular Tectonism also plays a role in the formation of large
islands, particularly along island forearcs adjoining con-
vergent plate boundaries where successive upthrusting of
reefs and shallow-water sediments can create large lime-
stone islands. Islands like Espiritu Santo (3885 km2) in
Vanuatu and Vava’u (260 km2) in Tonga were created in
this fashion (Greene and Wong 1988; Nunn 1998b). The distribution of large islands (>100 km2) in Fig. 3 is
closely linked to that of the sites of principal island-form-
ing processes along convergent plate boundaries and near
hotspots. From Tonga and Samoa through Fiji, Vanuatu
and Solomon islands to Papua New Guinea in the south-
west Pacific, there are numerous large islands associated Page 13 of 19 Nunn et al. Geosci. Lett. (2016) 3:7 In this classification, islands composed of less than
80 % volcanic material (and less than 80 % limestone)
are classified as composite, so the distribution of vol-
canic islands (see above) is a good guide only to recent/
contemporary volcanism. Island areash Composite islands of domi-
nantly volcanic composition occur mainly near conver-
gent plate boundaries in the western Pacific, particularly
in situations where volcanic islands sensu stricto have
been exposed to prolonged tectonic activity that may
have caused them to become conjoint with emerged reef
(limestone) or other sedimentary formations (reef island)
associated with interarc basin compression and uplift. Examples are common in Papua New Guinea and along
the relict island arcs in Solomon Islands and Vanuatu that
were isolated from proximal volcanism following a switch
in subduction direction around 15–11 Ma attributed to
the blockage of the ancient Vitiaz Trench by the Ontong
Java Plateau (Taylor 2006; Wessel and Kroenke 2000). with the presence of the long-active convergent bound-
ary between the Pacific and Indo-Australian plates here. Other large islands are dotted along the convergent
boundary in the western part of the region (Palau-Guam)
but most other large islands are associated with hotspots. These include clusters in the Galapagos and Hawaii as
well as ones at Easter Island and in French Polynesia. with the presence of the long-active convergent bound-
ary between the Pacific and Indo-Australian plates here. Other large islands are dotted along the convergent
boundary in the western part of the region (Palau-Guam)
but most other large islands are associated with hotspots. These include clusters in the Galapagos and Hawaii as
well as ones at Easter Island and in French Polynesia. y
The distribution of small islands (≤1 km2) is less
straightforward to interpret, since these can form in a
wide range of situations. What is easier to see in Fig. 3 is
that islands in areas away from plate boundaries appear
to be mostly intermediate in size (>1–100 km2). This is
a residual distribution explainable by the absence of
processes that encourage either very large or very small
islands to develop in such areas. With volcanic and com-
posite islands, this may be because there are few oppor-
tunities for large islands to form here because of the few
hotspots where point volcanism dominates; then, as such
islands are moved away from the hotspot, they generally
subside and are further reduced in size by denudation. The size of reef islands is ultimately limited by that of the
reef foundations on which they are built but more com-
monly by the availability of island-building sediments
(McLean and Kench 2015). Island areash Limestone islands are found only in places where uplift
has been occurring for prolonged periods. These include
intraplate locations where there are localized oceanic-
lithospheric swells, such as the South Pacific Superswell
which accounts for the elevation of atoll islands in the
Tuamotu Archipelago (French Polynesia) (McNutt and
Fischer 1987). The other major intraplate location in
which limestone islands are found is where their under-
pinning plates are flexing, typically because they are mov-
ing toward ocean trenches faster than the rate at which
they can be accommodated there; good examples are the
Loyalty islands (New Caledonia) (Dickinson 2013). Lime-
stone islands also rise from forearc zones along conver-
gent plate boundaries. In Fig. 4, these are visible in Tonga
and Vanuatu; the Lau island group (eastern Fiji), which
consists mostly of limestone islands, is a remnant forearc
isolated from proximal convergence by opening of the
back-arc Lau Basin (Nunn 1998a). Island types All the islands in the database were classified by type on
the basis of their dominant lithology and their elevation. The distributions of the islands by lithology, (maximum)
elevation, and type are discussed in the following three
subsections. Island elevationh The arbitrary distinction between high and low islands,
based on a 30-m elevation above mean sea level, is easy to
apply in developing a classification of islands and follows
a similar procedure employed to separate younger from
older volcanic islands. A finer division of islands based
on their elevation is possible from the database, despite
potential sources of error and use of a maximum value
rather than a measure of central tendency. Hence, for
the purposes of discussion, five elevation categories were
identified and their distributions are mapped in Fig. 5. The highest islands in the database (≥1000 m) are
generally either volcanic or composite and are found in
places where uncommonly voluminous eruptions have
occurred during the Quaternary; examples come from
Hawaii, the Galapagos Islands, the Marquesas and Tahiti
(French Polynesia), and Samoa (Fig. 5). The 23 % of high
islands (100–1000 m) are not only clustered in the same
places but are also found along the more active parts of
plate-convergence zones in the southwest Pacific, point-
ing to the importance of tectonics in elevating islands in
such places. i
Geologic history provides the context of which island
elevation is just one expression. In general, islands would
be expected to become lower as they become older. This
may be because they become more denuded by subaerial
processes and/or because in an oceanic location they are
invariably carried from a shallower area where island-
forming processes dominate to a deeper place where
island-building processes are usually absent. There are
exceptions to this but it is an idea with abundant theo-
retical and empirical support (Menard 1983, 1986). Island-building processes include primary processes—
such as intrusion and extrusion for volcanic (parts of)
islands—that may be periodically renewed, perhaps when
an older volcanic island becomes the site of younger
volcanism, as with Maui (Hawaii) and Savai’i (Samoa)
(Bergmanis et al. 2000; Hart et al. 2004). Such rejuvena-
tion invariably results in an increase in maximum eleva-
tion both because the lithosphere is locally reheated
and because of island mass increase (McNutt and Judge
1990). Examples include some of the Tuamotu islands
(French Polynesia) (Pirazzoli and Montaggioni 1988). Island lithologyh The distribution of the five lithological types recognized
for Pacific islands is shown in Fig. 4 with proportions of
each type in the key. Most islands are volcanic and their
distribution is explained almost entirely by their prox-
imity to places where (subaerial or submarine) volcan-
ism is occurring today. These include intraplate hotspots
that produce linear chains of volcanic islands, something
readily seen in Fig. 4 for the Hawaii, Marquesas (French
Polynesia), Society Islands (French Polynesia), and Samoa
hotspots. Linear groups of volcanic islands are also seen
along volcanic arcs parallel to convergent plate bounda-
ries in the southwest Pacific from Tonga through Vanuatu
to Solomon Islands. Linearity of volcanic island distribu-
tion in Fiji and Papua New Guinea is less pronounced
because of the multiple island arcs that have contrib-
uted to island origin in these large complex archipelagos. There is another clear volcanic island alignment parallel
to the adjoining ocean trench in the northern Mariana
islands. Around 1 % of islands are classified as having a con-
tinental lithology (Fig. 4). They are found exclusively in
part of New Caledonia where a sliver of continental crust
became isolated by Late Cretaceous-Eocene rifting asso-
ciated with the opening of the Tasman Sea (Cluzell et al. 2012). More than one-third of the islands in the database are
classified as reef islands and these tend to occur more
often in intraplate than plate boundary locations (Fig. 4). Most such islands are built on broad reef surfaces at
low-tide level and rise only 2–3 m above mean sea level,
a result of repeated episodes of large-wave deposition
and the leeward migration of deposits beyond the range
of normal wave erosion (McLean and Hosking 1991). Although such islands form on fringing reefs, those that
endure longest are generally found on more isolated Page 14 of 19 Nunn et al. Geosci. Lett. (2016) 3:7 a result of the ocean floor being thrust upwards, usually
episodically (Ota and Yamaguchi 2004). reefs, typically barrier or atoll reefs far from the shores
of higher islands. Most atoll reefs occur where (a line
of) volcanic islands have subsided within the coral seas
allowing their former location to continue to be marked
by a ring (atoll) reef on which islands (motu) may form. Thus, lines of reef islands rise from submerged vol-
canic islands in the central western Pacific, from Tuvalu
through (western) Kiribati to the Marshall Islands. Island elevationh Lithospheric plate flexure also affects the levels of islands
close to convergent plate boundaries in the Pacific, often
resulting in their rapid uplift as in the case of Niue and
the Loyalty islands (New Caledonia) (Dickinson 2013;
Nunn and Britton 2004) and subsequently their rapid
submergence as for several seamounts near the bottom
of the Tonga-Kermadec Trench (Watts et al. 2010). Yet
most of the tectonism that affects Pacific islands occurs
on the overriding plate at convergent plate boundaries as The lower islands in the database (<30 m) dominate
intraplate locations, illustrating the comparative impo-
tence of most island-building processes here compared
to convergent margins as well as the tendency for sink-
ing islands to be preserved as (atoll) reef islands long
after their volcanic foundations have been submerged. The (blue-colored) islands in the 5 to <30 m category are
scattered across the region (Fig. 5) yet concentrated in
the region of the South Pacific Superswell in French Poly-
nesia, speaking to the importance of such phenomena in
island elevation (McNutt and Fischer 1987). Island lithologyh Other
lines of reef islands follow hotspot traces in Micronesia
(northwest Pacific) and in French Polynesia (Duncan and
McDougall 1976). Lithology as a measure of resistance to erosion is a
major cause of elevation differences in islands of simi-
lar age. Comparatively hard-resistant rocks like emerged
reef limestones or various igneous rocks allow an island
to remain higher longer than when it is composed of low-
resistant (even unconsolidated) materials. Denudation is
the complement of lithology in this regard because it is
principally an expression of the climatic processes that
determine the pace of surface lowering of islands in par-
ticular places. Islands that experience heavy rainfall, espe-
cially during regular storms, will generally be reduced in
elevation faster than those located in drier parts of the
Pacific; this difference shows up when islands in the west-
ern Pacific that are often exposed to tropical cyclones are
compared to those in the east that are rarely so. Being an
outcome of both lithology and climate, natural vegeta-
tion generally increases resistance to erosion although its
absence, perhaps after fire, may heighten it. Studies of the
Hawaiian islands and Kadavu (Fiji) have linked current
island form to these factors (Li 1988; Terry 1999).h Island typeh The two most common island types in the Pacific are reef
(36 %) and volcanic high (31 %) islands; they have quite
contrasting distributions (Fig. 6). Reef islands are concentrated in intraplate locations,
most commonly in linear groups that mark lines of sub-
merged volcanic islands above which (atoll/barrier) reef
has grown. This situation is exemplified by several linear Page 15 of 19 Nunn et al. Geosci. Lett. (2016) 3:7 reef-island groups in the Federated States of Micronesia,
French Polynesia, Kiribati, Marshall Islands, and Tuvalu
as well as the western islands of the Hawaii group (Fig. 6). The large number of reef islands is explainable by their
tendency to develop on broad reef surfaces throughout
the low-latitude Pacific. Yet this tendency has also been
assisted by Late Holocene sea-level fall within the Pacific
that has seen some reef surfaces emerge by around 1 m
within the past few thousand years, providing foci for
sediment accumulation that has manifestly aided the
growth of large reef islands (Dickinson 2004); exam-
ples have been described from the Marshall Islands and
Tuvalu (McLean and Hosking 1991; Yamaguchi et al. 2009). Yet it is also worth noting that reef islands are
generally more vulnerable than other island types to size
reduction (and even erasure) under conditions of sea-
level rise; projections of this event occurring this cen-
tury have been made (Dickinson 2009), although there
are studies that demonstrate the dynamic nature of reef-
island shorelines and emphasizes the point that sea-level
rise does not uncritically equate with reef-island ‘disap-
pearance’ (Webb and Kench 2010; Woodroffe 2008). surprising, may also be explained by the general form of
volcanic islands, which invariably involves isolated higher
parts that resist erosion. It could be argued that it takes a
long time and/or unusually comprehensive denudation to
reduce a volcanic high to a volcanic low island. g
Composite high islands (6 %) are found almost solely
along convergent plate boundaries which demonstrates
that most are partly volcanic and share similar origins. Convergent plate boundaries are also those places where
tectonic forces are greatest in the Pacific and thus islands
that have been uplifted are common here. The composite
islands in southwest French Polynesia and the southern
Cook Islands include the classic makatea islands where
a volcanic island with a fringing reef has been uplifted,
producing a characteristic composite-lithology landscape
(Nunn 1994). Island typeh The makatea islands of the southern Cooks
owe their origin to localized lithospheric flexure asso-
ciated with the growth of the Rarotonga island volcano
(Spencer et al. 1987; Stoddart and Spencer 1987). Com-
posite low islands (1 %) have a similar distribution to
composite high islands. Contrary to the situation with volcanic islands (see
above), limestone low islands are more numerous (10 %)
than limestone high islands (7 %) in the Pacific (Fig. 6). This can be explained in two ways. First, uplift processes
are generally unable to raise limestone islands more than
30 m, something that is consistent with the stop-start
nature of uplift in many situations; examples where over
long time periods bursts of coseismic subsidence are sep-
arated by interseismic subsidence have been described
from Fiji and Vanuatu (Calmant et al. 1999; Nunn 1995). Second, the form of most limestone islands—character-
ized by flat (former reef) surfaces rather than peaks—
means that it may be less common to find any part of
them elevated above the 30-m threshold. f
Volcanic high islands are the next most common
island type in the Pacific. Part of this can be explained
by the fact that almost every Pacific island originated
as an ocean-floor volcano, so volcanic islands might be
expected to be common. Yet the fact that volcanic high
islands are so much more numerous than volcanic low
islands suggests that where ocean-floor volcanism is suf-
ficiently voluminous and enduring to produce an (emer-
gent) island, it is more often than not able to produce one
that reaches more than 30 m above sea level. Of course,
the conical form and structure of many volcanic islands
encourage this for ‘high’ in this classification which refers
to maximum elevation; a typical volcanic high island
need have only a small fraction of its area exceeding 30 m
for it to be so classified. It is also possible that the majority of limestone islands
in the high and low sub-categories have different origins,
the former being largely the product of uplift while the
latter are mostly a result of sea-level fall. Examination of
the distribution of limestone high and low islands (Fig. 6)
suggests this may indeed be the case. Limestone low
islands in intraplate situations are not generally found
near the high ones. Conclusions An earth-science-based classification of islands within
the Pacific Basin that is descriptive and readily applicable
to a variety of purposes while also capturing essential ele-
ments of island diversity has been developed. The classifi-
cation resulted from preparation of a database describing
the location, area, and type of 1779 islands, where island
type is determined as a function of the prevailing lithol-
ogy and maximum elevation of each island. Its essence
is that of the scheme adopted by Darwin and others who
almost two centuries ago attempted to make sense of
the diversity of island types they had encountered in the
world’s oceans (Darwin 1839; Wallace 1881). Compila-
tion of the database required definition of a island as a
discrete landmass within the Pacific Basin comprised of
contiguous areas of land ≥1 ha (0.01 km2) above mean
high-water level. Short-lived (< 20 years) transient islands
and reefs lacking islands are not included in the database. Various possible uses of both the database and clas-
sification can be envisaged. At a Pacific-wide scale, it is
expected that this classification will be useful to regional
planning on topics like exposure to disasters and vulner-
ability to climate change, particularly sea-level rise. In the
past, the development of regional initiatives in such areas
has been frustrated by the lack of data from the entire
population of Pacific islands as well as by biased meas-
urements of exposure/vulnerability arising from partisan
agendas. The classification presented here allows regional
planners to recognize these and develop coping/adaptive
strategies based on a complete and accurate dataset. This is not to say of course that the classification pre-
sented above can provide a complete solution for every
situation. For many regional purposes, it would need
elaboration through the addition of classificatory layers. For instance, while vulnerability to long-range tsunamis is
crudely a function of island type (especially lithology and
elevation), additional data referring to an island’s posi-
tion relative to its neighbors (near and far) as well as the
numbers of exposed inhabitants would be needed before
it might be usable at a regional (Pacific-wide) scale.i The principal aim of this classification was to assess the
spatial diversity of the geologic and geomorphic attrib-
utes of islands within the Pacific region. Island typeh Most of the former appear to be relict
reef surfaces from pre-Holocene interglaciations; exam-
ples come from parts of the Line islands [eastern Kiri-
bati—Schlanger et al. (1984)]. Only a few limestone high
islands are found in intraplate situations; they include
the islands of Nauru and Niue, which evolved because of
lithospheric flexure attributable, respectively, to volcano
loading and plate convergence (Hill and Jacobson 1989;
Nunn and Britton 2004). i
Figure 6 shows that volcanic high islands are found in
both intraplate and convergent plate boundary situations
in the Pacific. Intraplate volcanic high islands appear
exclusively associated with hotspots. Examples include
Easter Island, the Galapagos, Marquesas and Society
groups (French Polynesia), and Samoa; a hotspot expla-
nation for the origin of volcanic high islands in Microne-
sia (like Pohnpei) appears generally accepted, although
other explanations have been suggested (Dixon et al. 1984; Rehman et al. 2013). Volcanic high islands are also
numerous around convergent plate boundaries, particu-
larly where active subduction occurred during the Late
Quaternary. Volcanic low islands (8 %) tend to form near volcanic
high islands and their distribution has a similar expla-
nation (see above). Their comparative paucity, perhaps Limestone high islands appear proportionally more
numerous along convergent plate boundaries, reflecting Page 16 of 19 Nunn et al. Geosci. Lett. (2016) 3:7 Page 16 of 19 perhaps the long-term uplift they have experienced. Limestone low islands in such locations tend to be
either outliers of larger (higher) islands and/or farther
from places where uplift rates are highest. One exam-
ple of the latter situation is found in the Loyalty Islands
group (New Caledonia) which is rising obliquely across
a lithospheric flexure; limestone high islands like Maré
are near the crest of the flexure, limestone low islands
like Beautemps-Beaupré are just beginning to rise up its
flank (Dickinson 2013). Another example comes from the
(limestone) forearc in Tonga which adjoins the Tonga-
Kermadec Trench, at one point of which the (submerged)
Louisville Ridge is being subducted, resulting in anoma-
lous forearc uplift above (Dupont and Herzer 1985);
limestone islands like Tongatapu rising from the forearc
above the Louisville Ridge are high, while others like
most in the Ha’apai group are lower because they are not
so affected (Nunn 1998b). Further applications of the classificationh The purpose of this study was to develop a classification of
Pacific islands that could be used as a foundation for other,
more detailed, studies requiring objective measurement of
the diversity of island types within this vast region. Island typeh islands are far more vulnerable to water pollution, per-
haps like that associated with the 2011 collapse of the
Fukushima nuclear power plant that leaked radioactive
waters across a great expanse of the northern Pacific
Ocean (Johansen et al. 2015). Another use of this classification at both regional and
subregional scales would be to identify commonali-
ties between islands that might not otherwise be readily
apparent, particularly to development partners of Pacific
Island countries that are accustomed to dealing with
political rather than geographical entities. For decades,
most regional planning in the Pacific has used countries
as a highest-level divisor, thereby ensuring that much
research into the characteristics of particular islands and
island types to extraneous change would be duplicated—
repeated for nations sharing similar island types. As an
alternative to this, the classification developed in this
paper allows all islands of particular types (or groups of
types) to be readily identified without reference to politi-
cal boundaries, although these can be superimposed later
in any subsequent analysis. Conclusions It was intention-
ally set at a regional scale and based on two attributes:
five types of lithology (volcanic, limestone and non-
volcanic sedimentary, composite and continental) and
a distinction between high and low islands at elevations
greater or less than 30 m above mean sea level. Classifi-
cation of island type and the geographic distribution of
each attribute were achieved through development of a
procedure for classification; identification of data sources;
production of maps separately showing the distribution
of island locations, areas, lithology, elevation and type; i
Perhaps more uses of this classification could come at
subregional level, where the nature of islands within a
certain geographical area could be examined without ref-
erence to political boundaries. This would be useful when
considering transboundary environmental stressors not
only like disasters and various manifestations of climate
change but also pollution. Reef islands and low limestone Nunn et al. Geosci. Lett. (2016) 3:7 Page 17 of 19 Page 17 of 19 Bergmanis EC, Sinton JM, Trusdell FA (2000) Rejuvenated volcanism along the
southwest rift zone, East Maui, Hawai’i. Bull Volc 62(4–5):239–255 interpretation of the mapped distributions in the context
of available literature; and discussion of potential future
applications of the database and its derived classifica-
tion. The two key sets of attributes yielded eight island
types: volcanic high and low islands; limestone high and
low islands, composite high and low islands, reef islands
and continental islands. The most common types are reef
islands (36 %) and volcanic high islands (31 %), whereas
the least common are composite low islands (1 %). Con-
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l This paper derives from a project driven by the Department of Environment
in the Australian Government. We thank Lalage Cherry, Cameron Darragh,
Kiri Yapp, Sam Hussey-Smith, and Dominic Ransan-Cooper for their support. We also worked at various times with Geoscience Australia and thank their
staff for cooperation. We are grateful to Cate Macgregor, Subhashni Taylor,
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63(4):537–565. doi:10.2984/049.063.0405 Wallace AR (1881) Island life or, the phenomena and causes of insular faunas
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Monitoring the censored lognormal reliability data in a three-stage process using AFT model
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Monitoring the censored lognormal reliability data in a
three-stage process using AFT model Journal of Industrial Engineering International Provided in Cooperation with:
Islamic Azad University (IAU), Tehran Suggested Citation: Goodarzi, Azam; Amiri, Amirhossein; Asadzadeh, Shervin (2017) : Monitoring the
censored lognormal reliability data in a three-stage process using AFT model, Journal of Industrial
Engineering International, ISSN 2251-712X, Springer, Heidelberg, Vol. 13, Iss. 1, pp. 67-80,
https://doi.org/10.1007/s40092-016-0168-4 Suggested Citation: Goodarzi, Azam; Amiri, Amirhossein; Asadzadeh, Shervin (2017) : Monitoring the
censored lognormal reliability data in a three-stage process using AFT model, Journal of Industrial
Engineering International, ISSN 2251-712X, Springer, Heidelberg, Vol. 13, Iss. 1, pp. 67-80,
https://doi.org/10.1007/s40092-016-0168-4 Terms of use: Die Dokumente auf EconStor dürfen zu eigenen wissenschaftlichen
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gelten abweichend von diesen Nutzungsbedingungen die in der dort
genannten Lizenz gewährten Nutzungsrechte. http://creativecommons.org/licenses/by/4.0/ http://creativecomm Goodarzi, Azam; Amiri, Amirhossein; Asadzadeh, Shervin J Ind Eng Int (2017) 13:67–80
DOI 10.1007/s40092-016-0168-4 ORIGINAL RESEARCH Monitoring the censored lognormal reliability data
in a three-stage process using AFT model Azam Goodarzi1 • Amirhossein Amiri1 • Shervin Asadzadeh2 Received: 8 January 2016 / Accepted: 29 August 2016 / Published online: 8 September 2016
The Author(s) 2016. This article is published with open access at Springerlink.com Abstract Improving the product reliability is the main
concern in both manufacturing and service processes which
is obtained by monitoring the reliability-related quality
characteristics. Nowadays, products or services are the
result of processes with dependent stages referred to as
multistage processes. In these processes, the quality char-
acteristic in each stage is affected by the quality charac-
teristic in the previous stages known as cascade property. Two regression-adjusted control schemes are applied to
monitor the output quality variables of interest. Moreover,
censoring is among the main limitations while monitoring
the reliability-related quality characteristics, causing not to
record the real values of some observations. Hence, the
right censored observations are used to extend monitoring
schemes under both the fixed- and variable-competing
risks. In this paper, the accelerated failure time (AFT) is
used to relate the reliability-related quality characteristic
with lognormal distribution to the incoming variables. Then, two cause-selecting control charts are developed to
monitor outgoing quality variables when censoring hap-
pens in each reliability-related stage. The performance of
the control charts is evaluated and compared through extensive simulation studies under the censored and non-
censored scenarios. Keywords Accelerated failure time (AFT) model
Multistage process Cascade property Regression-
adjusted control schemes Fixed- and variable-competing
risks Keywords Accelerated failure time (AFT) model
Multistage process Cascade property Regression-
adjusted control schemes Fixed- and variable-competing
risks Introduction In addition, Steiner and
MacKay (2001) proposed exponentially weighted moving
average (EWMA) control chart based on the conditional
expected value to monitor the bond strength values which
are censored under the competing risk variable. Moreover,
the EWMA control chart is used to monitor the mean of
censored lifetimes when censoring occurs at fixed level
(Zhang and Chen 2004; Lu and Tsai 2008). Then, some
control charts are developed to monitor the time-to-failure
data in the presence of right censoring using rank tests (Li
et al. 2012; Li and Kong 2015). The base of these control
charts is using the rank statistics to derive the generic
formula for the operating characteristic functions of the
control chart. data are widely used. The reliability data have two main
features which make the monitoring of such data compli-
cated. The first one is that the reliability data often follow
the specific parametric distributions, namely, location scale
and log-location scale (Meeker and Escobar 1998). Among
them, the lognormal, Weibull, and extreme value are the
most commonly used distributions to model reliability data
(Lawless 2003). The second issue is that it is not always
possible to record the exact values of the reliability data
due to the lack of time, resources or expenses. This prob-
lem makes us to consider the censoring as another property
while dealing with reliability data. Furthermore, it would
be more complicated when the variable-competing risk
takes place. p
Many authors investigated the monitoring of the relia-
bility data in the literature. Padgett and Spurrier (1990)
developed Shewhart-type chart to detect the shift of a
percentile of lognormal and Weibull reliability data. Xie
et al. (2002) proposed a new technique to monitor the inter-
failure times which follow the exponential distribution. After that, the control charts were extended to monitor time
between r failures (Surucu and Sazak 2009). In addition,
Zhang et al. (2011a, b) designed the economic inter-failure
times control chart system when the times follow the
exponential
distribution. Nichols
and
Padgett
(2006)
designed a new bootstrap control chart for monitoring the
tensile strength of carbon fiber. Batson et al. (2006) sug-
gested a new approach to monitor mean time between
failures. This approach is based on transforming expo-
nential, lognormal, and Weibull failure time data to normal
distributed data. The CUSUM control chart has been
developed to monitor the time between events by Shafae
et al. (2014). Introduction In addition, Akhundjanov and Pascual (2015)
proposed the EWMA control chart to diagnose the small
shifts when a single measurement is taken per sampling
period. On the other hand, as discussed earlier, the products
usually come from processes with more than two stages. Shamsuzzaman
et
al. (2009)
developed
a
control
chart system to monitor the time between successive events
at different process stages in the multistage manufacturing
system. Asadzadeh and Aghaie (2012) proposed two con-
trol charts to monitor and improve the product reliability in
a two-stage process with this assumption that the reliability
data follow the Weibull distribution. Moreover, the out-
going quality characteristic is censored due to the presence
of both the fixed- and variable-competing risks. In addition,
Asadzadeh et al. (2013) proposed two regression-adjusted
control schemes based on Cox-Snell residuals. In their
paper, the control charts were extended to monitor the
residuals in the presence of the right censoring at fixed
level. In these papers, the AFT model is used to relate the
quality characteristics. Furthermore, Zhang et al. (2015)
investigated the effect of estimation error on the perfor-
mance of risk-adjusted survival time CUSUM scheme in
continuous time with the cardiac surgery data. They used
the AFT model in their research that can be applied in
multistage processes. Asadzadeh et al. (2014a, b) used the
Proportional Hazard and Frailty models to relate the quality
characteristics in a two-stage process. The monitoring
schemes were developed in the presence of the right cen-
soring at fixed level. In addition, Zhou et al. (2012) used
proportional hazards (PH) model to link upstream supply
chain quality/testing information as explanatory covariates
for the early detection of reliability problems which is
similar to the PH model for the multistage processes and
cascade property. In real situations, the observations may be censored at a
pre-specified level because of time and cost limitations. Moreover, the variable-competing risk may be considered
as well which censors the values of response quality vari-
ables. For example, it is not possible to record the values of
bond strength accurately when the tensile strength of
adhesive bond between vinyl fabric and PVC foam backing
is monitored and evaluated. This problem would happen
due to the failing of the foam backing sooner. Then, this
kind of censoring is introduced as a variable-competing
risk which cannot be easily neglected for the sake of
proposing optimal monitoring schemes. Introduction Manufacturing or service processes do not solely include
one-stage processes, and the products’ reliability is mostly
the result of several different process steps. In such mul-
tistage processes, cascade property is a principal feature in
which the outgoing quality characteristic is affected by the
changes in the incoming variables (Yang 1999; Tsung et al. 2008). To
deal
with
this
problem,
cause-selecting
chart (CSC) is first introduced by Zhang (1984). The CSCs
are most widely used to monitor and diagnose the multi-
stage processes. Two-stage processes with normal output
variable are reviewed by Wade and Woodall (1993). The
CSC can also be used to monitor more than two-stage
processes (Xiang and Tsung 2008). However, there exists a
critical issue that quality variables do not follow the normal
distribution in all the cases, while the major assumption
using the CSC monitoring procedure is normally dis-
tributed quality characteristic. Therefore, some other
regression-adjusted monitoring procedures based on the
generalized linear models (GLMs) have been proposed to
relax the normality assumption. These models have been
extended to include the exponential family distributions
(Jearkpaporn et al. 2003, 2007; Skinner et al. 2003). & Amirhossein Amiri
amiri@shahed.ac.ir
Azam Goodarzi
a.goodarzi@shahed.ac.ir
Shervin Asadzadeh
sh_asadzadeh@iau-tnb.ac.ir
1
Department of Industrial Engineering, Faculty of
Engineering, Shahed University, Tehran, Iran
2
Department of Industrial Engineering, Islamic Azad
University, North Tehran Branch, Tehran, Iran To improve the product reliability in manufacturing and
service processes, monitoring and evaluating reliability 12 123 123 J Ind Eng Int (2017) 13:67–80 68 Steiner and MacKay (2000). In addition, Steiner and
MacKay (2001) proposed exponentially weighted moving
average (EWMA) control chart based on the conditional
expected value to monitor the bond strength values which
are censored under the competing risk variable. Moreover,
the EWMA control chart is used to monitor the mean of
censored lifetimes when censoring occurs at fixed level
(Zhang and Chen 2004; Lu and Tsai 2008). Then, some
control charts are developed to monitor the time-to-failure
data in the presence of right censoring using rank tests (Li
et al. 2012; Li and Kong 2015). The base of these control
charts is using the rank statistics to derive the generic
formula for the operating characteristic functions of the
control chart. Steiner and MacKay (2000). Introduction It is significant that
the main assumption of all the above-mentioned researches
is that the observations were recorded with no fixed and
variable censoring, even though the location-scale and log-
location-scale distributions have been addressed. Censored
observations at fixed level are considered for monitoring in It should be noted that all the discussed processes are
assumed to have two stages and the outgoing quality
characteristic has been censored. However, there exists a
real case dealing with cotton spinning in which the three
quality characteristics are Fiber Length, Fiber Strength, and
Skein Strength. If the fiber length was to change, it would
lead to change in the fiber strength. Moreover, a change in
any of the two quality variables would affect the skein 123 123 J Ind Eng Int (2017) 13:67–80 69 strength of the spun cotton. Obviously, fiber strength and
skein strength are the reliability-related quality character-
istics which bring about censored observations for the
second and third stages. Considering the mentioned
example and the concept of variable-competing risk dis-
cussed in the case of adhesive bond between vinyl fabric
and foam backing, we address a three-stage process in
which the quality characteristics of both the second and
third stages evaluate and reflect the reliability of product. Thus, we use the residuals to construct the CSC and
monitor the outgoing quality characteristic. It is remarkable
that the proposed monitoring schemes are extended when
the censoring occurred in these stages. As mentioned
before, the lognormal distribution is widely used to model
the reliability data. Furthermore, it is assumed that these
reliability-related quality characteristics follow the log-
normal distribution. case. One is the accelerated failure time (AFT) model
which relates the parameters of output quality character-
istic to the identified covariates and the other one is the
proportional hazards (PH) models in which covariates
affect the associated hazard function (Padgett and Spurrier
1990). Since the AFT model is the most widely used
models among the parametric ones, in this paper, the AFT
model is considered to relate the parameter of the relia-
bility-related quality characteristics of both the third and
second stages to the quality characteristics in the previous
stages. As discussed in the previous section, we assumed that
the reliability-related quality characteristics of both the
third and second stages follow the lognormal distribution
due to extensive usages among the variety distributions
which can be used in survival regression models. Introduction There-
fore, the general probability density and survival function
of the lognormal distribution are given in Eqs. (1) and (2),
respectively, as follows: The structure of this paper is as follows: the problem
statement and the AFT model are elaborated in the next
section. The monitoring schemes under the absence and
presence of the fixed- and variable-competing risks are
proposed in Sect. 3. In Sect. 4, the performance of the
proposed control charts is studied and compared by
extensive simulation studies. Numerical example is pro-
vided in Sect. 5. Concluding remarks are presented in
Sect. 6. fðyÞ ¼
1
ffiffiffiffiffiffi
2p
p
ry
e
1
2
log yl
r
ð
Þ
2
y [ 0;
ð1Þ
and
sðyÞ ¼ 1 / log y l
r
:
ð2Þ fðyÞ ¼
1
ffiffiffiffiffiffi
2p
p
ry
e
1
2
log yl
r
ð
Þ
2
y [ 0;
ð1Þ ð1Þ and and sðyÞ ¼ 1 / log y l
r
:
ð2Þ ð2Þ For the parameterization, l is the location parameter and
r is the shape parameter. These parameters are the mean
and standard deviation of the associated normal distribu-
tion, respectively. In addition, /ð:Þ is the cumulative dis-
tribution function of normal standard distribution. It is
assumed in this paper that only the location parameters of
the quality characteristics in the second and third stages (l1
and l2) depend on covariates of the previous stages and
their dependency is shown as follows: Problem statement As illustrated in Fig. 1, suppose a three-stage manufac-
turing process in which the outgoing quality characteristic
is affected by quality variables of the previous stages. In
this process, the quality characteristics in the second and
third stages reflect the reliability of product which are
denoted by y1 and y2, respectively. Thus, censoring would
occur at fixed level due to the cost and time constraints in
any of the discussed stages. Besides, some of actual
observations would be censored due to the presence of
another censoring mechanism which is referred to as
variable-competing risk. As a result, two distinct compet-
ing risks, namely, fixed and variable, would happen in the
process which prohibits the exact records of some values
corresponding to the reliability-related quality character-
istics of the second and third stages. ly2jx;y1 ¼ a0 þ a1x þ a2 logðy1Þ;
ð3Þ
and
ly1jx ¼ b0 þ b1x:
ð4Þ ly2jx;y1 ¼ a0 þ a1x þ a2 logðy1Þ;
ð3Þ ð3Þ and ly1jx ¼ b0 þ b1x:
ð4Þ ð4Þ ly1jx ¼ b0 þ b1x: Then, the AFT-based survival functions are defined for
quality characteristics of both the third and second stages
as as sðy2jx; y1Þ ¼ 1 / log y2 a0 a1x a2 log y1
r2
;
ð5Þ
and
sðy1jxÞ ¼ 1 / log y1 b0 þ b1x
r1
;
ð6Þ sðy2jx; y1Þ ¼ 1 / log y2 a0 a1x a2 log y1
r2
;
ð5Þ ð5Þ Taking into account the dependency of reliability data to
the previous stages, the survival analysis regression models
have been used. There are two major approaches in this and sðy1jxÞ ¼ 1 / log y1 b0 þ b1x
r1
;
ð6Þ ð6Þ ~
(
,
)
x
x
x
N
1
1
1 ~
(
,
)
y
lognormal
2
2
2 ~
(
,
)
y
lognormal
Fig. 1 Three-stage process where b0, b1, a0, a1, and a2 are the parameters of regres-
sion models of two different reliability-related quality Fig. 1 Three-stage process 12 J Ind Eng Int (2017) 13:67–80 70 characteristics. In addition, l1,r1, l2, and r2 are considered
as the mean and standard deviation of the quality charac-
teristics in the second and third stages (y1 and y2). Regression-adjusted control schemes wi ¼ d
2r2
z
ð2 logðy2iÞÞ þ d 2l2i:
ð10Þ ð10Þ In this section, the control charts are designed under two
scenarios whether the process observations are censored or
not. Subsequently, we have proposed the monitoring
schemes in the following sub-sections for the sake of
finding out-of-control situations immediately. This statistic is set to detect decreasing shifts, even
though the CUSUM control procedure signals an out-of-
control status when its statistic exceeds the lower control
limit, LCL1. The LCL1 is set by simulation to achieve the
desired in-control average run length (ARL) criterion. Problem statement Without
loss of generality, the covariates most widely follow the
normal distribution, i.e., x Nðlx; rxÞ. Therefore, this fact
directs us toward using logðy1Þ, which transforms the dis-
tribution of the second-stage quality characteristic to nor-
mal
distribution,
to relate the parameter
of quality
characteristic of the third stages to the previous stage. the quality characteristic in the last stage. The vector of in-
control
parameter
is
shown
as
xi0 ¼ ða0 þ a1x þ a2
logðy1Þ; r2Þ, and the out-of-control parameter vector is
xi1 ¼ ða0 þ a1x þ a2 logðy1Þ d; r2Þ,
where
d
is
the
decreasing shift in the in-control location parameter. Tak-
ing our attention into the mean of survival time which is
calculated by following equation, it implies that changing
the location parameter leads to changing the mean of sur-
vival time: In the next section, the cause-selecting control strategies
are elaborated based on the AFT model. The two proposed
control charts are extended in the presence of both the
fixed- and variable-competing risks. Eðy2Þ ¼ exp l2 þ r2
2
2
:
ð9Þ ð9Þ Hence, the CUSUM score is given by the following
equation based on the lognormal density function of quality
characteristic in the third stage: Regression-adjusted control schemes in the absence
of censoring Since the proposed CUSUM control chart is CSC, the
next monitoring surveillance EWMA control chart is also
proposed based on the residuals to omit the effect of
covariates in the previous stages on the quality character-
istics in the last stage. The residuals are computed as
follows: In this sub-section, monitoring schemes are developed in the
absence of both the fixed- and variable-competing risks. Doing so, we concentrate on developing the control charts to
monitor the quality characteristics to detect decreasing shifts,
although it is easy to establish the proposed control charts for
the sake of detecting increasing and decreasing shifts simul-
taneously. Accordingly, two regression-adjusted control
schemes, including CUSUM and EWMA, are proposed. zi ¼ y2i Eðy2iÞ
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
Varðy2iÞ
p
;
ð11Þ ð11Þ where Eðy2iÞ and Varðy2iÞ are the mean and variance of the
quality characteristic in the third stage. Since the quality
characteristic of the last stage follows the lognormal dis-
tribution, the mean of survival time is given by Eq. (9) and
its variance is calculated as follows: Having represented the manufacturing process and the
corresponding distributions for quality characteristics, we
concentrate on developing CUSUM control chart to mon-
itor the response variable, first. The statistic of this pro-
posed control chart is constructed based on the likelihood
function. The likelihood function related to the ith obser-
vation in the absence of censoring is modified as Li ¼ fðyiÞ
that can be calculated by Eq. (1). Then, the one-sided
CUSUM statistic is given by Varðy2Þ ¼ expð2l2 þ r2
2Þðexpðr2
2Þ 1Þ:
ð12Þ ð12Þ By considering Eq. (3), the mean and variance of log-
normal quality characteristic in the third stage are calcu- y
g
q
g
normal quality characteristic in the third stage are calcu-
lated
as
Eðy2Þ ¼ ea0þa1xþa2 logðy1Þþ
r2
2
2
and
Varðy2Þ ¼
e2ða0þa1xþa2 logðy1ÞÞþr2
2 er2
2 1
. The proposed residual zi in
Eq. (11) is standardized; hence, the mean and standard
deviation of zi are equal to 0 and 1, respectively. Note that
changing the scale parameter of the last stage quality
characteristic l2 leads to changing in the mean of interest. Consequently, the one-sided EWMA statistic based on the
residuals in Eq. Regression-adjusted control schemes in the absence
of censoring Since
zi are standardized residuals, then LCL2 reduces to w ¼
y
if y c and y cr
wc
if c\y and c cr
wcr
otherwise
8
<
:
:
ð16Þ ð16Þ Assuming lognormal distribution, the CEV of the
quality characteristics observation censored at q is derived
by the following equation regardless of which competing
risk occurs LCL2 ¼ L
ffiffiffiffiffiffiffiffiffiffiffi
k
2 k
r
:
ð14Þ LCL2 ¼ L
ffiffiffiffiffiffiffiffiffiffiffi
k
2 k
r
: ð14Þ The one-sided EWMA control chart triggers a signal
when Qi\LCL2. The LCL2 is set, such that a desirable in-
control ARL is obtained. CEV(yÞ ¼ Eðyjy [ qÞ ¼
1
2 e
1
2r2þl þ 1
2 e
1
2r2þlerf
llog yþr2
ffiffi
2
p
r
SðqÞ
;
ð17Þ ð17Þ In the next sub-section, our proposed control charts are
developed when the censoring data exist. where S(q) is the survival function of the reliability-related
quality characteristic, and erf(.) is the error function that is
defined as follows: Regression-adjusted control schemes in the presence
of censoring erfðtÞ ¼
Zt
0
2ffiffiffip
p et2dt:
ð18Þ ð18Þ After proposing the monitoring schemes in the absence of
censoring mechanisms, we concentrate on developing the
control chart strategies in the presence of both the fixed- and
variable-competing risks. Moreover, we present the formu-
las regardless of which stage is considered. At the end, they
would be adopted to the quality characteristics y2 and y1. Subsequently, the CEV weight, which was presented in
Eq. (16), can be given by the following equation: Subsequently, the CEV weight, which was presented in
Eq. (16), can be given by the following equation: w ¼
y
if yc and ycr
1
2e
1
2r2þl þ 1
2e
1
2r2þlerf l logðcÞ þ r2
ffiffiffi
2
p
r
SðcÞ
if c\y andccr
1
2e
1
2r2þl þ 1
2e
1
2r2þlerf l logðcrÞ þ r2
ffiffiffi
2
p
r
SðcrÞ
otherwise:
8
>
>
>
>
>
>
>
>
>
<
>
>
>
>
>
>
>
>
>
: if yc and ycr As mentioned before, the variable-competing risk can
occur unwantedly which precludes the accurate recording of
quality characteristic values. We assume that this variable
follows the normal distribution. Therefore, the values of
reliability-related quality characteristics are recorded com-
pletely unless they reach the pre-determined limit denoted by
c or the failure related to variable-competing risk, which is
denoted by cr. Given that the existence of the fixed- and
variable-competing risks affect the actual values deleteri-
ously, all censored data can be replaced with their conditional
expected values (CEV) which are calculated by the following
formulas considering which competing risk occurs first ð19Þ ð19Þ Now, we can establish the CEV weights for the relia-
bility-related quality characteristic of the second stage by
considering y1, b0 þ b1x, and r1 instead of y, l and r,
respectively, in Eq. (18). Regression-adjusted control schemes in the absence
of censoring (11), is derived as follows to detect
decreasing shifts in the mean of the quality characteristic in
the third stage: lated
as
Eðy2Þ ¼ ea0þa1xþa2 logðy1Þþ
r2
2
2
and
Varðy2Þ ¼
e2ða0þa1xþa2 logðy1ÞÞþr2
2 er2
2 1
. The proposed residual zi in lated
as
Eðy2Þ ¼ ea0þa1xþa2 logðy1Þþ
r2
2
2
and
Varðy2Þ ¼
e2ða0þa1xþa2 logðy1ÞÞþr2
2 er2
2 1
. The proposed residual zi in c
i ¼ min 0; c
i1 wi
;
c
0 ¼ 0;
ð7Þ ð7Þ Eq. (11) is standardized; hence, the mean and standard
deviation of zi are equal to 0 and 1, respectively. Note that
changing the scale parameter of the last stage quality
characteristic l2 leads to changing in the mean of interest. Eq. (11) is standardized; hence, the mean and standard
deviation of zi are equal to 0 and 1, respectively. Note that
changing the scale parameter of the last stage quality
characteristic l2 leads to changing in the mean of interest. Consequently, the one-sided EWMA statistic based on the
residuals in Eq. (11), is derived as follows to detect
decreasing shifts in the mean of the quality characteristic in
the third stage: where wi is the CUSUM score proposed by Page (1954)
and is computed by wi ¼ log Lðxi1jy2iÞ
Lðxi0jy2iÞ
:
ð8Þ ð8Þ In this equation, xi0 and xi1 are defined as in-control and
out-of-control values of the parameters corresponding to J Ind Eng Int (2017) 13:67–80 71 wc ¼ CEV(yjy [ cÞ
or
wcr ¼ CEVðyjy [ crÞ;
ð15Þ Qi ¼ minfl0; kzi þ ð1 kÞQi1g
¼ minf0; kzi þ ð1 kÞQi1g;
ð13Þ ð15Þ ð13Þ where the CEV weights of the process are defined as
follows: where k is the smoothing parameter (0\k 1) and Q0 is
equal to the in-control mean of the residual statistic, i.e.,
Q0 ¼ 0. The general one-sided lower control limit of the
EWMA statistic is given by LCL2 ¼ l0 Lr
ffiffiffiffiffiffi
k
2k
q
. Since
zi are standardized residuals, then LCL2 reduces to where k is the smoothing parameter (0\k 1) and Q0 is
equal to the in-control mean of the residual statistic, i.e.,
Q0 ¼ 0. The general one-sided lower control limit of the
EWMA statistic is given by LCL2 ¼ l0 Lr
ffiffiffiffiffiffi
k
2k
q
. Regression-adjusted control schemes in the presence
of censoring Consequently, the CEV
weights of the third stage quality characteristic can be
obtained as follows when the location parameter l2 is
defined as a0 þ a1x þ a2 logðw1Þ: wi ¼ si log
1 U
lncl0
ry
1 U
lncðl0dÞ
ry
2
4
3
5 þ d
2r2
y
cið1 siÞð2lny 2l0 þ dÞ
þ ð1 ciÞð1 siÞlog
1 U
lncrl0
ry
1 U
lncrðl0dÞ
ry
2
4
3
5:
ð27Þ ð27Þ w2 ¼
y2
if
y2 c2 and y2 cr2
1
2 e
1
2r2
2þa0þa1xþa2 logðw1Þ 1 þ erf a0 þ a1x þ a2 logðw1Þ logðc2Þ þ r2
2
ffiffiffi
2
p
r2
Sðc2Þ
if
c2\y2 and c2 cr2
1
2 e
1
2r2
2þa0þa1xþa2 logðw1Þ 1 þ erf a0 þ a1x þ a2 logðw1Þ logðcr2Þ þ r2
2
ffiffiffi
2
p
r2
Sðcr2Þ
otherwise:
8
>
>
>
>
>
>
>
>
>
<
>
>
>
>
>
>
>
>
>
:
ð21Þ w2 ¼
y2
if
y2 c2 and y2 cr2
1
2 e
1
2r2
2þa0þa1xþa2 logðw1Þ 1 þ erf a0 þ a1x þ a2 logðw1Þ logðc2Þ þ r2
2
ffiffiffi
2
p
r2
Sðc2Þ
if
c2\y2 and c2 cr2
1
2 e
1
2r2
2þa0þa1xþa2 logðw1Þ 1 þ erf a0 þ a1x þ a2 logðw1Þ logðcr2Þ þ r2
2
ffiffiffi
2
p
r2
Sðcr2Þ
otherwise:
8
>
>
>
>
>
>
>
>
>
<
>
>
>
>
>
>
>
>
>
:
ð21Þ ð21Þ Moreover,
occurring
of
the
censoring
mechanism
changes the likelihood function of survival time. Hence,
the likelihood function associated with the ith observation
is defined as follows, where the probability density and the
cumulative distribution functions for y are shown by f and
F and by g and G for cr, respectively: The CUSUM statistic regarding the presence of censoring
is similar to Eq. (7). However, it is constructed based on the
CUSUM score in Eq. (27) to identify the decreasing shifts. The CUSUM statistic regarding the presence of censoring
is similar to Eq. (7). However, it is constructed based on the
CUSUM score in Eq. (27) to identify the decreasing shifts. Regression-adjusted control schemes in the presence
of censoring Thus, the CEV weights of the
second stage can be indicated as follows: w1 ¼
y1
if
y1 c1 andy1 cr1
1
2 e
1
2r2
1þb0þb1x þ 1
2 e
1
2r2
1þb0þb1xerf b0 þ b1x logðc1Þ þ r2
1
ffiffiffi
2
p
r1
Sðc1Þ
if
c1\y1 and c1 cr1
1
2 e
1
2r2
1þb0þb1x þ 1
2 e
1
2r2
1þb0þb1xerf b0 þ b1x logðcr1Þ þ r2
1
ffiffiffi
2
p
r1
Sðcr1Þ
otherwise,
8
>
>
>
>
>
>
>
>
>
<
>
>
>
>
>
>
>
>
>
:
ð20Þ if
y1 c1 andy1 cr1 ð20Þ 72 J Ind Eng Int (2017) 13:67–80 where c1 and cr1 are the pre-determinedlimit and thevariable-
competing risk of the second-stage quality characteristic. where c1 and cr1 are the pre-determinedlimit and thevariable-
competing risk of the second-stage quality characteristic. The proposed likelihood function is used to construct the
CUSUM score which is defined in Eq. (8). Constructing the CEV weights of the third-stage quality
characteristics is similar to the second one, but there is a As a consequence, the lognormal CUSUM score for the
censored observations is written as wi ¼ log ½ð1 Fðtijxi1ÞÞð1 GðtiÞÞsi ½½fðtijxi1Þð1 GðtiÞÞci½gðtiÞð1 Fðtijxi1ÞÞð1ciÞ1si
½ð1 Fðtijxi0ÞÞð1 GðtiÞÞsi ½½fðtijxi0Þð1 GðtiÞÞci½gðtiÞð1 Fðtijxi0ÞÞð1ciÞ1si
! :
ð26Þ ð26Þ At the end, this score is reduced to At the end, this score is reduced to difference in defining the location parameter, l2. Since the
censoring mechanism occurs in stage two as well, it is
indispensable to use the CEV weights of the second stage
for defining l2 specified in Eq. (3). Regression-adjusted control schemes in the presence
of censoring Its mean can be obtained as ð24Þ ti ¼ minðyi; cri; cÞ:
ð24Þ ti ¼ minðyi; cri; cÞ:
ð24Þ ti ¼ minðyi; cri; cÞ: Therefore, the likelihood function is written as follows: EðW2Þ ¼ y2 pðy2 c2 and y2 cr2Þ þ wc2
pðc2 y2 and c2 cr2Þ þ wcr2
pðcr2 y2 and cr2 c2Þ:
ð29Þ Li ¼ ½ð1 FðtiÞÞð1 GðtiÞÞsi ½½fðtiÞð1
GðtiÞÞci½gðtiÞð1 FðtiÞÞð1ciÞ1si:
ð25Þ ð29Þ ð25Þ
ð
Þ
2
pðcr2 y2 and cr2 c2Þ:
ð29Þ ð25Þ 123 J Ind Eng Int (2017) 13:67–80 73 þ
Z
y1
Z
x
1
2e
1
2r2
y2þa0þa1xþa2 logðy1Þ 1þerf
a0þa1xþa2 logðy1Þlogðc2Þþr2
y2
ffiffi
2
p
ry2
Sðc2Þ
2
664
3
775
2
1/y2
lnca0 a1xa2logðy1Þ
ry2
fxðxÞfy1ðy1Þdxdy1
Zþ1
c2
1
rcr2
ffiffiffiffiffiffi
2p
p
e
1
2
cr2lcr2
rcr2
2
dcr2
2
4
3
5
þ
Zc2
0
Z
y1
Z
x
1
2e
1
2r2
y2a0þa1xþa2 log y1
ð
Þ 1þerf
a0þa1xþa2 logðy1Þlogðcr2Þþr2
y2
ffiffi
2
p
ry2
Sðcr2Þ
2
664
3
775
2
1
rcr2
ffiffiffiffiffiffi
2p
p
e
1
2
cr2lcr2
rcr2
2
1/y2
lncr2 a0 a1xa2logðy1Þ
ry2
fxðxÞfy1ðy1Þdxdy1dcr2:
ð32Þ The variance of observations is computed based on the
traditional
definition;
VarðW2Þ ¼ EðW2
2Þ E2ðW2Þ. Finally, by calculating the probability in Eq. (29), the mean
and EðW2
2Þ can be indicated as follows: EðW2
2Þ ¼
Zc2
0
y2
2 fy2ðy2Þ ð1 Gcr2ðy2ÞÞdy2
þ W2
c2 ½1 Fy2ðc2Þ ½1 Gcr2ðc2Þ
þ
Zc
0
W2
cr2 gcr2ðcr2Þ ð1 Fy2ðcr2ÞÞdcr2:
ð30Þ ð30Þ The presented mean and variance of observations lead to
long time to set the lower control limit, LCL4 by simulation. Therefore, we propose the general mean and variance. The
significant advantage in applying the general mean and
variance is that the censoring mechanism in the second stage
does not influence on the last stage, although the simulation
error to set the lower control limit would increase. In
addition, the general mean and variance of quality charac-
teristic in the third stage are fixed for each observation. The
general EðW2Þ and EðW2
2Þ, to compute the variance of
observations, are indicated by the following formulas: ð32Þ Finally, obtaining the residuals, the one-sided EWMA
statistic is constructed. Regression-adjusted control schemes in the presence
of censoring Then, it is needed to set the lower control limit shown as
LCL3 and compared with the CUSUM statistic. Note that
the one-sided CUSUM control chart generates a signal when
this statistic is less than LCL3. Then, it is needed to set the lower control limit shown as
LCL3 and compared with the CUSUM statistic. Note that
the one-sided CUSUM control chart generates a signal when
this statistic is less than LCL3. Li ¼ ½ð1 FðcÞÞð1 GðcÞÞsi ½½fðyiÞð1
GðyiÞci½gðcriÞð1 FðcriÞÞð1ciÞ1si;
ð22Þ ð22Þ Another control scheme is EWMA control chart which
was described completely. There is a critical issue that the
proposed residual is not suitable in the presence of censor-
ing, because the observations are not the actual value and the
mean and variance of them would be changed. Hence, we
present a new residual based on the CEV weights as follows: in which si ¼
1
if
c yi and c cri
0
if
c [ yi and c [ cri
and
ci ¼
1
if
yi cri
0
if
yi [ cri:
ð23Þ ð23Þ z0
i ¼ W2i EðW2iÞ
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
VarðW2iÞ
p
;
ð28Þ ð28Þ For the simplicity, variable t is defined as For the simplicity, variable t is defined as For the simplicity, variable t is defined as
ti ¼ minðyi; cri; cÞ:
ð24Þ where EðW2iÞ and VarðW2iÞ are the mean and variance of
observations. Its mean can be obtained as where EðW2iÞ and VarðW2iÞ are the mean and variance of
observations. Regression-adjusted control schemes in the presence
of censoring The EWMA control chart signals an
alarm when the EWMA statistic falls below LCL4. LCL4 is
obtained by simulation to achieve a desired in-control
ARL. It should be noted that a three-stage process has been
studied in this paper. One may easily consider a multistage
process in which the response variable is dependent on
several preceding quality variables. Subsequently, the
parameters of the survival regression model can be mod-
eled in terms of all effective prior quality variables for a
multistage process with n stages as follows: EðW2Þ ¼
Zc2
0
Z
y1
Z
x
1
ry2
ffiffiffiffiffiffi
2p
p
e
1
2
lnðy2Þa0a1xalogðy1Þ
ry2
Zþ1
y2
1
rcr2
ffiffiffiffiffiffi
2p
p
e
1
2
cr2lcr2
rcr2
2
dcr2
2
64
3
75 fxðxÞ fy1ðy1Þdxdy1dy2
þ
Z
y1
Z
x
1
2e
1
2r2
y2þa0þa1xþa2 logðy1Þ 1 þ erf
a0þa1xþa2 log y1
ð
Þlog c2
ð
Þþr2
y2
ffiffi
2
p
ry2
Sðc2Þ
1 /y2
lnc a0 a1x a2 logðy1Þ
ry2
fxðxÞ fy1ðy1Þdxdy1
Zþ1
c2
1
rcr2
ffiffiffiffiffiffi
2p
p
e
1
2
cr2lcr2
rcr2
2
dcr2
2
4
3
5
þ
Zc2
0
Z
y1
Z
x
1
2e
1
2r2
y2þa0þa1xþa2 logðy1Þ
1 þ erf
a0þa1xþa2 logðy1Þlogðcr2Þþr2
y2
ffiffi
2
p
ry2
Sðcr2Þ
1
rcr2
ffiffiffiffiffiffi
2p
p
e
1
2
cr2lcr2
rcr2
2
1 /y2
lncr2 a0 a1x a2logðy1Þ
ry2
fxðxÞ fy1ðy1Þdxdy1dcr2;
ð31Þ
and
EðW2
2Þ¼
Zc2
0
Z
y1
Z
x
y2
1
ry2
ffiffiffiffiffiffi
2p
p
e
1
2
lnðy2Þa0a1xa2 logðy1Þ
ry2
Zþ1
y2
1
rcr2
ffiffiffiffiffiffi
2p
p
e
1
2
cr2lcr2
rcr2
2
dcr2
2
64
3
75fxðxÞfy1ðy1Þdxdy1dy2
ðÞ lyn1jx;y1;...;yn2 ¼ a0 þ a1x þ
X
n1
i¼2
ai logðyi1Þ:
ð33Þ ð33Þ Performance analysis This section analyzes and compares the performance of the
proposed control charts to determine which monitoring
method is better at detecting decreasing step shifts in the
response mean or equivalently the location parameter of
the lognormal distribution. It is remarkable that the simu-
lation runs are performed to calculate ARLs with 10,000
replicates. In addition, the lower control limit for all the
control charts is set through simulation to achieve the in-
control ARL of roughly 200. ð31Þ The simulation studies are done with assuming these
input parameters: k = 0.2, b0 ¼ 1:5, b1 ¼ 1, a0 ¼ 2,
a1 ¼ 0:5, a2 ¼ 0:5, x*N(2, 1), ry1 = 1.3, and ry2 = 1. Moreover, the presence of variable-competing risk is
assumed and we aware of its occurrence which follows a
normal distribution. The in-control parameters of normal
distribution and also the fixed-censoring level are selected,
respectively, to reach the situation in which the censoring and 12 J Ind Eng Int (2017) 13:67–80 74 Table 1 Comparison of the control charts in the absence of competing risk in the last stage
Control charts
Censoring rate in the
second stage (%)
d0
0
0.05
0.1
0.2
0.5
1
EWMA
0
200.53
144.15
109.04
65.68
22.86
9.76
20
200.76
146.43
110.50
67.04
23.09
9.77
50
200.35
152.08
116.81
73.11
25.65
10.17
80
200.92
155.42
122.62
79.74
28.33
10.62
CUSUM
0
200.61
139.54
101.05
58.24
19.43
8.20
20
200.78
144.10
104.43
59.82
19.53
8.11
50
200.67
149.62
114.10
70.23
23.19
8.50
80
200.08
153.91
121.94
79.95
27.44
9.16 Table 1 Comparison of the control charts in the absence of competing risk in the last stage rate becomes approximately 50 % for the third-stage quality
characteristic. Adopting a special value for competing risks
to achieve a special censoring rate of two fixed- and vari-
able-competing risks for both the second and third stages is
described in Appendices A and B, respectively. competing risks take place in the last stage. Three cen-
soring rates of 20, 50, and 80 % are assumed in both the
second and third stages. As discussed earlier, the proposed
EWMA control chart is independent of which censoring
rate happens in the second stage. Then, the ARL values are
shown in Table 2. The ARL values of the control charts are compared; under
the presence and absence of censoring in the quality charac-
teristic of the last stage. Performance analysis In addition, the independency of the
proposed cause-selecting control charts under no censoring
scenario against the shifts in the mean quality characteristics
of the first and second stages is shown in this section. The outcomes of performing simulation study indicate
that CUSUM control chart outperforms the EWMA control
chart. There is an exception that the EWMA control
chart performs better in the low and moderate (20 and
50 %) censoring rates when 80 % of outcomes of the
second-stage quality characteristic are censored under the
small shifts. In general, the performance of control charts
deteriorates as the censoring rate increases. A numerical example To evaluate and compare the performance of the proposed
monitoring procedures, a numerical example is used in this
section. It is remarkable that the skein strength and fiber
strength aim to reflect the product reliability in this case. We consider some scenarios to evaluate the performance of
the proposed EWMA and CUSUM control charts. In the Performance analysis under mean shifts
in the previous sub-processes In this sub-section, the ARL of the proposed control charts is
compared in the absence of both the fixed- and variable-
competing risks. Moreover, the presence of censoring in the
second stage is considered to show its effect on the perfor-
mance of the proposed control charts because of assuming
quality characteristic of the second stage as reliability data. The results of ARL are summarized in Table 1 by consid-
ering three censoring rates of low (20 %), moderate (50 %),
and high (80 %) in which the fraction of the fixed- and
variable-competing risks is assumed the same. In this section, we investigate that the proposed control
charts are CSC through simulation. In other words, any
shifts in the mean of quality characteristics in the first and
second stages do not affect the ARL of the CSC control
charts developed to monitor the quality characteristic in the
third stage. For this purpose, the performance of the pro-
posed control charts is studied under decreasing shifts of
EðXÞ crX or Eðy1Þ #ry1 in the mean of X and Y1. Table 3 presents the ARL results in the absence of cen-
soring with decreasing shifts, in the mean of the first- and
second-stage quality characteristics. As illustrated in Table 1, the ARL values of the
CUSUM control chart are less than the ARL values of
EWMA control chart under different shifts. It implies that
the proposed CUSUM control chart performs better in all
censoring rates in the second stage. Besides, censoring
scenario in the second stage has a detrimental effect on the
performance of control charts and leads to increasing the
corresponding out-of-control ARL values. Performance analysis under the presence
of censoring in the last stage The detection ability of the proposed control charts is
evaluated and compared when both the fixed- and variable- 75 J Ind Eng Int (2017) 13:67–80 J Ind Eng Int (2017) 13:67–80 Table 2 Comparison of the
control charts in the presence of
competing risk in the last stage
Control
charts
Censoring rate in
third stage (%)
Censoring rate in the
second stage (%)
d0
0
0.05
0.1
0.2
0.5
1
EWMA
20
0
200.26
155.90
120.44
78.78
30.11
12. 67
50
0
200.87
160.32
127.34
83.82
34.68
13.83
80
0
200.02
169.40
144.09
104.62
46.66
19.05
CUSUM
20
0
200.95
139.61
101.47
58.67
19.78
8.23
20
200.32
145.75
105.13
60.70
20.11
8.29
50
200.02
150.42
115.01
70.72
24.14
8.62
80
201.46
157.37
123.70
80.40
28.37
9.25
50
0
199.96
144.25
104.90
60.85
20.55
8.38
20
199.56
146.74
109.77
64.54
21.14
8.40
50
199.78
151.68
116.95
71.93
25.26
8.83
80
200.25
161.55
128.42
84.74
29.20
9.36
80
0
199.91
153.73
117.84
72.85
26.74
10.18
20
199.95
154.52
119.60
75.16
27.14
10.27
50
200.7
160.33
127.59
82.99
29.77
10.70
80
200.17
164.77
131.76
87.02
32.49
11.19
J Ind Eng Int (2017) 13:67–80
75 Table 2 Comparison of the
control charts in the presence of
competing risk in the last stage rate (80 %). In addition, the presence of censoring in both
the second and third stages is considered as the last
scenario. Table 3 ARLs of the CSC charts under shift in the mean of the first-
and second-stage quality characteristics Table 3 ARLs of the CSC charts under shift in the mean of the first-
and second-stage quality characteristics EðXÞ hrX
Eðy1Þ #ry1
h
EWMA
CUSUM
#
EWMA
CUSUM
0
200.53
200.61
0
200.53
200.61
0.5
202.53
200.22
0.5
199.81
199.89
1
203.29
201.15
1
203.16
202.06
1.5
201.42
199.61
1.5
201.05
199.72
2
200.22
199.95
2
202.19
201.66 In the illustrative example, the data are generated by the
parameters which are introduced at the previous sec-
tion. After generating 10 in-control samples, the data are
generated under the out-of-control status. This status is
occurred by a decreasing shift in the mean of quality
characteristic in the third stage which is equal to 0.2. The CUSUM and EWMA control charts for the data set
are shown in Fig. 2. Performance analysis under the presence
of censoring in the last stage The lower control limit of the EWMA
control chart is -0.4507, and the lower control limit of the
CUSUM control chart is -2.8. As shown in this figure, the
CUSUM control chart performs better than EWMA control
chart. first scenario, there are no censored data in both the second
and third stages. The second scenario includes high-cen-
sored data (80 %) in the second stage. In the third scenario,
censoring occurs in the third stage by the high-censoring first scenario, there are no censored data in both the second
and third stages. The second scenario includes high-cen-
sored data (80 %) in the second stage. In the third scenario,
censoring occurs in the third stage by the high-censoring Fig. 2 Proposed control charts under the absence of censoring Fig. 2 Proposed control charts under the absence of censoring Fig. 2 Proposed control charts under the absence of censoring 12 3 J Ind Eng Int (2017) 13:67–80 76 Fig. 3 Proposed control charts under the presence of high-censoring rate in the second stage
Fig. 4 Proposed control charts under the presence of high-censoring rate in the third stage Fig. 3 Proposed control charts under the presence of high-censoring rate in the second stage Fig. 3 Proposed control charts under the presence of high-censoring rate in the second stage Fig. 4 Proposed control charts under the presence of high-censoring rate in the third stage As illustrated in Fig. 3, the CUSUM control chart out-
performs the EWMA control chart in detecting decreasing
shift in the presence of censoring in the second stage. It
should be noted that the values of the fixed- and mean-
variable-competing risks are obtained by solving the
equations
in
Appendix
A,
i.e.,
c ¼ 0:1583
and
lcr ¼ 0:1685. In addition, the LCL of the EWMA and the
LCL of the CUSUM control charts are -0.44 and -2,
respectively. Figure 5 shows the control charts under the last scenario
when the data are censored in moderate rate (50 %) of the
last stage and the high rate (80 %) of the second stage. The
fixed and mean variable completing risks of the second
stage are similar to the previous cases, and for the third
stage, its values are 3.9 and 3.99, respectively. In addition,
the mean and variance of observations under the moderate
censoring rate are equal to 7.1075 and 71.6985, respec-
tively. Performance analysis under the presence
of censoring in the last stage Moreover, the lower control limit of the EWMA and
the lower control limit of the CUSUM control charts are
-0.68 and -1.9, respectively. The figure implies that the
performance of EWMA control chart is better than the
CUSUM control chart in this situation. Figure 4 shows the CUSUM and EWMA control charts
under the presence of high-censoring rate (80 %) in the
third stage, respectively. In this case, we have c = 3.8901,
lcr ¼ 3:9883, LCL = -2.28, and LCL = -0.6568. In
addition, the mean and variance of observations are
obtained generally based on Eqs. (31) and (32), whose
values are equal to 6.7356 and 31.7678, respectively. It is
clear that the CUSUM control chart performs better than
the EWMA control chart in this case. By comparing the figures, we conclude that as the
censoring rate decreases, the performance of control charts
deteriorates. In addition, the effect of censoring rate on the
performance of both EWMA and CUSUM control charts in
the third stage is more than the censoring rate in the second 123 J Ind Eng Int (2017) 13:67–80 77 Fig. 5 Proposed control charts under the presence of high-censoring rate in the second and third stages Fig. 5 Proposed control charts under the presence of high-censoring rate in the second and third stages stage. It means that the control charts perform better when
the censoring mechanism (80 %) is occurred in the second
stage instead of the third stage. performance of the control charts is larger when the cen-
soring happens in the third stage rather than the previous
stages. The authors suggest using the proposed methods of
this paper in a real case as a future research. Furthermore,
in this paper, we considered one quality characteristic in
each stage. However, sometimes, this assumption violates,
and there is more than one quality characteristic in each
stage. Therefore, developing control charts for monitoring
the multivariate lognormal quality characteristics in a
three-stage process could be considered as a future
research. In addition, the quality characteristics in different
stages may follow different distributions. This case can
also be considered as a fruitful area for future researches. Conclusion and future researches In this paper, a three-stage process with reliability-related
quality characteristic in the second and third stages was
considered. We assumed that the quality characteristics of
the second and third stages follow lognormal distribution
due to its vast application in real world. In addition, cen-
soring under both fixed- and variable-competing risks was
considered in this study. The AFT model, which is the most
widely used among the survival regression models, was
considered to relate the quality characteristics in three
stages. Then, we designed the EWMA and CUSUM cause-
selecting control charts based on this model and condi-
tional expected value to alleviate censoring mechanism. The performances of monitoring schemes were investi-
gated by the ARL values. The results of simulation study
confirmed that the censoring scenario in each stage dete-
riorates the performance of the proposed control charts. Moreover, the CUSUM control chart was usually better
than the EWMA control chart to detect shifts. The EWMA
control chart performs better just in the high-censoring rate
of the third stage when the censoring rate of the second
stage is 20 or 50 % simultaneously. Finally, it was shown
that the monitoring procedures are independent against
changing the mean shifts of the preceding stages. More-
over, an illustrative example was provided to show the
performance of control charts. It is clear that the censoring
would affect the performance of the control charts and
leads to deteriorating the performance of the control charts. It was also shown that the effect of the censoring on the Acknowledgments The authors are thankful to the respectful
anonymous referees for precious comments which led to improve-
ment in the paper. Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://crea
tivecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made. Appendix A: Determining the values of the fixed-
competing risk and the mean of variable-
competing risk corresponding to the quality
characteristic of the second stage to achieve
a specified percentage of censoring Appendix A: Determining the values of the fixed-
competing risk and the mean of variable-
competing risk corresponding to the quality
characteristic of the second stage to achieve
a specified percentage of censoring In a three-stage process assume that the quality character-
istics of the first, second, and third stages follow normal,
lognormal,
and
lognormal
distributions,
respectively,
shown by fx, fy1, and fy2, and the variable-competing risk of 12 J Ind Eng Int (2017) 13:67–80 78 PðCensoring due to the fixed competing riskÞ ¼ 0:25
PðCensoring due to the variable competing riskÞ ¼ 0:25:
ð37Þ the second stage follows normal distribution. To determine
the accurate values of the fixed-censoring level and the
mean of variable-competing risk of the second stage, the
following equations should be solved: the second stage follows normal distribution. To determine
the accurate values of the fixed-censoring level and the
mean of variable-competing risk of the second stage, the
following equations should be solved: ð37Þ c1 = 0.7378 and lcr1 ¼ 0:8698 are obtained by solving the
previous equations. PðCensoringduetothefixedcompetingriskÞ ¼ a PðCensoringduetothevariablecompetingriskÞ ¼ b; PðCensoringduetothevariablecompetingriskÞ ¼ b; ð34Þ ð34Þ Appendix B: Determining the values of fixed-
censoring level and the mean of variable-
competing risk to reach a specified percentage
of censoring corresponding to the quality
characteristic of the third stage where these formulas are computed as follows: PðCensoring due to the fixed competing riskÞ PðCensoring due to the fixed competing riskÞ ¼ Pðy1 [ c1 \ cr1 [ c1Þ ¼ Pðy1 [ c1Þ Pðcr1 [ c1Þ
¼
Z
x
½1 Fy1ðc1ÞfxðxÞdx ½1 Fcr1ðc1Þ
¼
Z
x
1 /y1
log c1 b0 b1x
ry1
1
rx
ffiffiffiffiffiffi
2p
p
e1
2
xlx
rx
ð
Þ
2
dx
Zþ1
c1
1
rcr1
ffiffiffiffiffiffi
2p
p
e
1
2
crlcr1
rcr1
2
dcr1
2
4
3
5;
ð35Þ In this Appendix, we calculate the accurate values of the
fixed-competing risk and the mean of the variable-
competing risk of the reliability-related quality charac-
teristic in the third stage. Therefore, all assumptions are
similar to Appendix A that the quality characteristics of
the first, second, and third stages follow normal, log-
normal, and lognormal distributions, respectively (shown
by fx, fy1, and fy2) in a three-stage process. The fixed-
competing risk value, c2, and the mean of the variable-
competing risk are normally distributed (denoted by fcr2). These variables are obtained by solving the following
equations: ð35Þ and PðCensoring due to the variable competing risk) ¼ Pðy1 [ cr1 \ cr1\c1Þ ¼ Pðy1 [ cr1 \ cr1\c1Þ ð
Þ
¼
Zc1
0
Zcr1
c1
fy1;cr1ðy1; cr1Þdcr1dy1 þ
Zþ1
c1
Zc1
0
fy1;cr1ðy1; cr1Þdcr1dy1
¼
Zc1
0
Zcr1
c1
fy1;cr1ðy1; cr1Þdcr1dy1 þ
Zþ1
c1
Zc1
0
fy1;cr1ðy1; cr1Þdcr1dy1
¼
Z
x
Zc1
0
/y1
log c1 b0 b1x
ry1
/y1
log cr1 b0 b1x
ry1
1
rcr1
ffiffiffiffiffiffi
2p
p
e
1
2
cr1lcr1
rcr1
2
1
rx
ffiffiffiffiffiffi
2p
p
e1
2
xlx
rx
ð
Þ
2
dcr1dx
þ
1
Zþ1
c1
1
rcr1
ffiffiffiffiffiffi
2p
p
e
1
2
crlcr1
rcr1
2
2
4
3
5
Z
x
1 /y1
log c1 b0 b1x
ry1
1
rx
ffiffiffiffiffiffi
2p
p
e1
2
xlx
rx
ð
Þ
2
dx:
ð36Þ PðCensoringduetothefixedcompetingriskÞ ¼ a PðCensoringduetothevariablecompetingriskÞ ¼ b: PðCensoringduetothevariablecompetingriskÞ ¼ b: ð38Þ Qual Reliab Eng
Int 19(6):477–491 PðCensoringduetothefixedcompetingriskÞ ¼ 0:25 Jearkpaporn D, Borror CM, Runger GC, Montgomery DC (2007)
Process monitoring for mean shifts for multiple stage processes. Int J Prod Res 45(23):5547–5570 PðCensoringduetothevariablecompetingriskÞ ¼ 0:25: ð41Þ Lawless JF (2003) Statistical models and methods for lifetime data. Wiley, New York After solving the above equations, c2 ¼ 3:8901 and lcr2 ¼
3:9883 are obtained. Li ZH, Kong ZH (2015) A generalized procedure for monitoring
right-censored
failure
time
data. Qual
Reliab
Eng
Int
31(4):695–705 Li Z, Zhou S, Sievenpiper C, Choubey S (2012) Statistical monitoring
of time-to-failure data using rank tests. Qual Reliab Eng Int
28(3):321–333 ð38Þ ð38Þ ð38Þ Subsequently, those equations are given by the follow-
ing equations: PðCensoring due to the fixed competing riskÞ PðCensoring due to the fixed competing riskÞ ð
g
p
g
Þ
¼Pðy2[c2\cr2[c2Þ
¼Pðy2[c2ÞPðcr2[c2Þ
¼
Z
y1
Z
x
1Fy2ðc2ÞfxðxÞfy1ðy1Þdxdy1 ½1Fcr2ðc2Þ
¼
Z
y1
Z
x
1/y2
logc2 a0 a1xi a2logðy1iÞ
ry2
fxðxÞfy1ðy1Þdxdy1
Zþ1
c2
1
rcr2
ffiffiffiffiffiffi
2p
p
e
1
2
cr2lcr2
rcr2
2
dcr2
2
4
3
5;
ð39Þ ¼Pðy2[c2\cr2[c2Þ
¼Pðy2[c2ÞPðcr2[c2Þ
Z Z ð36Þ For example, if it is aimed to have a total 50 % censored
observations with the equal censoring rate of the fixed- and
variable-competing risks, we can derive the following
equations by replacing 0.25 instead of a and b in Eq. ð38Þ (34)
as follows: ð39Þ and 123 123 79 J Ind Eng Int (2017) 13:67–80 PðCensoring due to the variable competing riskÞ ¼ Pðy2 [ cr2 \ cr2\c2Þ PðCensoring due to the variable competing riskÞ ¼ Pðy2 [ cr2 \ cr2\c2Þ PðCensoring due to the variable competing riskÞ ¼ Pðy2 [ cr2 \ cr2\c2Þ PðCensoring due to the variable competing riskÞ ¼ Pðy2 [ cr2 \ cr2\c2Þ PðCensoring due to the variable competing riskÞ ¼ Pðy2 [ cr2 \ cr2\c2Þ PðCensoring due to the variable competing riskÞ ¼ Pðy2 [ cr2 \ cr2\c2Þ
¼
Zc2
0
Zy2
0
fy2;cr2ðy2; cr2Þdcr2dy2 þ
Zþ1
c2
Zc2
0
fy2;cr2ðy2; cr2Þdcr2dy2
¼
Zc2
0
Zcr2
c2
fy2;cr2ðy2; cr2Þdcr2dy2 þ
Zþ1
c2
Zc2
0
fy2;cr2ðy2; cr2Þdcr2dy2
¼
Z
y1
Z
x
Zc2
0
/y2
log c2 a0 a1xi a2logðy1iÞ
ry2
/y2
log cr2 a0 a1xi a2logðy1iÞ
ry2
1
rcr2
ffiffiffiffiffiffi
2p
p
e
1
2
cr2lcr2
rcr2
2
1
rx
ffiffiffiffiffiffi
2p
p
e1
2
xlx
rx
ð
Þ
2
1
ffiffiffiffiffiffi
2p
p
ry2y2
e
1
2
logy2b0b1x
ry2
2
dcr2dxdy1
þ
1
Zþ1
c2
1
rcr2
ffiffiffiffiffiffi
2p
p
e
1
2
cr2lcr2
rcr2
2
2
4
3
5
Z
y1
Z
x
/y2
logc2 a0 a1xi a2logðy1iÞ
ry2
1
rx
ffiffiffiffiffiffi
2p
p
e1
2
xlx
rx
ð
Þ
2
1
ffiffiffiffiffiffi
2p
p
ry2y2
e
1
2
log y2b0b1x
ry2
2
dxdy1:
ð40Þ ¼
Zc2
0
Zy2
0
fy2;cr2ðy2; cr2Þdcr2dy2 þ
Zþ1
c2
Zc2
0
fy2;cr2ðy2; cr2Þdcr2dy2
¼
Zc2
0
Zcr2
c2
fy2;cr2ðy2; cr2Þdcr2dy2 þ
Zþ1
c2
Zc2
0
fy2;cr2ðy2; cr2Þdcr2dy2 ¼
Zc2
0
Zy2
0
fy2;cr2ðy2; cr2Þdcr2dy2 þ
Zþ1
c2
Zc2
0
fy2;cr2ðy2; cr2Þdcr2dy2
¼
Zc2
0
Zcr2
c2
fy2;cr2ðy2; cr2Þdcr2dy2 þ
Zþ1
c2
Zc2
0
fy2;cr2ðy2; cr2Þdcr2dy2 ð40Þ As mentioned in Appendix A, if we would like to have a
total of 50 % censored data with equal censoring rate of the
fixed- and variable-competing risks, for instance, Eq. (38)
would be as follows: Batson RG, Jeong Y, Fonseca DJ, Ray PS (2006) Control charts for
monitoring field failure data. Qual Reliab Eng Int 22(7):733–755 Jearkpaporn D, Montgomery DC, Runger GC, Borror CM (2003)
Process monitoring for correlated gamma distributed data using
generalized-linear model-based control charts. References Lu W, Tsai T (2008) Exponentially weighted moving average control
chart for gamma distribution with type I censoring. In:
Proceedings of the 3rd international conference on innovative
computing information and control, pp 128–131 Akhundjanov SH, Pascual F (2015) Moving range EWMA control
charts for monitoring the Weibull shape parameter. J Stat
Comput Simul 85(9):1864–1882 Meeker WQ, Escobar LA (1998) Statistical methods for reliability
data. Wiley, New York Asadzadeh Sh, Aghaie A (2012) Improving the product reliability in
multistage manufacturing and service operations. Qual Reliab
Eng Int 28(4):397–407 Nichols MD, Padgett WJ (2006) A bootstrap control chart for Weibull
percentile. Qual Reliab Eng Int 22(2):141–151 Asadzadeh Sh, Aghaie A, Niaki STA (2013) AFT regression-adjusted
monitoring of reliability data in cascade processes. Qual Quant
47(6):3349–3362 Padgett WJ, Spurrier JD (1990) Shewhart-type charts for percentiles
of strength distributions. J Qual Technol 22(4):283–288 Page
ES
(1954)
Continuous
inspection
schemes. Biometrika
41:100–115 Asadzadeh S, Aghaie A, Shahriari H, Niaki STA (2014a) The application
of proportional hazards and frailty models to multistage processes
surveillance. Int J Adv Manuf Technol 74(1–4):461–470 Shafae M, Dickinson R, Woodall W, Camelio J (2014) Cumulative
sum control charts for monitoring Weibull-distributed time
between events. Qual Reliab Eng Int 31(5):839–849 Asadzadeh Sh, Aghaie A, Shahriari H (2014b) Using frailty models to
account for heterogeneity in multistage manufacturing and
service processes. Qual Quant 48(2):593–604 Shamsuzzaman M, Xie M, Goh TN, Zhang HY (2009) Integerated
control chart system for time between-events monitoring in a 123 12 3 80 J Ind Eng Int (2017) 13:67–80 multistage manufacturing system. Int J Adv Manuf Technol
40(3–4):373–381 Zhou C, Chinnam RB, Korostelev A (2012) Hazard rate models for
early detection of reliability problems using information from
warranty databases and upstream supply chain. Int J Prod Econ
139(1):180–195 Skinner KR, Montgomery DC, Runger GC (2003) Process monitoring
for multiple count data using generalized linear model-based
control charts. Int J Prod Res 41(6):167–1180 Steiner SH, MacKay RJ (2000) Monitoring processes with highly
censored data. J Qual Technol 32(3):199–208 Azam Goodarzi received her BS degree in Industrial Engineering
from Alzahra University in Tehran. In addition, she holds an MS
degree in Industrial Engineering from Shahed University in Tehran in
2014. Her research interests are Statistical Quality Control, Reliabil-
ity, and Survival Data Analysis. Steiner SH, MacKay RJ (2001) Monitoring processes with data
censored owing to competing risks by using exponentially
weighted moving average control charts. References App Stat 50:293–302 Surucu B, Sazak HS (2009) Monitoring reliability for a three-
parameter
Weibull
distribution. Reliab
Eng
Syst
Saf
94(2):503–508 Amirhossein Amiri is an Associate Professor at Shahed University in
Iran. He holds a BS, MS, and Ph.D. in Industrial Engineering from the
Khajeh Nasir University of Technology, Iran University of Science
and Technology, and Tarbiat Modares University in Iran, respec-
tively. He is now a head of Industrial Engineering Department at
Shahed University in Iran and a member of the Iranian Statistical
Association. His research interests are statistical quality control,
profile monitoring, and Six Sigma. He has published many papers in
the area of statistical process control in high-quality international
journals, such as Quality and Reliability Engineering International,
Communications in Statistics, Computers and Industrial Engineering,
Journal of Statistical Computation and Simulation, Soft Computing,
and so on. He has also published a book with John Wiley and Sons in
2011 entitled the Statistical Analysis of Profile Monitoring. Tsung F, Li Y, Jin M (2008) Statistical process control for multistage
manufacturing and service operations: a review and some
extensions. Int J Serv Oper Inf 3(2):191–204 p
Wade MR, Woodall WH (1993) A review and analysis of cause-
selecting control charts. J Qual Technol 25(3):161–169 g
Xiang L, Tsung F (2008) Statistical monitoring of multi-stage
processes
based
on
engineering
models. IIE
Trans
40(10):957–970 Xie M, Goh TN, Ranjan P (2002) Some effective control chart pro-
cedures
for
reliability
monitoring. Reliab
Eng
Syst
Saf
77(2):143–150 Yang CM (1999) Economic process management and its application
on bank industry. Qual Quant 33(4):381–394 Zhang GX (1984) A new type of control charts and theory of
diagnosis with control charts. In: World quality congress
transactions. Milwaukee, WI: American Society for Quality
Control, 3, pp 175–185 Shervin Asadzadeh is an Assistant Professor in Islamic Azad
University, North Tehran Branch. He holds his Ph.D. in Industrial
Engineering from the K.N. Toosi University of Technology. He has
been teaching Statistical Methods, Design of Experiments, Time
Series Analysis, Statistical Quality Control, Quality Management and
Productivity, and Engineering Statistics at the K.N. Toosi University
of Technology, Allameh Tabataba’i University, and Islamic Azad
University. His primary research interests include Statistical Quality
Control, Reliability, Survival Data Analysis, Applied Statistics,
Robust Statistics, and Simulation. He is a member of National Elites
Foundation of Iran, Iranian Institute of Industrial Engineering, and
Iranian Statistical Society. References Zhang L, Chen G (2004) EWMA charts for monitoring the mean of
censored Weibull lifetimes. J Qual Technol 36(3):321–328 Zhang HY, Shamsuzzaman M, Xie M, Goh TN (2011a) Design and
application of exponential chart for monitoring time-between-
events data under random process shift. Int J Adv Manuf
Technol 57(9–12):849–857 Zhang HY, Xie M, Goh TN, Shamsuzzaman M (2011b) Economic
design of time-between-events control chart system. Comput Ind
Eng 60(4):485–492 Zhang M, Xu Y, He Z, Hou X (2015) The effect of estimation error on
risk- adjusted survival time cusum chart performance. Qual
Reliab Eng Int. doi:10.1002/qre.1849 123 123 123
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English
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Mortality Causes of Autoimmune Rheumatic Diseases
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IntechOpen eBooks
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cc-by
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Abstract Autoimmune Rheumatic Diseases (ARDs) are chronic multisystemic diseases that
have a low prevalence (estimated to range from 4 to 5% of the general population),
and the impact on mortality in ARDs is lower (mortality reported in ARDs is 0.3 to
2.1) in general statistics worldwide compared to other diseases with higher prevalence
such as arterial hypertension (HBP) or diabetes mellitus (DM). The objective of
this review is to update the concepts regarding mortality associated with ARD, and
the most relevant studies and review were included. The causes of mortality among
ARDs vary widely between geographic areas and cannot be generalized, although
the most important frequency reported is in rheumatoid arthritis (RA), systemic
lupus erythematosus (SLE) and scleroderma (SSc). It has recently been reported
that measuring the standardized mortality rate (SMR) identifies the inflammatory
diseases with increased risk: 4.80 in systemic vasculitis (SV), 2.9 in SLE, and 1.44 in
RA. The causes of death are regularly associated with acute events (infections and
respiratory and cardiovascular diseases) and less frequent related to the disease sever-
ity. Other reported associated factors have been age, duration, type of presentation of
the disease, and socioeconomic status. We found that the variation between the main
reported causes is little; significantly higher mortality (five times more) has been
found in the regional analysis in Latin America compared to that in Europe. The most
important factor in the last decades is the habitual use of drugs that increase the risk
of immunosuppression and infection. Keywords: autoimmune rheumatic diseases, systemic vasculitis, systemic lupus
erythematosus, rheumatoid arthritis, standardized mortality ratio Chapter
Mortality Causes of Autoimmune
Rheumatic Diseases Maynor Herrera-Méndez 1. Introduction Autoimmune rheumatic diseases have a low prevalence of associated mortal-
ity (0.3 to 2.1) [1]. When the mortality of ARDs is compared with more prevalent
diseases (DM and BHP), the statistical impact is lower worldwide, and consequently,
the study of its causes has been limited. y
The objective of this review is to update the concepts on mortality associated with
ARD, and we searched for studies of the last 50 years in the main databases (Pubmed,
Cochrane, Google academic, and Scopus) using the words autoimmune disease,
rheumatic disease, and mortality in autoimmune disease and then looking for causes
of mortality in VS, SLE, and RA. 1 Probing Selected Autoimmune Diseases for Focused Perspectives The information analyzed comes from databases of better organized institutions
and complemented with the analysis of studies that describe in detail the causes of
mortality in the different latitudes; sixty-five reports (cases, observational studies,
and database analysis) of mortality and rheumatic disease were reviewed, including
at the end 25 that contain the most important information. Recently was informed that measure the standardized mortality ratio risk identi-
fied at population in risk. In general terms, the group of rheumatic diseases has a risk
of 2.03 (95% CI: 1.79–2.29) and has been reported particularly for those considered as
inflammatory. The highest risk reported was 4.80 in systemic vasculitis, followed by
2.9 in systemic lupus erythematosus and 1.44 in rheumatoid arthritis [2]. Some studies also report that patients with diseases such as Myositis (MI), SSc,
and Sjogren’s syndrome (SS) may have a significant risk, which is not yet considered
among the most important because its prevalence is lower. g
p
p
The causes of death are regularly associated with acute events, infections, cardio-
vascular and lung diseases, and a lower probability with the clinical spectrum of the
disease. Other related factors reported are age and duration of the disease, the severity of
presentation, and the adverse effects relating to the treatment administered [3–5]. The comorbidities, specialty cardiovascular, metabolic, and respiratory are the most
important [6–8]. Most recently, use of novelty pharmacies (monoclonal antibodies, Jak inhibi-
tors) in combination with glucocorticoids and immunosuppressive drugs has been
proposed as the factor that increases the risk [9–12]. However, it should be noted that
thanks to this therapeutic innovation, ARD patients have improved their quality and
life expectancy. 2.1 General features The notion of autoimmune diseases in humans’ dates to the beginning of the 20th
century, and currently, more than 40 human diseases have been described that could
have this name. The first widely described autoimmune disease with underlying pathophysiologi-
cal mechanisms was Hemolytic Anemia, described in 1925 by Lederle [13]. However,
autoimmune diseases were not officially recognized as such until the 1940s–50s, and
until then, the reports on mortality in these diseases were scarce. The first congress on
these diseases was held in 1965. In this line of time (Figure 1), the main events related to autoimmune diseases are
identified. 2.2 Background The study of rheumatic diseases focused on morbidity, loss of function, instabil-
ity caused by joint damage, and reduced quality of life. Nonetheless, in 1955, it was
estimated that the 5-year survival in patients with SLE, vasculitis, and polymyositis
was only 5%, and it was considered part of the “natural history” of the disease; it was
considered that the systemic inflammatory process and the progression of disease
were the cause of death [14]. 2 Mortality Causes of Autoimmune Rheumatic Diseases
DOI: http://dx.doi.org/10.5772/intechopen.1002549 Mortality Causes of Autoimmune Rheumatic Diseases
DOI: http://dx.doi.org/10.5772/intechopen.1002549 Figure 1. Line of time. As early as 1953, Cobb et al. described the course of death in 130 patients with
rheumatoid arthritis who were followed for nine years. The first cause of death
reported in 25% was infection, 24% heart disease and 13% various forms of nephrop-
athy [15]; besides, in similar study, Mitchell reported a decrease in 5–15 years life
expectancy in this patient [16]. p
y
p
In 1976, Urowitz and his collaborators suggested bimodality as the cause of death
in SLE: 1. Early death with an active disease that requires high doses of glucocorti-
coids is regularly associated with infections and 2. A late death in an inactive disease,
which has required many years of glucocorticoid intake and whose outcome is heart
disease [17]. 2.3 Current development in mortality trends Due to the wide variation in mortality in rheumatic diseases, it has been necessary
to develop precise and weighted analyses that calculate the risk; one of them is the
SMR. This is obtained by calculating the relationship between the deaths observed in
a cohort and those expected in a group of the same size from the general population
(in the same area and standardized by age and sex). Toledano et al. measured the SMR in rheumatic diseases and performed a meta-
analysis in 2012, finding 32 studies that were analyzed (16 in RA, 7 in SC, 5 in SLE,
and 2 in Vasculitis). They reported that the highest risk is in inflammatory diseases,
vasculitis 4.8, SLE 2.9, and RA 1.4, and reported that the leading cause of mortality
is cardiovascular diseases, followed by pulmonary, renal, and infectious diseases
[2]. In ARDs with an inflammatory component, SMR 2.03 is higher and becomes
evident when compared with that of fibromyalgia, an entity without an inflammatory
burden, with 0.49 [18]. Recently, in 2020, Scherlinger and collaborators have carried out a review of the
mortality of rheumatic diseases between 2001 and 2014; the six main diseases, SLE,
SSC, MI, SS, Vasculitis, and MCTD (mixed connective tissue disease), reported
according to the World Health Organization were included. The number of deaths
per million inhabitants was estimated using the ASMR (aged-standardized mortality
rate), with the most important data being SLE 2.68 and SSc 1.48. The following were
reported during 2014 (reports from 35 to 85 countries): 6418 deaths in SLE, 4287 in
SSc, 1313 in MI, 438 in SS, 235 in MCTD, and 1350 in Vasculitis [19]. 3 Probing Selected Autoimmune Diseases for Focused Perspectives It is found that the ASMR is up to five times higher in the countries of Latin
America compared to the in the countries of Europe, not finding this relationship
with countries of other continents [19]. This could be related to inequity in the provi-
sion of medical care and the quality of health services in each of the countries that
consistently report mortality. 3.1 Background ARDs are a heterogeneous group of diseases, characterized by an immune dysreg-
ulation resulting in inflammation and multi-organ involvements that can conditioned
the died. The immune dysregulation involves tissues, cells, and molecules that usually
participated in immune response, especially against infectious agents [20]. Usually factors involved in this dysregulation are several: Ambiental, genetic,
infectious diseases, and most recent changes in the microbiome and hygienic
hypothesis. Because of this initial stimulus, the immune response has become unbalanced, and
our own tissues become the target of action, named horror autotoxicus for Paul Erlich
in 1901 [20]. This imbalance is reflected in the different pathways that participate in the
immune response, which react to the presence of an “autoantigen” that is not recog-
nized as their own (loss of self-tolerance), because the peptide sequences are similar
and can be confused with those of foreign agents. 3.2 Hypothesis and ARD development In recent years, different hypotheses have been accepted as triggers for an autoim-
mune disease; the most important are [21]: Exposure of cryptic antigens, overexpres-
sion of MHC (Major Histocompatibility Complex) and costimulatory molecules,
neoantigen (microorganism + self-antigen), super antigen (polyclonal activation of T
lymphocytes), and molecular mimicry. y
p
y
y
Initially, the autoantigen is caught for antigen presenting cells (APC) and pre-
sented to the T cells in the thymus, in response the most autoreactive T cells that
undergo apoptosis, but a small percentage of them enter the general circulation and
escape the usual controls: clonal anergy (in peripheral lymphoid tissue), phenom-
enon of suppression by Treg cells and immunological ignorance (whose mechanisms
are still unknown). These reactive T cells are responsible for activating B cells that produce and release
specific antibodies and proinflammatory cytokines, which in turn are responsible for
amplifying inflammation and triggering the phenomena of tissue destruction and the
systemic manifestations of autoimmune diseases, which can be perpetuated and cause
death of the patient [21]. p
Figure 2 illustrates the process of generalized inflammation, with the possible
triggers for an ARD and the factors that contribute to death as the outcome. Timely
therapeutic intervention (pharmacological or non-pharmacological) can change the
outcome. 4 Mortality Causes of Autoimmune Rheumatic Diseases
DOI: http://dx.doi.org/10.5772/intechopen.1002549 Mortality Causes of Autoimmune Rheumatic Diseases
DOI: http://dx.doi.org/10.5772/intechopen.1002549 p
g
577
p
549
i 4.2 Analysis of identified risks The vascular compromise suggested the close relationship between the constant
inflammatory states that affect the blood vessels, especially in the heart, kidney, and
the central and peripheral nervous system. The importance of therapeutic intervention is vital; however, it is not free of
complications. This could explain the bimodal presentation of mortality reported by
Urowitz [17]. The use of corticosteroids, immunosuppressants such as cyclophosphamide, and,
recently, monoclonal antibodies decreases the risk of progression of the disease by
controlling inflammation but increases the risk of infection. g
In the case of metabolic diseases, especially DM type II could be secondary at use
of glucocorticoids and lifestyle patterns no modified that increase the risk of death
secondary to immunosuppression and vascular diseases associated. The use of con-
comitant drugs to control glucose levels and blood pressure continuously contributes
to a good control of the disease and a better outcome. 5. Therapeutics and ARD mortality Survival of patients with ARD in the last 5 decades has improved significantly due
to the use of medications such as glucocorticoids, immunosuppressants, monoclonal
antibodies, and concomitant drugs (antihypertensive agents, antibiotics, etc.). However, for the extended use of glucocorticoids, there has also been an increase
in the risk of developing infections. Saag et al. reported an 8 times increased risk
(OR) compared to those who do not use them [9]. Petri et al. reported a higher risk of infection when comparing patients with SLE
with and without hospitalization, with doses of prednisone higher than 10 mg/day
[7], and Listing et al. in RA found that doses of glucocorticoids above 5 mg were
associated with an increase in mortality [10]. In general terms, an increased risk of death has not been demonstrated with the
use of immunosuppressants; two authors, Bultink and Listing, reported a lower risk
of mortality (not statistically significant) in patients with SLE intake any immuno-
suppressant drugs [10, 11], and Mok et al. highlight that the use of immunosuppres-
sants was not associated with increased survival in patients with SLE; however, the
risk of infections increases considerably [12]. 4.1 Background and reports In recent years, data collection through population-based studies has increased; it
is the most widely used method for analyzing the prevalence of comorbidities. y
y
g
p
However, it is pertinent in the analysis to establish that it requires consider-
able resources and the self-filling of documents by the patients, which implies
the probability of making errors in the identification of the main pathologies,
especially in the case of ARDs, which are diseases with great clinical and complex
heterogeneity. The report of various studies has assessed the co-occurrence of ARD with other
diseases at the time of death, regularly using death certificate data, among the most
frequent is include to cardiovascular diseases, pulmonary disorders, coagulation and
hemorrhagic disorders, and renal failure. We have reported in our center the mortality-associated comorbidities in
ARD; those of the highest prevalence were HBP at 38.9% and DM type II at 11.3%. Specifically, in SLE, 50% reported HBP, 8.7% antiphospholipid syndrome (APS)
associated with thrombosis, and 6.5% chronic kidney disease (CKD). In RA, the main
association was HBP in 26.9% and DM-II in 23% of cases. In 24.3%, no comorbidity
was reported [22]. These data are like those reported by another investigators: Panoulas et al. and
Petri et al. reported in RA and SLE the double the prevalence of HBP in comparison
with the general population [6, 7]. g
p p
Sabio et al. reported HBP in 40% of patients with SLE compared with the 11% of
controls of the same age [8]. Other authors also include minor comorbidities, musculoskeletal disease, geni-
tourinary system, blood diseases, and diseases of the skin and subcutaneous tissue, 5 Probing Selected Autoimmune Diseases for Focused Perspectives and confirm the classic cause, cardiovascular diseases (55.5%), followed by diseases
of the respiratory system (35.9%), endocrine and metabolic disorders (21.6%), and
neoplasms (20.9%) [23]. 6. Mortality around the world In recent years, the number of studies on the causes of mortality in ARDs has
increased, although the methodologies used are diverse; the results of most stud-
ies show infections as the main cause, followed by cardiovascular and pulmonary
diseases. Some studies have also described disease activity as the cause of death, and it
stands out that at least two of them report the cause of death as missing or unknow 6 Mortality Causes of Autoimmune Rheumatic Diseases
DOI: http://dx.doi.org/10.5772/intechopen.1002549 Mortality Causes of Autoimmune Rheumatic Diseases
DOI: http://dx.doi.org/10.5772/intechopen.1002549 Mortality Causes of Autoimmune Rheumatic Diseases
DOI: http://dx.doi.org/10.5772/intechopen.1002549 Recently, in a population study in Greece, Bournia et al. have described that
mortality in rheumatic diseases occurs more frequently compared to the general
population, preferably in young patients and the prevalence is higher in the main
inflammatory diseases (SLE, SV and SSC) [24]. Author/Country
Year study/
patients number
Mortality causes (%)
Reference/comments
Mok et al./Hong
Kong
1999–2008/2486
Infections (28%)
CV complications (18%)
Cancer (16%)
Disease activity (7%)
Renal failure (6%)
Pulmonary causes (6%)
GI/Hepatic complications (4%)
Accidents,
injury, or poisoning (including
suicide) (0.7%)
Missing (9%)
[5]
Toledano et al./
Spain
2012
SMR high risk
Infections 11.3
CV 3
Pulmonary causes 2.33
[2]
Meta-analysis
Garen et al./
Norwegian
1999–2015/279
CV (27%)
SV (28%)
Cancer (CTD 25%-SV 27%),
Pulmonary causes (CTD
20%-SV10%)
Infections (CTD 9%-SV 16%). [25]
Ten-year follow
Mitratza et al./
Netherlands
2013–2017/ 3335
CV (55.5%)
Respiratory causes (35.9%)
Endocrine and metabolic disorders
(21.6%)
Cancer (20.9%)
Infections (16%)
Musculoskeletal system 3.38
Genitourinary system 2.73
Influenza 2.71
Blood diseases 2.02
Skin and subcutaneous tissue
diseases 1.95
Infectious diseases 1.85
[23]
Dadoniene J et al./
Lithuanian
2013–2019/950
CV (47%)
Cancer (23%)
Respiratory causes (6%)
Musculoskeletal system (5%)
Other causes (16%)
[26]
Patients with ARD
have a higher risk of
mortality and lower
life expectancy
Leonardo H et al./
Guatemala
2009–2019/185
Infections (56%)
Activity Disease (16%)
CV (8%)
Other (7%)
Unknown (12%)
[22]
CV, cardiovascular; SMR, standardized mortality ratio; CTD, Connective Tissue Disease; SV, Systemic vasculitis. T bl CV, cardiovascular; SMR, standardized mortality ratio; CTD, Connective Tissue Disease; SV, Systemic vasculitis Table 1. 7 Probing Selected Autoimmune Diseases for Focused Perspectives It is possible that factors such as the quality of care provided by health services
and the socioeconomic conditions of the patients are not correctly recorded, and
for now, it is not possible to establish a direct relationship with these factors that are
mentioned anecdotally as contributors to the mortality of the ARDs. The consensus is that effective therapeutic modalities such as treat-to-target and
effective control of comorbidities improve survival and, with experience obtained
with this methodology in RA and EA during the years 2015–2019, should be extended
to all ARDs [24]. Table 1 summarizes the most important studies in recent years and identifies the
main causes. Most agree in reporting that infections, CV and pulmonary complica-
tions, and disease activity are the leading causes of death. It also allows you to view the comorbidities that are reported as a product of the
same disease, associated with the disease or consequence of the therapeutic used,
such as HBP, endocrine and metabolic diseases, kidney failure, liver complications,
and cancer. 7. Conclusion We found that the variation between the main reported causes is little; the results
of most studies show infections as the main cause, followed by cardiovascular and
pulmonary diseases. p
y
The risk is largely different among various ARDs, due to diverse underlying
pathogenetic mechanisms and the degree of vital organ involvement, as well as due to
different therapeutic regimes used and different age of onset of each disease. The higher mortality (five times more) that has been reported in Latin America
compared to in Europe is significantly important. It could be related to the quality and
inequity in the provision of health services; this analysis is beyond this review. The most important factor in the last decades is the habitual use of drugs that
increase the risk of immunosuppression and infection. It is common that the ARDs
are characterized by increased morbidity and mortality, due to disease-specific fac-
tors and iatrogenic damage (e.g., drug-related side effects) and the associated comor-
bidity as cardiovascular disease, malignancy, and increased risk for infections. The routine electronic or manual recording systems for death, admissions, and
hospital discharge statistics provide only a partial picture of the prevalence of ARDs
and could be the reason why these diseases have a low lethality and hospitalization
frequency. Conflict of interest The author does not have conflict of interests to declare. Author details Maynor Herrera-Méndez y
Internal Medicine Department, Rheumatology Unit - Roosevelt Hospital, Post
Graduate School of San Carlos University of Guatemala, Guatemala *Address all correspondence to: drherreram@gmail.com Mortality Causes of Autoimmune Rheumatic Diseases
DOI: http://dx.doi.org/10.5772/intechopen.1002549 Mortality Causes of Autoimmune Rheumatic Diseases
DOI: http://dx.doi.org/10.5772/intechopen.1002549 Mortality Causes of Autoimmune Rheumatic Diseases
DOI: http://dx.doi.org/10.5772/intechopen.1002549 Funding This research did not receive any specific funding from public sector, commercial
sector, or not-for-profit agencies. 8 © 2023 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of
the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided
the original work is properly cited. 9 Probing Selected Autoimmune Diseases for Focused Perspectives Probing Selected Autoimmune Diseases for Focused Perspectives Probing Selected Autoimmune Diseases for Focused Perspectives References [1] Thomas SL, Griffiths C, Smeeth L,
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Meyer A, Felten R, et al. Worldwide
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immunosuppressive therapies on survival
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DOI: http://dx.doi.org/10.5772/intechopen.1002549 2014;66:2915-2920. DOI: 10.1002/
art.38796 autrev.2020.102531 [20] Plotz PH. Autoimmunity: The history
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Práticas sociais com crianças e adolescentes: o impacto dos conselhos tutelares
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Psicologia
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198 198 1 O artigo integra a tese de
doutorado
intitulada
“Conselho
Tutelar
e
Comunidade: o Impacto dos
Conselhos nas Práticas
Sociais com Crianças e
Adolescentes”, apresentada
ao Programa de Pós-
Graduação em Psicologia
da PUCRS, em 2004, para
obtenção do título de Doutor
em Psicologia, com apoio da
CAPES. Práticas Sociais com Crianças
e Adolescentes: O Impacto
dos Conselhos Tutelares1 Práticas Sociais com Crianças
e Adolescentes: O Impacto
dos Conselhos Tutelares1
Social Practices With Children And Adolescents: The
Impact Of The Guardian Council
Artigo
Katia Regina Frizzo
Universidade do
Oeste de Santa Catarina
Jorge Castellá Sarriera
Pontifícia
Universidade Católica do
Rio Grande do Sul
1 O artigo integra a
doutorado
inti
“Conselho
Tute
Comunidade: o Impa
Conselhos nas Pr
Sociais com Cria
Adolescentes”, apres
ao Programa de
Graduação em Psi
da PUCRS, em 200
obtenção do título de
em Psicologia, com a
CAPES. PSICOLOGIA CIÊNCIA E PROFISSÃO, 2006, 26 (2), 198-209 Social Practices With Children And Adolescents: The
Impact Of The Guardian Council 1 O artigo integra a tese de
doutorado
intitulada
“Conselho
Tutelar
e
Comunidade: o Impacto dos
Conselhos nas Práticas
Sociais com Crianças e
Adolescentes”, apresentada
ao Programa de Pós-
Graduação em Psicologia
da PUCRS, em 2004, para
obtenção do título de Doutor
em Psicologia, com apoio da
CAPES. PSICOLOGIA CIÊNCIA E PROFISSÃO, 2006, 26 (2), 198-209 199 PSICOLOGIA CIÊNCIA E
PROFISSÃO, 2006, 26 (2), 198-209 Resumo: As práticas sociais constituem um importante campo de reflexão
e pesquisa dentro da Psicologia comunitária latino-americana. As práticas
sociais com crianças e adolescentes envolvem principalmente as famílias
e as instituições educativas, de saúde e outras. O presente artigo apresenta
uma pesquisa realizada com a finalidade de verificar o impacto produzido
pela ação do Conselho Tutelar no Município sobre as práticas familiares e
institucionais com crianças e adolescentes. Foi realizado um levantamento
das estatísticas de atendimento de 2000 a 2003 (quatro anos) do Conselho
Tutelar de um Município do meio-oeste do Estado de Santa Catarina. Também foram realizadas algumas entrevistas com profissionais de
diferentes setores de atividade que interagem freqüentemente com o
Conselho para verificar suas opiniões sobre o mesmo. Os resultados
indicaram um impacto maior nas práticas sociais familiares e nas práticas
sociais institucionais de saúde. O impacto sobre as práticas institucionais
educacionais e de trabalho não é relevante. Resumo: As práticas sociais constituem um importante campo de reflexão
e pesquisa dentro da Psicologia comunitária latino-americana. As práticas
sociais com crianças e adolescentes envolvem principalmente as famílias
e as instituições educativas, de saúde e outras. O presente artigo apresenta
uma pesquisa realizada com a finalidade de verificar o impacto produzido
pela ação do Conselho Tutelar no Município sobre as práticas familiares e
institucionais com crianças e adolescentes. Práticas Sociais com Crianças
e Adolescentes: O Impacto
dos Conselhos Tutelares1 Foi realizado um levantamento
das estatísticas de atendimento de 2000 a 2003 (quatro anos) do Conselho
Tutelar de um Município do meio-oeste do Estado de Santa Catarina. Também foram realizadas algumas entrevistas com profissionais de
diferentes setores de atividade que interagem freqüentemente com o
Conselho para verificar suas opiniões sobre o mesmo. Os resultados
indicaram um impacto maior nas práticas sociais familiares e nas práticas
sociais institucionais de saúde. O impacto sobre as práticas institucionais
educacionais e de trabalho não é relevante. Palavras-chave: práticas sociais, conselho tutelar, comunidade. Abstract: The social practices constitute an important field of reflection
and research within Latin American communitarian Psychology. The social
practices with children and adolescents comprehend manly the families
and educational institutions, health institutions and others. This article
presents a research conducted with the purpose of verifying the impact
produced by the action of the guardian council in the community on the
family and institutional practices with children and adolescents. A raising
of service statistics of the guardian council of a middle-west district of the
State of Santa Catarina was done from 2000 to 2003 (four years). Some
interviews were also made with professionals from different areas that
frequently interact with the council to verify their opinions on it (the
council itself). The results indicated a bigger impact on family social
practices and on institutional social health practices. The impact on
institutional educational and work practices is not relevant. Key words: social practices, guardian council, community. p
Key words: social practices, guardian council, community. comunitária que desenvolve e divulga as
pesquisas da área. O campo das práticas sociais tem se revelado
um campo fértil para as pesquisas em
Psicologia social no País, nos últimos tempos. Após a “virada” da Psicologia social na década
de 80, tornou-se importante investigar a
produção das condições sociais de existência
e isso se dá no terreno das práticas sociais. A ABRAPSO cumpriu importante papel no
desenvolvimento da área e constituiu-se um
grupo de trabalho sobre a Psicologia social Entendemos por prática social toda prática de
interação (= relação) entre sujeitos sociais,
em que uns acabam produzindo efeitos em
outros, planejada e conscientemente, ou
não. A Psicologia social comunitária
e seu campo de atuação aconteça, produz um efeito, e, portanto, é
uma prática. As práticas de educação dos
filhos, as práticas pedagógicas e as práticas
de submissão e poder nas relações de
trabalho são exemplos de práticas sociais que
produzem efeitos calculados; elas visam a
determinados resultados. Outras práticas,
como as que se desenvolvem nas relações
de gênero, não são necessariamente
controladas, mas apontam um sentido de
reprodução social de estereótipos e
preconceitos. Não há, portanto, como afirma
Guareschi (1997), neutralidade na ação. O desenvolvimento da Psicologia comunitária
no Brasil e na América Latina seguiu o
caminho da identificação com os problemas
sociais como aqueles que mereciam sua
atenção privilegiada e com os setores
populares que lutavam por uma sociedade
mais justa como os segmentos de
desenvolvimento do seu campo de atuação
(Lane, p.1996). Como uma área cuja
identidade não estava definida, escorregou
nas práticas clínicas como primeira alternativa
de ancoragem de sua identidade, mas logo
encontrou o caminho da pesquisa e da
intervenção participante como forma mais
adequada de sua inserção nesse novo campo
(Freitas, p.1996). No presente estudo, abordamos as práticas
sociais voltadas para as crianças e
adolescentes, tanto dentro da família como
na escola e em outras instituições sociais. Por exemplo, a prática de bater nos filhos
para atingir determinados objetivos de
mudança de comportamento (violência física)
pode levar a uma intervenção do Conselho
Tutelar, caso haja denúncia por parte de
vizinhos e/ou outras pessoas. A prática de
denunciar também é uma prática social, pois
exige a ação do sujeito em favor da defesa
de outro/os. É nossa convicção que certas
práticas só aprendemos mesmo em
comunidade ou pela intervenção externa à
família, através de organismos e/ou
instituições. Trata-se de uma aprendizagem
social produzida pelas interações sociais no
desenvolvimento do sujeito. Participar de
uma reunião pode não ser uma prática
comum, muito menos ainda se exige a nossa
participação, entretanto, muitos aprendem
a participar dentro de reuniões promovidas
pela igreja, pelas escolas, pelas associações
de bairro. 200 200 200 Práticas Sociais com Crianças e Adolescentes: O Impacto dos Conselhos Tutelares Práticas Sociais com Crianças
e Adolescentes: O Impacto
dos Conselhos Tutelares1 Segundo Guareschi (1997, p.13), “é
impossível não agir”, pois mesmo a omissão
ou a ação passiva, quando impede que algo Após a “virada” da Psicologia social na década
de 80, tornou-se importante investigar a
produção das condições sociais de existência
e isso se dá no terreno das práticas sociais. A ABRAPSO cumpriu importante papel no
desenvolvimento da área e constituiu-se um
grupo de trabalho sobre a Psicologia social A Psicologia social comunitária
e seu campo de atuação O desenvolvimento
da Psicologia
comunitária no
Brasil e na América
Latina seguiu o
caminho da
identificação com
os problemas
sociais como
aqueles que
mereciam sua
atenção
privilegiada e com
os setores
populares que
lutavam por uma
sociedade mais
justa como os
segmentos de
desenvolvimento
do seu campo de
atuação Os vários enfoques utilizados pela Psicologia
social comunitária latino-americana (Montero,
1994) demonstram seu compromisso com a
emancipação das classes populares, com a
defesa dos direitos humanos e da qualidade
de vida da população, e com metodologias
de intervenção e de pesquisas que não só
privilegiem a participação dos atores
envolvidos mas também considerem o saber
daqueles a quem se dirige o trabalho como
constituinte do conhecimento a ser gerado
sobre ele. Nesse sentido, podemos dizer que
uma quantidade razoável de trabalhos foram
produzidos visando contribuir para a
transformação da realidade latino-americana
e brasileira, com atuações que vão desde a
formação de lideranças na área da saúde e
trabalho social, estudos sobre habitação e
planejamento urbano e diagnósticos locais,
até a construção de centros e programas de
intervenção
psicológico-comunitária
(Wiesenfeld e Sánchez, 1995; Jacób e
Torrens, 1998; Sarriera, 2000). A
multiplicidade de trabalhos atesta, sem
dificuldade, que a Psicologia comunitária O desenvolvimento comunitário apresenta-
se como um campo de práticas associativas
e de participação social nos fóruns ou
instâncias onde se dão decisões sobre o
coletivo social. PSICOLOGIA CIÊNCIA E PROFISSÃO, 2006, 26 (1), 4-11 201 PSICOLOGIA CIÊNCIA E
PROFISSÃO, 2006, 26 (2), 198-209 Katia Regina Frizzo & Jorge Castellá Sarriera constitui um campo de saber e de atuação, e
que sua identidade está fortemente calcada
nos pressupostos da emancipação humana e
nas formas de construção conjunta do
conhecimento. ● a comunidade, entendida como o campo
onde se dá a atuação do CT, e onde o sistema
produz impactos duradouros na vida social. Ao abordar o Conselho Tutelar da perspectiva
de uma instituição (Nasciutti, 1996, p.114),
estamos considerando que as instituições
sejam “espaços de mediação entre a vida
individual e a vida coletiva”. Ao analisá-lo
como um sistema (Campanini e Luppi, 1996),
estamos enfatizando o ambiente dinâmico e
o sistema de trocas em que se insere. O
desenvolvimento comunitário seria o efeito
sistêmico da atuação de um subsistema
responsável pela defesa da ordem
democrática e de seus imperativos de
participação (Aragão, p. 2001). A Psicologia social comunitária
e seu campo de atuação Dentre os diversos enfoques para a realização
do nosso trabalho, escolhemos o marco
ecológico pelas possibilidades de exploração
metodológica das relações do Conselho
Tutelar, enquanto instituição e, portanto,
sistema, com o seu entorno, o meio
circundante. É importante considerar que,
pelas características do processo de sua
construção e legitimação, o Conselho Tutelar
possui uma relação muito estreita com a
comunidade de que faz parte, pois,
diferentemente de outras instituições sociais,
ele tem uma base territorial fixa e uma
atuação delimitada a ela, seja o Município
ou uma região dentro dele. “No entender
tradicional do que
é agir, ou exercer
uma prática,
somente há
“prática” quando
interfiro
positivamente. O
agir
“passivamente”
não seria prática,
apesar de, em
alguns casos,
acontecer muita
coisa exatamente
porque “permiti”,
ou “me omiti”. Já
na visão da
sociedade como
um rio, como algo
construído sobre
“relações”, que
implicam sempre
algo dinâmico,
mudança,
contradição, é
impossível “não
agir”. Isto é,
sempre há uma
prática, de nossa
parte...” “No entender
tradicional do que
é agir, ou exercer
uma prática,
somente há
“prática” quando
interfiro
positivamente. O
agir
“passivamente”
não seria prática,
apesar de, em
alguns casos,
acontecer muita
coisa exatamente
porque “permiti”,
ou “me omiti”. Já
na visão da
sociedade como
um rio, como algo
construído sobre
“relações”, que
implicam sempre
algo dinâmico,
mudança,
contradição, é
impossível “não
agir”. Isto é,
sempre há uma
prática, de nossa
parte...” A construção dos indicadores
de atendimento A utilização de indicadores na pesquisa social
serve para tornar evidentes aqueles processos
de mudança provocados por algum tipo de
intervenção social. Segundo Armani (2000,
p.58), “um indicador é um instrumento de
medição usado para indicar mudanças na
realidade social que nos interessa”. Não
existem, portanto, indicadores “a priori”, pois
devemos inicialmente definir em qual
realidade esperamos verificar tais mudanças
e quais mudanças pretendemos medir. O enfoque ecológico-contextual foi tematizado
por Bronfenbrenner (1996) como uma
compreensão sistêmica do desenvolvimento
humano. A utilização desse enfoque tem sido
crescente, pois possibilita analisar os diferentes
níveis e ambientes de que participa uma pessoa
para contextualizar seus problemas e/ou as
possibilidades de intervenção. Guareschi Assim, nossa compreensão focalizará, neste
estudo, o Conselho Tutelar em três níveis: Para a compreensão do que estamos
denominando por “práticas sociais”, tomamos
como referência a reflexão de Guareschi
(1997, p. 13) que nos diz: ● como um microssistema, com um
conjunto de atividades, função e atribuições
específicas; o enfoque sistêmico ajudar-nos-
á a abordá-lo desde uma perspectiva do CT
enquanto instituição (sistema); “No entender tradicional do que é agir, ou
exercer uma prática, somente há “prática”
quando interfiro positivamente. O agir
“passivamente” não seria prática, apesar de,
em alguns casos, acontecer muita coisa
exatamente porque “permiti”, ou “me omiti”. Já na visão da sociedade como um rio, como ● as relações do sistema CT com outros
microssistemas, como a família, a escola, os
serviços públicos de saúde, habitação,
segurança e outros; trata-se da relação do
sistema com seu ambiente; 202 Práticas Sociais com Crianças e Adolescentes: O Impacto dos Conselhos Tutelares violações do direito à VIDA E SAÚDE, de que
trata o Capítulo I dos Direitos Fundamentais
elencados no Estatuto da Criança e do
Adolescente (artigos 7
o a 14
o). Na rubrica
2.0.00.0, temos mais um bloco de sete
violações do direito à LIBERDADE, RESPEITO,
DIGNIDADE, e assim por diante. Com base
nesse sistema de registro, construímos uma
forma de produção dos indicadores sobre as
práticas sociais. O sistema foi desenvolvido
nacionalmente para fins exclusivos de
utilização pelos Conselhos Tutelares, com o
objetivo de padronizar os dados de
atendimento. algo construído sobre “relações”, que
implicam sempre algo dinâmico, mudança,
contradição, é impossível “não agir”. Isto é,
sempre há uma prática, de nossa parte...” Assim, entendemos que o Conselho Tutelar
institui uma nova prática entre outras práticas
que podem ser consideradas no trato das
questões da infância. As práticas educativas,
por exemplo, constituem um tipo de prática
social na infância. As diversas práticas
profissionais, então, constituem outro tipo de
práticas sociais. Segundo nossa hipótese de trabalho,
pretendemos demonstrar que os Conselhos
Tutelares produzem mudanças em algumas
dimensões da realidade social, que
denominamos conceitualmente de “práticas
sociais”, já que ele interage com diversos
setores da sociedade e produz algum impacto
nas práticas sociais desses setores. Guareschi Para fins de análise, utilizaremos como
referência duas experiências ou dimensões
da realidade social que são marcadas pela
presença de padrões estruturados de
comportamentos estreitamente relacionados
ao exercício de um papel social, que
configuram práticas sociais e que são as
práticas sociais familiares e as práticas sociais
institucionais. Katia Regina Frizzo & Jorge Castellá Sarriera Katia Regina Frizzo & Jorge Castellá Sarriera Conceito
Variáveis
Indicadores
1) Melhora nas práticas de
*índices de aprisionamento ;
cuidado e educação dos filhos;
*índices de violência física;
*índices de violência psicológica;
*índices de ausência de convívio familiar;
*índices de ausência de infraestrutura. Práticas
2) Criação de ambientes
*índices de inadequação do convívio familiar;
sociais nas
familiares mais harmônicos;
*índices de inadequação do convívio familiar;
famílias
*índices de atos atentatórios à cidadania;
*índices em que o agente violador é a família
(conjunto) ou outros familiares;
3) Melhoria das relações
*índices em que os pais aparecem como
parentais com as crianças
agente violador;
e adolescentes. Figura. 1. Quadro conceitual das práticas sociais familiares Conceito
Variáveis
Indicadores
1) Melhora nas práticas de
*índices de aprisionamento ;
cuidado e educação dos filhos;
*índices de violência física;
*índices de violência psicológica;
*índices de ausência de convívio familiar;
*índices de ausência de infraestrutura. Práticas
2) Criação de ambientes
*índices de inadequação do convívio familiar;
sociais nas
familiares mais harmônicos;
*índices de inadequação do convívio familiar;
famílias
*índices de atos atentatórios à cidadania;
*índices em que o agente violador é a família
(conjunto) ou outros familiares;
3) Melhoria das relações
*índices em que os pais aparecem como
parentais com as crianças
agente violador;
e adolescentes. Figura. 1. Quadro conceitual das práticas sociais familiares Figura. 1. Quadro conceitual das práticas sociais familiares As práticas sociais institucionais estão relacionadas, por sua vez, com o exercício dos papéis
profissionais e com as dinâmicas próprias das instituições do campo social, onde são executados
serviços públicos destinados ao atendimento das necessidades básicas dos cidadãos (políticas
públicas de educação, saúde, habitação, etc.) e/ou programas destinados a um segmento social
necessitado de proteção especial (programas de proteção especial, segundo o ECA). Conceito
Variáveis
Indicadores
1)práticas
*índices de não atendimento médico ;
institucionais na
*índices de atendimento médico deficiente;
área de saúde;
*índices de prejuízos por ação ou omissão de agente externo;
*índices de práticas hospitalares e ambulatoriais irregulares;
*índices de irregularidades na garantia da alimentação;
*índices de atos atentatórios à vida;
*índices em que o agente violador é o posto de saúde;
Práticas
2) práticas
*índices de impedimento de acesso à educação;
sociais
institucionais
*índices de impedimento de permanência no sist. Método Para observar e medir o impacto da ação dos
Conselhos Tutelares sobre as práticas sociais
voltadas para crianças e adolescentes,
realizamos uma análise das estatísticas de
atendimento de um Conselho Tutelar
durante um período de quatro anos. As práticas sociais familiares constituem
aqueles comportamentos característicos dos
papéis familiares. Assim, pai e mãe têm por
dever educar os filhos e utilizam seus próprios
métodos educativos para educá-los. Também
têm o dever de prover o sustento da casa,
ainda que contem com a ajuda de outros
adultos da família, adolescentes e/ou
membros da família extensa para exercer
adequadamente o seu papel (Osório, 1996). Como se tratam de conceitos subjetivos que
indicam qualidades, buscamos tornar essas
dimensões visíveis e operacionalizáveis em
termos de indicadores, usando algumas
estatísticas produzidas pelo Conselho Tutelar
“A” através do sistema SIPIA (Sistema de
Informações para a Infância e a
Adolescência). Pelo SIPIA, os direitos violados
são categorizados de acordo com o que
dispõe o Estatuto da Criança e do
Adolescente. Assim, por exemplo, sob a
rubrica 1.0.00.0, temos um bloco de seis Podemos visualizar a forma de produção dos
indicadores sobre as práticas sociais nas
famílias pelo seguinte quadro demonstrativo,
onde a terceira coluna representa o conjunto
das estatísticas extraídas diretamente do SIPIA
de 1999 a 2003: PSICOLOGIA CIÊNCIA E PROFISSÃO, 2006, 26 (1), 4-11 203 PSICOLOGIA CIÊNCIA E
PROFISSÃO, 2006, 26 (2), 198-209 Katia Regina Frizzo & Jorge Castellá Sarriera escolar;
institucionais
na área
*índices de ausência de condições educacionais adequadas;
educacional;
*índices de cultura/esporte/lazer;
*índices de ausência ou impedimento do uso de equipamentos;
*índices em que o agente violador é escola/creche;
3) práticas
*índices de exploração do trabalho infantil;
institucionais no
*índices de condições adversas de trabalho;
mercado de
*índices de inobservância da legislação trabalhista;
trabalho;
*índices de ausência de condições para a formação e
desenvolvimento;
*índices em que o agente violador é
estabelecimento comercial. Figura. 2. Quadro conceitual das práticas sociais institucionais Figura. 2. Quadro conceitual das práticas sociais institucionais Práticas sociais nas famílias Através dessas dimensões das práticas sociais,
pretendemos demonstrar que a intervenção
do Conselho Tutelar na realidade social
produz algum impacto no sentido de reduzir
ou aumentar esses índices. Em alguns casos,
o impacto é tão insignificante que podemos
dizer mesmo que é nulo. A família constitui o primeiro grupo social de
uma criança. Nela formam-se os hábitos e
comportamentos que apresentam mais
resistência à mudança externa, pois baseiam-
se em relações afetivas intensas e marcadas
pelas relações de dependência entre os filhos
e seus pais ou adultos cuidadores (Brant de
Carvalho et al., 1995). Além da realização desse trabalho com
indicadores, foram realizadas entrevistas com
profissionais do campo social que interagem
freqüentemente com o Conselho Tutelar, de
modo a buscar a percepção externa sobre o
trabalho do Conselho e algumas indicações
das possíveis mudanças na vida social a partir
da existência e do contato com o Conselho
Tutelar. As práticas de educação dos filhos são
transmitidas de geração em geração através
de tradições familiares, que só são
questionadas quando outros grupos ou
instituições sociais confrontam seus saberes e
práticas com as práticas familiares. É o caso da
escola, por exemplo, que pode constituir uma
fonte de aprendizagem para os pais e para as
crianças no que se relaciona às práticas
adequadas de cuidado com os filhos. É o caso,
também, da intervenção do Conselho Tutelar,
que pode qualificar como inadequadas certas
práticas pedagógicas familiares tradicionais,
obrigando os pais, principalmente, a mudar
certos hábitos domésticos de cuidado e
educação dos filhos. É o caso da violência
física, da negligência, da violência psicológica,
por exemplo. Para auferir o impacto produzido pelo
Conselho Tutelar, foi preciso também
determinar limites dentro dos quais passamos
a considerar o impacto nos indicadores, que
foram estabelecidos nos seguintes níveis: ● (0) inexistente – entre 0% e 20% de variação
no número de atendimentos; ● (0) inexistente – entre 0% e 20% de variação
no número de atendimentos; ● (1) irrelevante – entre 20% e 40% de
variação; ● (2) significativo – entre 40% e 70% de
variação; Na análise das práticas sociais familiares,
podemos dizer que, do total de oito
indicadores considerados, cinco apresentaram
impacto positivo, e apenas três, impacto
negativo. O impacto médio é de 1,75, ou seja,
um impacto irrelevante, mas próximo de
tornar-se um impacto significativo. Indicadores Figura. 2. Quadro conceitual das práticas sociais institucionais 204 Práticas Sociais com Crianças e Adolescentes: O Impacto dos Conselhos Tutelares Práticas sociais nas famílias As variáveis
que apresentaram um impacto positivo, ou
seja, apresentaram sensível diminuição nos
últimos quatro anos, foram as práticas de
cuidado e educação dos filhos. Os índices
que mais contribuíram para a mudança foram: ● (3) importante – acima de 70% de variação. Os indicadores de impacto também foram
classificados de acordo com o sentido do
impacto em: impacto negativo, quando a
variação foi no sentido do aumento no número
de atendimentos, e impacto positivo, quando
a variação indicou a diminuição do número
de registros (Pereira, 2001). A violência
psicológica
geralmente está
associada à
violência física,
pois apresenta
índices muito
semelhantes. Figura. 3. Práticas sociais familiares Figura. 3. Práticas sociais familiares Figura. 3. Práticas sociais familiares Resultados e discussão ● violência física (28%); Através da análise dos indicadores, pôde-se
constatar que o Conselho Tutelar produz
mudanças nas práticas sociais em níveis
diferenciados nas famílias e nas instituições. ● violência psicológica (69%); PSICOLOGIA CIÊNCIA E PROFISSÃO, 2006, 26 (1), 4-11 205 PSICOLOGIA CIÊNCIA E
PROFISSÃO, 2006, 26 (2), 198-209 Tipo de
Perfil das
2000
2001
2002
2003 Var.%
Nível
Qual. prática
violações
00 - 03
do impacto
Do impacto
social
práticas de
aprisionamento
05
04
05
07
40%
1
Neg. cuidado e
violência física
39
39
33
28
28%
1
Pos. educação
violência
dos filhos
psicológica
23
11
28
07
69%
2
Pos
ausência de
convívio
familiar
82
54
52
25
69%
2
Pos
ausência de
infraestrutura
-
04
17
02
200%
3
Neg. ambiente
inadequação do
familiar
conv. familiar
179
199
143
276
54%
2
Neg. atos atentatórios
à cidadania
68
71
60
30
56%
2
Pos
relações
parentais
agente violador
327
318
327
253 24%
1
Pos. : pais
Figura. 3. Práticas sociais familiares
Como mostra a figura 3, houve uma diminuição
sensível de registros de atendimento nesses
itens, possivelmente por constituírem aspectos
bastante visíveis das relações familiares, como
é o caso da violência física e da violência
psicológica, bem como da ausência de convívio
familiar, que nada mais é do que o abandono
dos filhos à própria sorte. A violência
psicológica geralmente está associada à
violência física, pois apresenta índices muito
semelhantes. Outro fator que pode ter
contribuído para a redução desses índices é a
inclusão das famílias em programas de apoio
socio-familiar, que aportem recursos materiais
e/ou educativos para a mudança nas práticas
educativas domésticas. Entretanto, podemos
relacionar esse fator como resultante
indiretamente da intervenção do Conselho,
haja vista que uma das suas funções é
justamente monitorar a política de
atendimento, sugerindo a criação de
programas para atendimento dos direitos
fundamentais, expressos na lei. Práticas sociais institucionais
As práticas sociais examinadas neste trabalho
não abrangem todas as instituições que
desenvolvem programas e/ou atividades com
crianças e adolescentes. Elas restringem-se
apenas a alguns setores de atividade, como a
saúde e educação, pois são itens que
permitem a comparabilidade dos dados,
enquanto as entidades e programas são
incomparáveis devido à inconstância de sua
existência e atendimento, configurando a
saúde e a educação políticas públicas que
menos sofrem descontinuidade nas suas
ações. As práticas sociais institucionais apresentaram
menos indicadores com impacto positivo. Resultados e discussão Do
total de quinze indicadores, apenas 1/3 (cinco)
apresentaram impacto positivo, enquanto
todos os demais apresentaram impacto
negativo. O impacto médio é de 2,53, o que
consideramos um impacto significativo,
embora no sentido predominantemente
negativo. PSICOLOGIA CIÊNCIA E
PROFISSÃO, 2006, 26 (2), 198-209
A violência
psicológica
geralmente está
associada à
violência física,
pois apresenta
índices muito
semelhantes. Katia Regina Frizzo & Jorge Castellá Sarriera Katia Regina Frizzo & Jorge Castellá Sarriera Tipo de
Perfil das
2000
2001
2002
2003 Var.%
Nível
Qual. prática
violações
00 - 03
do impacto
Do impacto
social
práticas de
aprisionamento
05
04
05
07
40%
1
Neg. cuidado e
violência física
39
39
33
28
28%
1
Pos. educação
violência
dos filhos
psicológica
23
11
28
07
69%
2
Pos
ausência de
convívio
familiar
82
54
52
25
69%
2
Pos
ausência de
infraestrutura
-
04
17
02
200%
3
Neg. ambiente
inadequação do
familiar
conv. familiar
179
199
143
276
54%
2
Neg. atos atentatórios
à cidadania
68
71
60
30
56%
2
Pos
relações
parentais
agente violador
327
318
327
253 24%
1
Pos. : pais Práticas sociais institucionais Como mostra a figura 3, houve uma diminuição
sensível de registros de atendimento nesses
itens, possivelmente por constituírem aspectos
bastante visíveis das relações familiares, como
é o caso da violência física e da violência
psicológica, bem como da ausência de convívio
familiar, que nada mais é do que o abandono
dos filhos à própria sorte. A violência
psicológica geralmente está associada à
violência física, pois apresenta índices muito
semelhantes. Outro fator que pode ter
contribuído para a redução desses índices é a
inclusão das famílias em programas de apoio
socio-familiar, que aportem recursos materiais
e/ou educativos para a mudança nas práticas
educativas domésticas. Entretanto, podemos
relacionar esse fator como resultante
indiretamente da intervenção do Conselho,
haja vista que uma das suas funções é
justamente monitorar a política de
atendimento, sugerindo a criação de
programas para atendimento dos direitos
fundamentais, expressos na lei. As práticas sociais examinadas neste trabalho
não abrangem todas as instituições que
desenvolvem programas e/ou atividades com
crianças e adolescentes. Elas restringem-se
apenas a alguns setores de atividade, como a
saúde e educação, pois são itens que
permitem a comparabilidade dos dados,
enquanto as entidades e programas são
incomparáveis devido à inconstância de sua
existência e atendimento, configurando a
saúde e a educação políticas públicas que
menos sofrem descontinuidade nas suas
ações. As práticas sociais institucionais apresentaram
menos indicadores com impacto positivo. Do
total de quinze indicadores, apenas 1/3 (cinco)
apresentaram impacto positivo, enquanto
todos os demais apresentaram impacto
negativo. O impacto médio é de 2,53, o que
consideramos um impacto significativo,
embora no sentido predominantemente
negativo. 206 Práticas Sociais com Crianças e Adolescentes: O Impacto dos Conselhos Tutelares práticas insti-
tucionais no
mercado de
trabalho
As práticas sociais institucionais que
apresentaram uma diminuição sensível no
registro de atendimentos localizam-se
principalmente entre os indicadores de saúde,
demonstrados na figura 4. O impacto sobre
as práticas dessa área é significativo, com
média 2,71. Sofreram alguma diminuição dos
índices de atendimento, com impacto positivo
na área da saúde, as seguintes situações
registradas no SIPIA:
Entre as práticas sociais registradas na
educação, apenas o item “ausência de
condições educacionais adequadas”
apresentou uma diminuição nos registros
(61%), enquanto os outros três indicadores
apresentaram resultados negativos. O impacto
é irrelevante, com média 1,75. Práticas sociais institucionais Ressalte-se que
dois importantes direitos são registrados nesse
bloco, e apresentaram aumento dos registros
nos quatro anos examinados configurando
práticas
insti-
tucionais
na área
da saúde
Tipo de
perfil das
2000 2001 2002 2003 Var.%
Nível do Qual. do
prática
violações
00 - 03 impacto impacto
social
não atendimento médico
12
23
03
-
100%
3
Positivo
atendimento médico
deficiente
11
04
05
01
91%
3
Positivo
prejuízos por ação/omissão ag. externo
09
03
05
04
55%
2
Positivo
práticas hospitalares e
ambulatoriais irregulares;
03
-
01
-
100%
3
Positivo
irregularidade na garantia
da alimentação;
-
01
-
08
100%
3
Negativo
atos atentatórios à vida;
02
-
-
09
350%
3
Negativo
agente violador: posto
de saúde;
06
08
05
09
50%
2
Negativo
práticas insti-
impedimento de acesso;
25
20
03
34
36%
1
Negativo
tucionais na
impedimento de permanência; 06
09
01
08
33%
1
Negativo
área da
ausência de condições
educação
educacionais adequadas;
39
47
03
15
61%
2
Positivo
agente violador: esc./creche; 01
04
01
10
900%
3
Negativo
exploração trabalho infantil;
-
05
04
2
200%
3
Negativo
inobservância da legislação;
01
01
02
02
100%
3
Negativo
ausência de condições;
-
05
01
02
200%
3
Negativo
agente violador: est. comercial. -
09
06
03
300%
3
Negativo
Figura 4. Práticas sociais institucionais Figura 4. Práticas sociais institucionais Entre as práticas sociais registradas na
educação, apenas o item “ausência de
condições educacionais adequadas”
apresentou uma diminuição nos registros
(61%), enquanto os outros três indicadores
apresentaram resultados negativos. O impacto
é irrelevante, com média 1,75. Ressalte-se que
dois importantes direitos são registrados nesse
bloco, e apresentaram aumento dos registros
nos quatro anos examinados, configurando
flagrante desrespeito aos direitos fundamentais
estabelecidos constitucionalmente: acesso e
permanência na escola. O quadro preocupa,
principalmente porque a educação concentra
as expectativas sociais de melhora do quadro
de exclusão social com que sofrem muitas
famílias em razão de pobreza e de dificuldades As práticas sociais institucionais que
apresentaram uma diminuição sensível no
registro de atendimentos localizam-se
principalmente entre os indicadores de saúde,
demonstrados na figura 4. O impacto sobre
as práticas dessa área é significativo, com
média 2,71. Práticas sociais institucionais Sofreram alguma diminuição dos
índices de atendimento, com impacto positivo
na área da saúde, as seguintes situações
registradas no SIPIA: ● não atendimento médico (100%); ● atendimento médico deficiente (91%); ● prejuízos por ação/omissão de agente
externo (55%) e ●práticas hospitalares e ambulatoriais
irregulares (100%). ●práticas hospitalares e ambulatoriais
irregulares (100%). 207 PSICOLOGIA CIÊNCIA E
PROFISSÃO, 2006, 26 (2), 198-209 Katia Regina Frizzo & Jorge Castellá Sarriera a ela associadas, como a falta de qualificação,
o trabalho infantil e o envolvimento em
problemas com a Lei por falta de perspectivas
positivas em relação à inserção no mundo do
trabalho. pedagógica. A professora entrevistada, com
dezoito anos de experiência profissional e cujo
contato com o Conselho Tutelar é “quase
semanal”, disse que não desenvolve atividades
diretamente com crianças e adolescentes, “só
pedagógicas”, o que parece restringir sua
responsabilidade frente aos graves problemas
que ela própria aponta nas crianças e
adolescentes de hoje: “falta de concentração,
hiperatividade e falta de limites”. No mercado de trabalho, os resultados são
paradoxais, pois o impacto na área é
importante (média 3,0) e negativo, mas,
considerando-se o baixo número de registros
desse grupo de indicadores nos quatro anos
examinados, não se pode afirmar que o quadro
preocupe. É flagrante o descompasso: em lugar de se
colocar
como
recurso
educativo
complementar à família nesses aspectos, a
escola se descompromete, recusando sua
responsabilidade na mudança da realidade de
vida das crianças e adolescentes. A avaliação
sobre o trabalho do Conselho Tutelar no
Município não surpreende: é negativa, “deixa
muito a desejar”, segundo a professora, que
parece não confiar muito no trabalho do
Conselho. Um exame atento dos indicadores e de seus
respectivos números, entretanto, não nos
permite afirmar com clareza que a variação se
deve exclusivamente à intervenção do
Conselho Tutelar. Na verdade, são muitos os
fatores concorrentes para produzir tais
resultados: por um lado, temos, na série de
quatro anos examinados, uma gestão inteira
(2000-2002) e uma parte de outra gestão,
quando todos os conselheiros foram
renovados. Essa realidade, porém, é
representativa dos problemas dos Conselhos. Outro aspecto que limita o alcance explicativo
da pesquisa é a interferência dos fatores de
variação individual dos conselheiros. Entretanto, optamos por não introduzir tais
variáveis neste estudo, uma vez que
distorceriam o seu foco principal, que pretende
ser sobre o Conselho Tutelar como instituição
e sistema, e não sobre os conselheiros,
considerados individualmente. Práticas sociais institucionais Por outro lado, os profissionais da saúde
entendem de forma mais positiva o trabalho
do Conselho, mas consideram precária a sua
preparação para o trabalho de responsabilidade
que executam. A assistente social, com
experiência no trabalho de assistência a famílias
economicamente carentes em dois Municípios,
considera que o Conselho seja muito
importante no Município, mas os conselheiros
“são pouco preparados”, o que deixa a atuação
de alguns aquém do desejado. Já a enfermeira,
com três anos de experiência, sente a falta do
retorno do Conselho diante dos casos
acompanhados. Já foi procurada pelo Conselho
e também teve de procurá-lo em diversas
situações, e comenta que “eles deveriam dar
o retorno sobre a situação, a gente fica
querendo saber”. Ela insiste que não é para
controlar o trabalho do Conselho, mas que
“todo mundo quer saber como ficou a situação
daquela criança, ou daquela família”,
evidenciando a necessidade dos profissionais
de saber dos encaminhamentos dados pelo
Conselho aos atendimentos. 208 Práticas Sociais com Crianças e Adolescentes: O Impacto dos Conselhos Tutelares Essa característica de pouco comprometimento
com a prestação de contas do seu trabalho
também foi evidenciada por Sierra (2003) nos
Conselhos do Rio de Janeiro. Por outro lado,
podemos perceber na fala da enfermeira que,
ao atuar ao lado do Conselho junto a uma
família, a uma criança, enfim, ao interagir com
o Conselho, desenvolve-se uma expectativa
que é própria dos profissionais do campo
técnico-científico, de reciprocidade na relação,
de compartilhamento das responsabilidades
frente às soluções e de expectativas de sucesso
da ação que alimenta cotidianamente a auto-
estima dos profissionais e a confiança no seu
processo de formação. eles tentam fazer um bom trabalho, mas falta
formação para isso, e que o trabalho do
Conselho é um trabalho “que exige muito”, ou
seja, que precisa ser valorizado. Percebe-se, dessa forma, que os profissionais
já assimilaram o papel do Conselho Tutelar na
comunidade e entendem o seu valor. Suas
críticas se dirigem, via de regra, aos conselheiros,
cuja qualificação é posta em dúvida frente à
magnitude da importância que adquire o
Conselho na sua tarefa de zelar pelos direitos
da criança e do adolescente. O problema da
qualificação parece o nó da questão, portanto. A identidade do Conselho está estreitamente
relacionada à aquisição de habilidades, por um
lado, e ao processo de legitimação, por outro. O delegado tem outra experiência com o
Conselho. Com vinte e sete anos de
experiência profissional, ele diz que começou
a interagir com os Conselhos desde a aprovação
do Estatuto, desde que eles foram instalados. Sente que os conselheiros são “pouco
preparados”, apesar da “boa vontade”. Seu
contato com o Conselho Tutelar é freqüente,
mas não sistemático. A polícia está sempre à
disposição do Conselho para fazer qualquer
abordagem domiciliar que envolva algum risco. Segundo o delegado, “a gente sempre ajuda,
assim, quando tem um caso complicado, um
pai bêbado, ou que bateu no filho e tá agressivo... então a gente vai junto, acompanha o
Conselho”. Outra fonte de contatos é através
dos atos infracionais cometidos por
adolescentes, quando o Conselho é chamado
à delegacia para fazer encaminhamentos. Outro fator a ser considerado é que a maioria
dos profissionais não recebe formação específica
sobre direitos da criança e adolescente nem
informações adequadas sobre o Conselho
Tutelar. Somente o delegado e os comissários
da infância e juventude disseram ter recebido
alguma formação sobre esses aspectos. A visão dos profissionais A fala dos profissionais é significativa, pois
apresenta claras distinções a partir do lugar de
onde cada um se posiciona na rede social de
apoio à infância. Assim, as crianças são vistas pelos educadores
apenas como “alunos”, o que parece qualificar
a relação que se estabelece na escola como
uma relação única e exclusivamente Recebido 19/04/05 Aprovado 11/03/06 Recebido 19/04/05 Aprovado 11/03/06 LANE, Sílvia Tatiana Maurer. Histórico e Fundamentos da Psicologia
Comunitária no Brasil. In Campos, Regina Helena de Freitas. Psicologia Social Comunitária: da Solidariedade à Autonomia. Petrópolis, RJ: Vozes, 1996, pp.17-34. ARAGÃO, Elizabeth Maria Andrade. Recorte de uma Realidade: em
Busca da Construção de Cidadania. In Novo, Helerina A.; Souza, Lídio
de ; Andrade, Angela Nobre de. Ética, Cidadania e Participação. Debates no Campo da Psicologia. Vitória: EDUFES: CCHN
Publicações, 2001, pp. 43-60. Referências MONTERO, Maritza. Psicología Social Comunitaria. México:
Universidad de Guadalajara, 1994. ARMANI, Domingos. Como Elaborar Projetos? Guia Prático para
Elaboração e Gestão de Projetos Sociais. Porto Alegre: Tomo
Editorial, 2000. NASCIUTTI, Jacyara C. Rochael. A Instituição como Via de Acesso à
Comunidade. In Campos, Regina Helena de Freitas. Psicologia Social
Comunitária: da Solidariedade à Autonomia. Petrópolis, RJ: Vozes,
1996, pp. 100-126. BRANT DE CARVALHO, Maria do Carmo et al. A Família
Contemporânea em Debate. São Paulo: EDUC/Cortez, 1995. OSÓRIO, Luiz Carlos. Família Hoje. Porto Alegre: Artes Médicas,
1996. CAMPANINI, Annamaria y LUPPI, Francesco. Servicio Social y
Modelo Sistémico. Buenos Aires: Paidós,1996. PEREIRA, Júlio César Rodrigues. Análise de Dados Qualitativos:
Estratégias Metodológicas para as Ciências da Saúde, Humanas
e Sociais. 3
a ed. São Paulo: EDUSP/FAPESP, 2001. 208 Os
demais nunca tiveram em sua formação alguma
disciplina ou conteúdos que os preparassem
para o trabalho com o Conselho. Na prática,
entretanto, hoje eles incorporam alguns
procedimentos que antes não existiam sempre
que têm de interagir com o Conselho Tutelar. Entendemos que o impacto produzido pelo
Conselho nas práticas sociais demonstra-se
através dos dados apresentados. A existência
de um órgão que participa da esfera pública na
defesa dos direitos da criança e do adolescente
implicou e está implicando mudanças nas
práticas dos profissionais, na prática de muitos
setores de atividades e das famílias também. Com certeza, a consciência sobre os direitos
da infância está mais difundida que alguns anos
atrás, antes da existência dos Conselhos. Entretanto, cabe a todo percurso coerente de
investigação reconhecer os limites do
conhecimento como algo inerente ao processo
de construção científica. Muitas perguntas ficam
ainda sem resposta diante desta pesquisa, e
suscitam novas investigações que pretendemos
empreender. Os técnicos do sistema judiciário são os que
têm mais contato sistemático com o Conselho
tutelar. A assistente social diz: “a gente está
sempre em contato, tem que estar, pois fazemos
muito acompanhamento de casos juntos”,
referindo-se a casos de violência, abandono e
negligência, em que os pais correm o risco de
perder o pátrio poder. O Conselho Tutelar
acompanha as famílias através da aplicação de
medidas de apoio sociofamiliar. As comissárias
de menores também tem bastante contato
com o Conselho. Compartilham com a
assistente social a percepção de que falta
qualificação para os conselheiros atuarem, que 209 209 PSICOLOGIA CIÊNCIA E
PROFISSÃO, 2006, 26 (2), 198-209 Katia Regina Frizzo
Doutoranda PICDT/CAPES em Psicologia da PUCRS. UNOESC – Universidade do Oeste de Santa Catarina
Rua Getúlio Vargas, 2125 Bairro Flor da Serra
89.600-000 – Joaçaba – SC Fone: 49-551-2000 Fax: 49-551-2004
E-mail: kátia.frizzo@unoesc.edu.br Jorge Castellá Sarriera
Professor orientador. PUCRS – Pontifícia Universidade Católica do Rio Grande do Sul
Av. Ipiranga, 6681Prédio 11 – 9o andar, sala 929. 90619-900 – Porto Alegre – RS Fone: 51-3320-3633
E-mail: sarriera@terra.com.br Jorge Castellá Sarriera
Professor orientador. PUCRS – Pontifícia Universidade Católica do Rio Grande do Sul
Av. Ipiranga, 6681Prédio 11 – 9o andar, sala 929. 90619-900 – Porto Alegre – RS Fone: 51-3320-3633
E-mail: sarriera@terra.com.br ESTATUTO DA CRIANÇA E DO ADOLESCENTE. Lei No. 8.069. FREITAS, Maria de Fátima Quintal de. Psicologia na Comunidade,
Psicologia da Comunidade e Psicologia (Social) Comunitária: Práticas
da Psicologia em Comunidade nas Décadas de 60 a 90, no Brasil. In
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da Solidariedade à Autonomia. Petrópolis, RJ: Vozes, 1996, pp. 54-
80. FREITAS, Maria de Fátima Quintal de. Psicologia na Comunidade,
Psicologia da Comunidade e Psicologia (Social) Comunitária: Práticas
da Psicologia em Comunidade nas Décadas de 60 a 90, no Brasil. In
Campos, Regina Helena de Freitas. Psicologia Social Comunitária:
da Solidariedade à Autonomia. Petrópolis, RJ: Vozes, 1996, pp. 54-
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Comunitária: Estudos Atuais. Porto Alegre: Sulina, 2000, pp.373-
395. SARRIERA, Jorge Castellá. O Modelo Ecológico-Contextual em
Psicologia Comunitária. In Sarriera, Jorge Castellá. Psicologia
Comunitária: Estudos Atuais. Porto Alegre: Sulina, 2000, pp.373-
395. SIERRA, Vania Morales. Conselho Tutelar: uma Identidade em
Questão. http://www.abmp.org.br. Portal_ABMP_Publicação 530,
2003. GUARESCHI, Pedrinho. Qual a “Prática” da Psicologia Social da
ABRAPSO? In Zanella, Andréa et al. Psicologia e Práticas Sociais. Porto Alegre: ABRAPSOSUL, 1997, pp.11-16. WIESENFELD, Esther y SÁNCHEZ, Euclides. Psicología Social
Comunitaria: Contribuciones Latinoamericanas. Caracas: Fondo
Editorial Tropycos, 1995. JACOB, Mariane Krause y TORRÉNS, Andrea Jaramillo. Intervenciones Psicológico-Comunitarias en Santiago de Chile. Santiago: Pontificia Universidad Católica de Chile, 1998. JACOB, Mariane Krause y TORRÉNS, Andrea Jaramillo. Intervenciones Psicológico-Comunitarias en Santiago de Chile. Santiago: Pontificia Universidad Católica de Chile, 1998.
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THE IMPACT OF CYBERSECURITY STANDARD GUIDELINES ON THE CULTURE OF CYBERSECURITY
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Zenodo (CERN European Organization for Nuclear Research)
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Keywords: Cybersecurity Culture, Cybersecurity standard guidelines. Keywords: Cybersecurity Culture, Cybersecurity standard guidelines. ISSN 2348-1218 (print)
International Journal of Interdisciplinary Research and Innovations ISSN 2348-1226 (online)
Vol. 10, Issue 4, pp: (68-72), Month: October 2022 - December 2022, Available at: www.researchpublish.com ISSN 2348-1218 (print)
International Journal of Interdisciplinary Research and Innovations ISSN 2348-1226 (online)
Vol. 10, Issue 4, pp: (68-72), Month: October 2022 - December 2022, Available at: www.researchpublish.com Research Publish Journals THE IMPACT OF CYBERSECURITY
STANDARD GUIDELINES ON THE
CULTURE OF CYBERSECURITY 1Jamilu Garba, 2Jasber Kaur Abstract: This paper examines factors that significantly affect the use of cybersecurity culture by banks in Nigeria. One of these factors was the cybersecurity standard guidelines. This study adopted a quantitative approach to
reaching its objectives by using questioner that provides a framework for building hypotheses. Fifty participants
from different banks in Nigeria participated in the study. After reviewing several relevant studies, a five-point Likert
scale questionnaire was designed to collect the required data, which was analyzed using SPSS. Hypotheses were
tested to see which results could then be generalized. The result showed the regression coefficients of the variable
cybersecurity standard guidelines explain (79.2%) of the variance in the culture of cybersecurity this interpretation
is statistically significant at the level (0.05), and the table shows that the values of the regression coefficients were
positive and statistically significant between the cybersecurity standard guidelines and culture of cybersecurity (β =
0.813; t = 26.356; p = 0.000); The square of the overall correlation coefficient R2 between cybersecurity standard
guidelines and culture of cybersecurity was (0.792), which means that the independent variable affects 79.2% of the
variance in the culture of cybersecurity as the dependent variable. Thus, we accept the H1 hypothesis. The
researchers recommend the use of new variables and factors in studying the relationships that affect cybersecurity
and studying the extent of culture that people have about this concept in different environments and countries, in
addition to different sectors. ISSN 2348-1218 (print)
International Journal of Interdisciplinary Research and Innovations ISSN 2348-1226 (online)
Vol. 10, Issue 4, pp: (68-72), Month: October 2022 - December 2022, Available at: www.researchpublish.com (p
)
International Journal of Interdisciplinary Research and Innovations ISSN 2348-1226 (online)
Vol. 10, Issue 4, pp: (68-72), Month: October 2022 - December 2022, Available at: www.researchpublish.com However, the guidelines were examined in terms of how it should be written, how to encourage the users to follow the
guidelines, and how the guidelines influence an organization's cybersecurity culture. The cybersecurity guidelines is
considered as a significant strategy for monitoring cybersecurity in organizations and businesses. Veiga et al (2021) carried
out a survey to determine the impact of cybersecurity standard guidelines on cybersecurity culture by relating organizations
with security standard guidelines and those without security standard guidelines. The finding shows that cybersecurity has
been increased in organizations where cybersecurity standard guidelines implemented. The foundation for shared cybersecurity values and beliefs among users is provided by cybersecurity standard guidelines
(Yıldırım and Mackie 2019). Users are expected to follow cybersecurity standard guidelines that communicate desired
cybersecurity behaviour (Mannan and Van Oorschot 2018). Users are often under the impression that implementing or
adhering to cybersecurity standard guidelines will reduce cyberthreats (Gcaza and von Solms 2017). Users who follow
cybersecurity standard guidelines may be able to protect their data from cyberthreats and risks. Virus attacks occurred in
80% of the organizations surveyed because users were unaware of or failed to follow cybersecurity standard guidelines. Cybersecurity standard guidelines have been identified as one of the factors influencing the development of an effective
cybersecurity culture by researchers. Hengstler and Pryazhnykova (2021) used the Theory of Planned Behaviour to develop
a research model to identify the factors that influence the intention to follow standard computer security policy. The
researcher discovered a connection between having standard computer security guidelines and the intention to follow them,
which is mediated by attitude and belief. This model was later tested by Acuna (2018), who discovered that computer
security guidelines influenced users' intention to follow computer security regulations, and this relationship was mediated
by cybersecurity culture's attitude and belief. Furthermore, Veiga (2017) conducted an empirical study over an eight-year
period to assess cybersecurity culture in twelve countries. As a result, the current paper will decide to use a mixed method
approach and focuses on developing a reliable and valid cybersecurity culture measurement model by identifying the human
factors that influencing cybersecurity culture. 2. THE RESEARCH QUESTION, AIM, AND HYPOTHESIS Based on the background of the study and a review of the previous literature, the following question was formulated: ased on the background of the study and a review of the previous literature, the following question was fo • Is there an impact of cybersecurity standard guidelines on the culture of cybersecurity? The aim of this question is: • Is there an impact of cybersecurity standard guidelines on the culture of cybersecurity? • Is there an impact of cybersecurity standard guidelines on the culture of cybersecurity? The aim of this question is: • Studying the relationship and influence between cybersecurity standard guidelines and cybersecurity culture. • Studying the relationship and influence between cybersecurity standard guidelines and cybersecurity culture. Through the background of the study, the study question, and the aim of the study, the following hypothe hrough the background of the study, the study question, and the aim of the study, the following hypothesis • H1: There is a statistically significant effect of the influence of cybersecurity standard guidelines on the culture of
cybersecurity. • H1: There is a statistically significant effect of the influence of cybersecurity standard guidelines on the culture of
cybersecurity. 3. CONCEPTUAL FRAMEWORK
4. THEORETICAL FRAMEWORK
Cybersecurity
Standard
Guidelines
Culture Of
Cybersecurity 3. CONCEPTUAL FRAMEWORK 3. CONCEPTUAL FRAMEWORK Cybersecurity
Standard
Guidelines Culture Of
Cybersecurity 1. BACKGROUND Cybersecurity guidelines is the organization document usually drafted by chief cybersecurity officer of the organization to
prevent or mitigate cybersecurity vulnerability (Yani, 2016; Lubua & Pretorius, 2019). The creation of cybersecurity culture
will begin with the creation of the organization’s cybersecurity guidelines, followed by the creation of cybersecurity
architecture and detailed cybersecurity blueprint (Liu et al., 2020). The organization cybersecurity effort can be succeeded
only if it operate in conjunction with the organizational cybersecurity standard guidelines (Papazoglou 2019). Without
cybersecurity standard guidelines, the organization will be unable to meet the cybersecurity needs of the various
communities of interest including management from all communities, general staff and public users (Masrek et al., 2017). According to Nasir et al., (2020) organization with lack cybersecurity standard guidelines clearly shows that it lacking
proper cybersecurity guidance, and showing a low level of senior management commitment to cybersecurity. Cybersecurity
standard guidelines should be regularly reviewed at the organizational level to determine whether it satisfied cybersecurity
needs and incorporated into the working environment so that it becomes a part of the individual's daily activities (Alshaikh,
2020 & Uchendu et al., 2021). The Individuals can use cybersecurity standard guidelines to learn about the acceptable level
of cybersecurity behaviour required to keep personal data secure (Tolah et al., 2017) . Page | 68 Research Publish Journals Research Publish Journals 5. METHODOLOGY This study selected fifty participants from banks in nigeria to be participants. The data was obtained online (online
questionnaires on the www.surveyshare.com site), and the questionnaire includes one part this section contains twenty basic
designs that are linked to cybersecurity standard guidelines and the culture of cybersecurity ISSN 2348-1218 (print)
International Journal of Interdisciplinary Research and Innovations ISSN 2348-1226 (online)
Vol. 10, Issue 4, pp: (68-72), Month: October 2022 - December 2022, Available at: www.researchpublish.com ISSN 2348-1218 (print)
International Journal of Interdisciplinary Research and Innovations ISSN 2348-1226 (online)
Vol. 10, Issue 4, pp: (68-72), Month: October 2022 - December 2022, Available at: www.researchpublish.com (p
)
International Journal of Interdisciplinary Research and Innovations ISSN 2348-1226 (online)
Vol. 10, Issue 4, pp: (68-72), Month: October 2022 - December 2022, Available at: www.researchpublish.com 7. HYPOTHESES TESTING This research proposed hypothesis in an attempt to examine the relationships among the factors of the proposed model. Here, the mean values of the variables are determined within the factors or constructs. The values obtained were then
evaluated for correlation. All hypothesis tests show that there is a positive relationship between structures. A positive
correlation ranging from zero and satisfying the above minimum criteria thus supports both the hypothesis and the
relationship. We seek to test a hypothesis (H1: There is a statistically significant effect of the influence of cybersecurity standard
guidelines on the culture of cybersecurity). To check this hypothesis, linear regression coefficients were extracted and the
following table shows these results: Table 2: Hypotheses Testing
Sample
Non-standard
transactions
β
T value
Statistical
significance
R
R2
F value
significance
Reg. S. E
Fixed
1.284
.124
19.102
.000
.847
.792
753.222
.000*
.802
.031
.813
26.356
.000* Table 2 shows the regression coefficients of the variable cybersecurity standard guidelines explain (79.2%) of the variance
in the culture of cybersecurity this interpretation is statistically significant at the level (0.05), and the table shows that the
values of the regression coefficients were positive and statistically significant between the cybersecurity standard guidelines
and culture of cybersecurity (β = 0.813; t = 26.356; p = 0.000); The square of the overall correlation coefficient R2 between
cybersecurity standard guidelines and culture of cybersecurity was (0.792), which means that the independent variable
affects 79.2% of the variance in the culture of cybersecurity as the dependent variable. Thus, we accept the H1 hypothesis. 6. INSTRUMENT’S RELIABILITY the test determines the reliability. Gay and Airasian (2005) mentioned reliability determine the level at which the test
measures what is constant. Furthermore, the basic consistent quality of the internal consistency of the information contained
in the study is estimated using Alpha Cronbach's (Cronbach, 1984). Alpha Cronbach's value can be expanded in a number
of things or a natural relationship. Table 1: Scale Reliability Questionnaire
Variable
N. of Items
Alpha (a)
Cybersecurity Standard Guidelines
10
0.892
Culture Of Cybersecurity
10
0.914
7. HYPOTHESES TESTING Table 1: Scale Reliability Questionnaire Table 1: Scale Reliability Questionnaire Research Publish Journals 4. THEORETICAL FRAMEWORK A theoretical framework refers to the structure that can hold or support research works (Uchendu et al., 2021). It will help
to explain why the problem in the research exists. And the theoretical framework that will guides the researcher to determine
what things will be measure, and what statistical relationships will be looking for. The theory of reasoned action (TRA)
developed by Fishbein & Ajzen, (1975), and Later, Ajzen (1991) extended the theory to theory to planned behavior by
adding perceived behavioral control. The primary goal of the theory of reasoned action and theory of planned behaviour is
to explain the relationship between attitudes and behaviors and has shown significant results in predicting behavioral
intentions and some committed behaviors (Fishbein and Ajzen 1975). Page | 69 Research Publish Journals REFERENCES [1] Adele, Veiga. 2017. “Employees Who Had Read the Information Security Policy Accepted Manuscript ( Unedited ).”
Information & Computer Security (June 2016). [2] Ajzen, I. (1991). The theory of planned behavior. Organizational behavior and human decision processes, 50(2), 179-
211. [3] Ajzen, I., & Fishbein, M. (1975). A Bayesian analysis of attribution processes. Psychological bulletin, 82(2), 261. [4] Alshaikh, Moneer. 2020. “Computers & Security Developing Cybersecurity Culture to Influence Employee Behavior :
A Practice Perspective.” computer & security 98. [5] Chowdhury, Noman H., Marc T.P. Adam, and Timm Teubner. 2020. “Time Pressure in Human Cybersecurity
Behavior: Theoretical Framework and Countermeasures.” Computers & Security 97: 101963. [6] Cronbach, L. J. (1984). A research worker's treasure chest. Multivariate behavioral research, 19(2-3), 223-240. [7] Gcaza, Noluxolo, and Rossouw von Solms. 2017. “A Strategy for a Cybersecurity Culture: A South African
Perspective.” Electronic Journal of Information Systems in Developing Countries 80(1): 1–17. [8] Hengstler, Sebastian, and Natalya Pryazhnykova. 2021. “Reviewing the Interrelation Between Information Security
and Culture : Toward an Agenda for Future Research.” 16th International Conference on Wirtschaftsinformatik, March
2021, Essen, Germany (March): 1–26. [9] Liu, Cai, Stephen Nicholas, and Jian Wang. 2020. “The Association between Protection Motivation and Hepatitis b
Vaccination Intention among Migrant Workers in Tianjin, China: A Cross-Sectional Study.” BMC Public Health
20(1): 1–10. [10] Lubua, Edison Wazoel, and Philip Pretorius. 2019. “Cyber-Security Policy Framework and Procedural Compliance in
Public Organisations.” Proceedings of the International Conference on Industrial Engineering and Operations
Management Pilsen, Czech Republic, July 23-26, 2019 (August). [11] Mannan, Mohammad, and P. C. Van Oorschot. 2018. “Security and Usability: The Gap in Real-World Online
Banking.” Proceedings New Security Paradigms Workshop: 1–14. [12] Mark, Evans, Yevseyeva Iryna, and Janickle Helge. 2019. “Published Incidents and Their Proportions of Human
Error.” Information & Computer Security 23(2): 145–60. [13] Masrek, Mohamad Noorman, Qamarul Nazrin Harun, and Muhammad Khairulnizan Zaini. 2017. “Information
Security Culture for Malysian Public Organization: A Conceptual Framework.” Proceedings of INTCESS 2017 4th
International Conference on Education and Social Sciences (February): 156–66. [14] Nasir, Akhyari et al. 2020. “Information Security Culture Model for Malaysian Organizations : A Review.”
International Journal of Advanced Trends in Computer Science and Engineering (1). [15] Nel, F., & Drevin, L. (2019). Key elements of an information security culture in organisations. Information &
Computer Security. [16] Onwuegbuzie, A. J., & Leech, N. L. (2005). ISSN 2348-1218 (print)
International Journal of Interdisciplinary Research and Innovations ISSN 2348-1226 (online)
Vol. 10, Issue 4, pp: (68-72), Month: October 2022 - December 2022, Available at: www.researchpublish.com ISSN 2348-1218 (print)
International Journal of Interdisciplinary Research and Innovations ISSN 2348-1226 (online)
Vol. 10, Issue 4, pp: (68-72), Month: October 2022 - December 2022, Available at: www.researchpublish.com in different environments and countries, in addition to different sectors. However, the guidelines were examined in terms
of how it should be written, how to encourage the users to follow the guidelines, and how the guidelines influence an
organization's cybersecurity culture. The cybersecurity guidelines are considered as a significant strategy for monitoring
cybersecurity in organizations and businesses. The researchers recommend the use of new variables and factors in studying
the relationships that affect cybersecurity and studying the extent of culture that people have about this concept in different
environments and countries, in addition to different sectors. 8. CONCLUSION The main objective of cybersecurity standard guidelines research in the context of cybersecurity is to approach, identify,
and finally analyze how an individual's cybersecurity standard guidelines , beliefs, and attitudes can affect cybersecurity,
either directly or indirectly, intentionally, or unintentionally. Understanding the root of the problem may lead to effective
solutions, such as implementing policies and guidelines, along with training programs that can help in mitigating
cybersecurity vulnerabilities. However, people of different cybersecurity standard guidelines in the modern world needs to
have private information such as password and private information developed to be essentially private and undisclosed, but
the sharing of private information is a common practice in some groups or family. This is because of social attitude and
trust with whom to share private information with. Cybersecurity standard guidelines played an important role in whether
private information would be shared or not. The researcher suggests and recommends the use of new variables and factors
in studying the relationships that affect cybersecurity, and studying the extent of culture that people have about this concept Page | 70 [24] Zwilling, Moti et al. 2020. “Cyber Security Awareness, Knowledge and Behavior: A Comparative Study.” Journal of
Computer Information Systems 00(00): 1–16. https://doi.org/10.1080/08874417.2020.1712269. REFERENCES Taking the “Q” out of research: Teaching research methodology courses
without the divide between quantitative and qualitative paradigms. Quality and Quantity, 39(3), 267-295. P
| 71
[17] Papazoglou, Grammatiki Emmy. 2019. “Society and Culture : Cultural Policies Driven by Local Authorities as A
Factor in Local Development — The Example of the Municipality of Xanthi-Greece.” International Journal of Social
Science and Humanity: 2625–39. Page | 71 Page | 71 Research Publish Journals ISSN 2348-1218 (print)
ISSN 2348-1226 (online) ISSN 2348-1218 (print)
International Journal of Interdisciplinary Research and Innovations ISSN 2348-1226 (online)
Vol. 10, Issue 4, pp: (68-72), Month: October 2022 - December 2022, Available at: www.researchpublish.com [18] Tolah, A, S M Furnell, and M Papadaki. 2017. “A Comprehensive Framework for Cultivating and Assessing
Information Security Culture.” Proceedings of the Eleventh International Symposium on Human Aspects of
Information Security & Assurance (HAISA 2017) (Haisa): 52–64. [19] Uchendu, Betsy, Jason R.C. Nurse, Maria Bada, and Steven Furnell. 2021. “Developing a Cyber Security Culture:
Current Practices and Future Needs.” Computers and Security 109. [20] Veiga, Adéle da, Liudmila V. Astakhova, Adéle Botha, and Marlien Herselman. 2020. “Defining Organisational
Information Security Culture – Perspectives from Academia and Industry.” Computers & Security: 101713. https://doi.org/10.1016/j.cose.2020.101713. [21] Yani, Yanyan M. 2016. “Cybersecurity Policy and Its Implementation in Indonesia Muhamad Rizal.” Journal of
ASEAN Studies 4(1): 61–78. [22] Yıldırım, M., and I. Mackie. 2019. “Encouraging Users to Improve Password Security and Memorability.”
International Journal of Information Security 18(6): 741–59. https://doi.org/10.1007/s10207-019-00429-y. [23] Zimmermann, Verena, and Karen Renaud. 2019. “Moving from a ‘human-as-Problem” to a ‘human-as-Solution”
Cybersecurity Mindset.” International Journal of Human Computer Studies 131: 169–87. https://doi.org/10.1016/
j.ijhcs.2019.05.005. [24] Zwilling, Moti et al. 2020. “Cyber Security Awareness, Knowledge and Behavior: A Comparative Study.” Journal of
Computer Information Systems 00(00): 1–16. https://doi.org/10.1080/08874417.2020.1712269. Page | 72 Page | 72 Research Publish Journals
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English
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Construction of the Optimization Model of Cargo Transport Network
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E3S web of conferences
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1 Introduction Low carbon is closely related to transportation. As an
important part of the transportation industry, bulk cargo
transportation has formed the four modes of bulk
transportation, bulk transportation, bulk unloading and
bulk storage for a long time, which pollutes the port and
urban environment to a large extent. As a green and
efficient mode of transportation, container transportation
has developed rapidly. In addition, it can reduce the trade
imbalance of container liner shipping lines, effectively
reduce the transportation cost of cargo owners, and thus
reduce the logistics cost of the whole society. Based on
this background, it is particularly important to discuss and
study the transportation problem of "changing scattered to
centralized". Construction of the Optimization Model of Cargo Transport
Network ng Yanyan1,a*, Tian Benfang1,b, Wang Qingyang2,c ,Liu Donghui1,Gao Yunfei2,Li Chuang2 and Zhang Yiwe 1Logistic engineering Department, Shandong Jiaotong University, Jinan Shandong Province, 250357, China
2Traffic engineering Department, Shandong Jiaotong University, Jinan Shandong Province, 250357, China
3Traffic equipment and control engineering Department, Shandong Jiaotong University, Jinan Shandong Province, 250357, China Abstract. The transportation is gradually integrated into the circulation system of bulk goods, and
developing and growing. Transportation has the advantages of high efficiency, high quality and green
environment, In time, efficiency and cost than the traditional bulk transport has certain advantages. This paper
analyzes the cost of logistics transportation network, which mainly includes transportation cost in transit,
transit cost, time cost of cargo transportation and special cost. This paper discusses in detail the transportation
cost, transit cost, time cost and carbon consumption cost of different transportation modes in the process of
"scattered transformation", and constructs the optimization model of" scattered transformation "transportation
network with the least comprehensive transportation cost including the above costs. related to transportation. In network optimization,
sometimes they do not consider the carbon costs brought
by carbon tax collection, which leads to the lack of
rationalization of transport networks. Carbon emissions
can not be reduced effectively. Transportation is different
from
bulk
transportation. The
optimization
of
transportation network technology needs to consider
multiple nodes and transportation modes. The conversion
speed between different transportation modes directly
affects the overall transportation efficiency. How to
optimize the transportation network is very important. https://doi.org/10.1051/e3sconf/202126103013 https://doi.org/10.1051/e3sconf/202126103013 E3S Web of Conferences 261, 03013 (2021)
ICEMEE 2021 Corresponding author: 1652214425@qq.com
b3266156130@qq.com
c1181359962@qq.com
© The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0
(http://creativecommons.org/licenses/by/4.0/). EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0
icenses/by/4.0/). 2 Analysis on Optimization of Cargo
Transport Network Suppose there are multiple trans-shipment points
Distribution Network between the O of the starting point
and the destination D, in the actual transport network,
these nodes represent the form of road container transit
stations, container ports, railway container handling
stations and air ports. s modes of transport are available
between the connected nodes, with different modes of
transport having different carbon costs, transport costs and
time, Here we do not consider the multi-way distribution
of goods, and any mode of transport between the two
nodes has a corresponding determination of the transport
path, and the node allows the transfer of goods, in the
event of transit needs a certain transit time, resulting in a
certain transfer cost. Under the constraint of not exceeding
the transportation time limit, the enterprise is required to
select the network node that needs the way, and at the same
time, it is necessary to decide which transportation mode
to select for each transportation section, so as to minimize The transportation of "loose change" is beneficial to
reduce the trade imbalance of container liner route, and
can effectively reduce the transportation cost of cargo
owners, thus reducing the logistics cost of the whole
society. Transportation can meet the requirements of small
batch, multi-variety and different quality grade, and can
use various means of transportation of railway, waterway
and highway to meet different time requirements, so that
multimodal
transport
can
be
further
developed. Multimodal transport can shorten the transportation time,
reduce the transportation cost to a large extent, reduce the
road congestion, improve the quality of transportation, and
the environmental protection benefit is remarkable, which
can not only achieve the improvement of energy efficiency,
but also reduce the noise pollution and CO2 emissions. Some
enterprises
that
carry
out
"scattered"
transportation only pay attention to the costs directly * Corresponding author: a1652214425@qq.com
b3266156130@qq.com
c1181359962@qq.com https://doi.org/10.1051/e3sconf/202126103013 E3S Web of Conferences 261, 03013 (2021)
ICEMEE 2021 E3S Web of Conferences 261, 03013 (2021) the comprehensive transportation cost of the enterprise. The comprehensive transportation cost includes carbon
cost, in-transit transportation cost, transit cost, cargo
transportation time cost and special cost. The construction
of transport network map is indispensable in the research
of low-carbon transport network optimization. 2 Analysis on Optimization of Cargo
Transport Network the
network is described by a directed graph G (a, N)
according to graph theory, in which the N is the collection
of each node of the "scattered transformation set" freight
network, which is the main gathering place, transit place
and receiving place; A is the arc set of the transportation
line in the network diagram, the transportation line is the
flow channel of the "scattered" goods produced by the
network node, and the "scattered" box quantity in the
transportation route is the capacity of the arc in the
diagram. The transport network diagram is shown in
figure 1. network diagram, In order to simplify the network
diagram, we can set up virtual nodes to represent direct
transportation. A point represents the transit node, the B
point represents the virtual node, and the direct
transportation through the virtual node B is called cross-
level transportation. The network diagram can directly
reflect the transit time, transit carbon emissions and transit
costs of goods, one side represents a mode of transport. Fig. 3. Extended Network Diagram Fig. 1. Caption of the Figure 1. Below the figure. Fig. 3. Extended Network Diagram 3 Optimization Model of Cargo
Transport Network Besides the basic freight, the freight
cost of container railway also includes the surcharge of
railway electrification, the equal share of new road and the
miscellaneous expenses of railway freight. The above cost
can be converted into the basic freight BSr. of railway
transportation according to the actual transportation Formula (1) to (2) represents the optimal function of
integrated transportation cost, The Ce is carbon cost, the
CT is the sum of transportation cost and transit cost, the
CV is the time cost of goods transportation, and the CI is
the special cost. Constraint (3) indicates that the total time
of
shipment
(including
transportation
time
and
replacement time) does not exceed the time required by
the customer. Constraints (4) to (6) guarantee a complete
path from the starting point to the end. Constraints (7)
ensure the continuity of the whole course of cargo
transportation. Constraint (8) indicates that only one mode
of transport is allowed between two transport nodes, that
is, the volume can not be divided. Constraint (9) indicates
that if transit occurs at a node, only one mode of transport
is allowed to be converted to another. Constraint (10)
indicates that the number of full transshipment of "bulk
transfer" does not exceed G. maximum number of
transshipment. In constraint condition (11),1 means that
the transport mode is adopted between the node and the
node, otherwise it is 0;1 means that the node changes from
the mode of transport to the mode of transport, otherwise
it is 0. The node can be i to the node according to the actual
calculation method of container railway freight combined
with the characteristics of "scattered collection" cargo
transportation j and the railway transportation cost
calculation method is shown in the following formula
(here k=2, for railway transportation) y
p
)
𝐶
ൌ𝜆∗𝑄∗𝐵𝑆∗𝐷
(13) (13) BSr means the basic freight rate, yuan / TEU km; for
railway transportation (3) At present, the shipping cost of container waterway
mainly includes package rate, discount rate and uniform
rate. Container
transportation
mostly
adopts
liner
transportation, because liner transportation generally uses
dry rate, so this paper adopts container dry rate, dry rate
varies with route. In addition, considering the imbalance
of container route cargo flow, in the return container ship
with high no-load rate, according to the actual situation,
the use of "scattered transfer" transport bulk goods can be
given a certain freight discount. 3 Optimization Model of Cargo
Transport Network The
transportation
cost
includes
carbon
cost,
transportation cost, transit cost, transportation time cost
and special cost. Taking the sum of the above cost as the
objective function, the optimization model of low-carbon
transport network for multimodal transport of goods is
obtained: Fig. 1. Caption of the Figure 1. Below the figure. If the transport mode conversion is carried out at a
node, the conversion process is shown in figure 2. The
solid frame represents a transport node, each node can
choose three modes of transport, namely, road, railway,
waterway. 𝑚𝑖𝑛𝑍ൌ𝐶𝐶்𝐶𝐶ூ (1)
𝑚𝑖𝑛𝑍ൌ𝑐∑
∑
ሺ𝜆∗𝑄∗𝐷
ே
,ୀଵ
௦
,ୀଵ
∗𝑈𝐶∗𝑥
𝜆∗
𝑄∗𝑈𝑍∗𝑦
ሻ∑
∑
ሺ𝐶
ே
,ୀଵ
ௌ
,ୀଵ
∗𝑥
𝜆∗𝑄∗
𝑅𝐸
∗𝑦
ሻ∑
∑
ሺ𝐶
ே
,ୀଵ
ௌ
,ୀଵ
∗𝑥
𝜆∗𝑄∗
𝑅𝐸
∗𝑦
𝜆∗𝑄∗ሺ𝑌ை𝑌ሻ (2) (1) Fig. 2. Transport Node Transhipment Chart. (2) The constraints are: The constraints are: ∑
∑𝑇
,
∗𝑥
∑∑
𝑡
,∈ே
𝑦
𝑇 𝑖, 𝑗∈ ∑
∑𝑇
,
∗𝑥
∑∑
𝑡
,∈ே
𝑦
𝑇 𝑖, 𝑗∈
𝑁; 𝑘, 𝑚∈𝑆 (3)
∑𝑥,ାଵ
െ∑𝑥ିଵ,
ൌ1 𝑖ൌ𝑂, 𝑘∈𝑆 (4)
∑𝑥,ାଵ
െ∑𝑥ିଵ,
ൌ0 𝑖∈𝐼, 𝑘∈𝑆 (5)
∑𝑥,ାଵ
െ∑𝑥ିଵ,
ൌെ1 𝑖ൌ𝐷, 𝑘∈𝑆 (6)
𝑥
∗𝑥
ൌ𝑦
𝑖, 𝑗, ℎ∈𝑁; 𝑘, 𝑚∈𝑆 (7)
∑
∑
𝑥,ାଵ
∈ௌ
∈ே
ൌ1 𝑖ൌ1,2 … , 𝑁െ1 (8)
∑
𝑦
,∈ே
1 𝑖∈𝑁 (9)
∑∑
𝑦
,∈ே
𝐺 𝑖∈𝑁; 𝑘, 𝑚∈𝑆 (10)
𝑥
, 𝑦
∈ሼ0,1ሽ 𝑖, 𝑗∈𝑁, 𝑘, 𝑚∈𝑆 (11) 𝑁; 𝑘, 𝑚∈𝑆 𝑁; 𝑘, 𝑚∈𝑆 Fig. 2. Transport Node Transhipment Chart. If we need to describe the change of transportation
mode at a certain node in detail, we can combine figure 1
and figure 2 to establish another commonly used extended
network diagram as shown in figure 3. The transport
network diagram extends the urban node according to the
type of mode available at the time of arrival. A virtual final
destination D1. is needed to ensure the integrity of the 2 E3S Web of Conferences 261, 03013 (2021)
ICEMEE 2021 https://doi.org/10.1051/e3sconf/202126103013 basis of the rates of container freight and the number of
boxes used as stipulated in the Schedule of Freight Rates
of Railway Goods. 3 Optimization Model of Cargo
Transport Network The node can be i to the
node according to the actual calculation method of
waterway freight j and the waterway transportation cost
calculation method is shown in the following formula
(here k=3 represents waterway transportation): 4.1 Cost of transportation in transit (1) Transportation cost of road transportation. ( )
p
p
Because "bulk transfer" transportation is to transport
bulk goods into containers, the transportation cost of "bulk
transfer" can be calculated by reference to container
transportation cost. Container road freight can be divided
into short distance transportation and long distance
transportation according to the length of transportation
distance. The short distance freight rate is calculated
according to the principle of long distance decline, while
the long distance freight rate is calculated according to the
basic freight rate. The "scattered change" freight
transportation discussed in this paper refers to long
distance transportation. The container road transportation
uses the unified basic freight rate in our country, of which
20 ft standard box is 6.00 yuan / box kilometer 40 ft
standard box is 9.00 yuan / box kilometer, allowing
provinces and municipalities to make the basic freight rate
of container road transportation in this area according to
the local actual situation. The node i to the node can be
obtained according to the calculation method of the actual
container road freight combined with the characteristics of
the "scattered change set" cargo transportation j and the
road transportation cost calculation method is shown in
the following formula (here k=1, for road transportation):
𝐶
𝜆
𝐵𝑆
𝐷
(12) y
p
)
𝐶
ൌ𝜆∗𝑄∗𝐸
∗𝛼 (14) (14) The E indicates that the node i to the node j adopts the
single box freight rate k by the mode of transportation. Here k=3 represents the waterway transportation, and the
yuan / TEU;α is the discount of the freight rate,%; 5 Conclusion The optimization scheme of transportation network
considering time limit and carbon emission is better. In
this paper, cross-node transport is considered compared
with most of the current transport network optimization
models, in the future, we can study the network
optimization problem considering that the transportation
of goods between two nodes is shared by two or more
modes of transportation. 4.2 Transfer Cost of Bulk Transfer Transportation includes many kinds of transportation
modes, which involve the transformation of transportation
mode. The reasonable selection of nodes for the
conversion of transportation mode and the transfer of
goods to another mode of transportation at the node can
effectively reduce the comprehensive cost of the whole
process. The main function of the transfer node is to
realize the transfer of goods by road, railway and
waterway, which will produce the transfer cost. In order to
simplify the calculation and more intuitively show the
functional relationship between freight volume and transit
cost, the method of calculating transit cost in this paper is
as follows: )
(12) (
,
p
)
𝐶
ൌ𝜆∗𝐵𝑆∗𝐷
(12) 𝐶
ൌ𝜆∗𝐵𝑆∗𝐷
𝐶௭ൌ𝜆∗𝑄∗𝑅𝐸
(15) (15) BSh means the basic freight rate for road transportation,
yuan / TEU km; BSh means the basic freight rate for road transportation,
yuan / TEU km; The cost of transportation mode is equal to the sum of
the transportation cost in transit between nodes and the
transit cost at nodes, that is: y
(2) Transportation cost of "scattered reform" railway. At present, domestic container railway tariff charging
methods include dry rate and average rate. The Rules on
Freight Rates of Railway Goods stipulate that the basic
freight rates of container goods shall be calculated on the 3 https://doi.org/10.1051/e3sconf/202126103013 E3S Web of Conferences 261, 03013 (2021) E3S Web of Conferences 261, 03013 (2021)
ICEMEE 2021 𝐶்ൌ∑
∑
ሺ𝐶
ே
,ୀଵ
ௌ
，ୀଵ
∗𝑥
𝜆∗𝑄∗𝑅𝐸
∗𝑦
ሻ
(16) 2. Miao Linzhan. Discussion on the Design of a New
Type of Loading and Unloading System of
"Dispersion and Reforming Collection "[ J].] Hoisting
transport machinery. 2020(19) (16) 3. Zhao Duofa. Equipment [J].] for port's "loose
assembly" unpacking operation. Containerization. 2019 (02) 4.3 Time cost of transportation of "bulk" goods The time cost of transporting goods includes the time
value of the capital of the goods, which is related to the
opportunity cost of the funds occupied by the goods in
transit, it also includes reducing the loss of goods, market
price changes, packaging costs, storage costs and other
capital values. For the time cost of transportation of goods,
it is generally related to the mode or route of transportation,
the distance of transportation, the type of goods, the
transportation interval or direction, and so on. When
evaluating the time cost of cargo transportation, the above
factors should be quantified. Therefore, the calculation
formula of transportation time cost of goods is as follows:
ௌ
ே 4. Xie
Shouhong,
Cai
Haiya,
Xia
Gangxiang. Calculation and Influencing Factors of Carbon
Emission in China Transportation Industry [J].]
1Resources and environment in arid areas. 2016(05). 5. Sun
Jiaqing. A
Some
Thoughts
on
Coal
Transportation under the Concept of Green Logistics
[J].] of "Dispersion and Reform Coal China, 2013
(09):13-15 6. Duan Xueyan, Fan Chen. Optimization of Container
Transport Network of Xiaonehe River in Yangtze
River Delta under Low Carbon Economy [J].] China
Navigation ,2016(01):110-114 𝐶ൌ𝜆∗𝑄∗𝑇𝑉∗ሺ𝑇
ௌ
,ୀଵ
ே
,ୀଵ
∗𝑥
𝑡
𝑦
(17 7. Zhang Junwei. A Study on Route Rationalization of
Full Container Transport Network in Multimodal
Transport [D].] Beijing Jiaotong University ,2011. (17) From the point of view of the time value of
transportation and the interest on the funds during the
period, the cost coefficient of the time of transportation of
the goods is calculated. The formula TV the time cost
coefficient of the goods is as follows: 𝑇𝑉ൌ
ೝ∗ூோ
ଷହ∗ଶସ (18) (18) 4.4 Special Cost of Scattered Reform The transportation of bulk goods will be carried in
containers, so there will be a certain packing fee and
transfer fee O the starting point. The one-way heavy box
transportation is considered here, so the return fee will not
be considered. At the end D there will be a certain cost of
taking out and cleaning. The cost is related to the quantity
of the box and can be calculated by the following formula:
𝐶ூൌ𝜆∗𝑄∗ሺ𝑌ை𝑌ሻ (19) (19) The YO indicates the packing fee and the transfer fee
O the starting point, Yuan / box, and the YD means the cost
of taking out and cleaning the terminal D, Yuan / box. References 1. Miao Hongyun. A Study on Optimization of Low-
carbon Transport Network for Goods [D].] Wuhan
University of Technology ,2018 4
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Genome Sequence and Annotation of Helicobacter pylori Strain Hp238, Isolated from a Taiwanese Patient with Mucosa-Associated Lymphoid Tissue Lymphoma
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Genome announcements
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Genome Sequence and Annotation of Helicobacter pylori Strain
Hp238, Isolated from a Taiwanese Patient with Mucosa-Associated
Lymphoid Tissue Lymphoma eng-Yen Kao,a Jenn-Wei Chen,b Yi-Ting Huang,a Shew-Meei Sheu,c Bor-Shyang Sheu,c Jiunn-Jong Wua,b Cheng-Yen Kao,a Jenn-Wei Chen,b Yi-Ting Huang,a Shew-Meei Sheu,c Bor-Shyang Sheu,c Jiunn-Jong Wua,b
Department of Medical Laboratory Science and Biotechnology, National Cheng Kung University, Tainan, Taiwana; Center of Infectious Disease and Signaling Research,
National Cheng Kung University, Tainan, Taiwanb; Department of Internal Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwanc Department of Medical Laboratory Science and Biotechnology, National Cheng Kung University, Tainan, Taiwana; Center of Infectio
National Cheng Kung University, Tainan, Taiwanb; Department of Internal Medicine, College of Medicine, National Cheng Kung Univ We present the complete genome sequence of Helicobacter pylori strain Hp238, isolated from a Taiwanese patient with gastric
mucosa-associated lymphoid tissue lymphoma. Importantly, H. pylori strain Hp238 can multiply in THP-1 cells after internaliza-
tion through the induction of autophagosome formation. These genome data will help to identify genes associated with H. pylori in-
tracellular multiplication and pathogenesis. Received 4 January 2015 Accepted 13 January 2015 Published 19 February 2015
Citation Kao C-Y, Chen J-W, Huang Y-T, Sheu S-M, Sheu B-S, Wu J-J. 2015. Genome sequence and annotation of Helicobacter pylori strain Hp238, isolated from a T
patient with mucosa-associated lymphoid tissue lymphoma. Genome Announc 3(1):e00006-15. doi:10.1128/genomeA.00006-15. Copyright © 2015 Kao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license. Address correspondence to Jiunn-Jong Wu, jjwu@mail.ncku.edu.tw. Citation Kao C-Y, Chen J-W, Huang Y-T, Sheu S-M, Sheu B-S, Wu J-J. 2015. Genome sequence and annotation of Helicobacter pylori strain Hp238, isolated fr
patient with mucosa-associated lymphoid tissue lymphoma. Genome Announc 3(1):e00006-15. doi:10.1128/genomeA.00006-15. Copyright © 2015 Kao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license. Address correspondence to Jiunn-Jong Wu, jjwu@mail.ncku.edu.tw. yright © 2015 Kao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license. ess correspondence to Jiunn-Jong Wu, jjwu@mail.ncku.edu.tw. H H
elicobacter pylori is a Gram-negative spiral-shaped mi-
croaerophilic bacterium that infects 50% of the population
worldwide (1). Persistent infection with H. pylori increases the risk
of developing gastroduodenal diseases, including peptic ulcer, du-
odenal ulcer, and gastric adenocarcinoma (2, 3). The most com-
mon histopathological features of gastric malignancies are adeno-
carcinoma and lymphoma of mucosa-associated lymphoid tissue
(MALT). Genome Sequence and Annotation of Helicobacter pylori Strain
Hp238, Isolated from a Taiwanese Patient with Mucosa-Associated
Lymphoid Tissue Lymphoma It has been calculated that the risk of gastric adenocar-
cinoma and MALT lymphoma in H. pylori-infected individuals is
3- to 6-fold higher than in those who are uninfected (4). Genome
diversity, especially involving disease-associated genes, is highly
associated with different gastroduodenal diseases, since H. pylori
has coevolved with human populations (5). The study of whole-
genome sequences from strains isolated from patients with MALT
lymphoma is necessary to better understand the pathogenesis of
the disease and to identify virulence genes useful in the identifica-
tion of patients with higher risk. B38 (NC_012973.1). The contigs were then remapped according
to a previous study (7), and the results showed two scaffolds with
67 gaps. Finally, gaps were filled in through PCR and direct se-
quencing. The Hp238 genome has a size of 1,586,473 bp and a GC
content of 38.7%. Sequences were annotated at NCBI (http://www.ncbi.nlm.nih
.gov/genome/annotation_prok; strain Hp238) or with the RAST
prokaryotic genome annotation server (http://www.nmpdr.org
/FIG/wiki/view.cgi/Main/RAST). RAST annotation results showed
that the genome contained 92.3% coding regions and 1,663 genes,
including39RNAgenes.Theaveragelengthforprotein-codinggenes
was found to be 897 bp. The results showed the presence of major
virulence markers, including cagA (typing of the repeating EPIYA
motifs revealed the ABD type), vacA (typing of s and m regions, s1c/
m2), and babA. Moreover, genome and gene comparison analy-
sis by RAST with other fully and partially sequenced strains
demonstrates that this genome is most similar to the H. pylori
51 strain isolated in eastern Asia (Korea), followed by H. pylori
strains Shi470 and 35A. We report the genome sequence of H. pylori strain Hp238,
isolated from a 57-year-old Taiwanese man diagnosed with
MALT lymphoma. In a previous study, we determined that
Hp238 can multiply in THP-1 cells after internalization
through the induction of autophagosomes formation (6). Al-
though CagA and VacA participate in Hp238 multiplication in
THP-1 cells, whether other virulence factors are involved in
this multiplication remained unclear. This genome sequence
will provide valuable information for a better understanding of
bacterial pathogenesis. Particularly in Taiwan, this is the first
report of a genome sequence of H. pylori. Nucleotide sequence accession number. The complete ge-
nome sequence of strain Hp238 has been deposited in GenBank
under the accession number CP010013. ACKNOWLEDGMENTS We thank Robert Jonas for helpful comments on this manuscript. We also
thank Chien-Yi Tung and Jia-Ping Pan for helping on comparative
genomic analysis. This study was supported by grants NSC101-2320-B-006-020-MY3
and NSC101-2320-B- 006-029-MY3 from the National Science Council,
Taiwan. The Hp238 strain was sequenced using the 454 junior plat-
form (Roche, Germany), generating a library containing
252,098 single reads with an average length of 405 bp and a
33-fold average coverage. The genome sequence was generated
by a de novo assembly using the GS Assembler version 2.6 soft-
ware (Roche). This strategy provided 80 large contigs. As a
reference, we employed the genome sequence of H. pylori strain crossmark smark genomea.asm.org
1 January/February 2015
Volume 3
Issue 1
e00006-15 5. Yamaoka Y, Graham DY. 2014. Helicobacter pylori virulence and cancer patho-
genesis. Future Oncol 10:1487–1500. http://dx.doi.org/10.2217/fon.14.29. p
g
7. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W,
Lipman DJ. 1997. Gapped BLAST and psi-blast: a new generation of pro-
tein database search programs. Nucleic Acids Res 25:3389–3402. http://
dx.doi.org/10.1093/nar/25.17.3389. 3. Rauws EA, Tytgat GN. 1990. Cure of duodenal ulcer associated with
eradication of Helicobacter pylori. Lancet 335:1233–1235. http://dx.doi.org/
10.1016/0140-6736(90)91301-P. g
p
g
6. Wang YH, Wu JJ, Lei HY. 2009. The autophagic induction in Helicobacter
pylori-infected macrophage. Exp Biol Med (Maywood) 234:171–180.
http://dx.doi.org/10.3181/0808-RM-252. 2
genomea.asm.org 4. Kim SS, Ruiz VE, Carroll JD, Moss SF. 2011. Helicobacter pylori in the
pathogenesis of gastric cancer and gastric lymphoma. Cancer Lett 305:
228–238. http://dx.doi.org/10.1016/j.canlet.2010.07.014. Orentreich N, Sibley RK. 1991. Helicobacter pylori infection and the risk of
gastric carcinoma. N Engl J Med 325:1127–1131. http://dx.doi.org/
10.1056/NEJM199110173251603. Orentreich N, Sibley RK. 1991. Helicobacter pylori infection and the risk of
gastric carcinoma. N Engl J Med 325:1127–1131. http://dx.doi.org/
10.1056/NEJM199110173251603.
3. Rauws EA, Tytgat GN. 1990. Cure of duodenal ulcer associated with
eradication of Helicobacter pylori. Lancet 335:1233–1235. http://dx.doi.org/
10.1016/0140-6736(90)91301-P.
4. Kim SS, Ruiz VE, Carroll JD, Moss SF. 2011. Helicobacter pylori in the
pathogenesis of gastric cancer and gastric lymphoma. Cancer Lett 305:
228–238. http://dx.doi.org/10.1016/j.canlet.2010.07.014. January/February 2015
Volume 3
Issue 1
e00006-15 REFERENCES 1. Calvet X, Ramirez Lázaro MJ, Lehours P, Mégraud F. 2013. Diagnosis
and epidemiology of Helicobacter pylori infection. Helicobacter 18(Suppl
1):5–11. http://dx.doi.org/10.1111/hel.12071. 2. Parsonnet J, Friedman GD, Vandersteen DP, Chang Y, Vogelman JH, 2. Parsonnet J, Friedman GD, Vandersteen DP, Chang Y, Vogelman JH, Genome Announcements genomea.asm.org
1 Kao et al. Orentreich N, Sibley RK. 1991. Helicobacter pylori infection and the risk of
gastric carcinoma. N Engl J Med 325:1127–1131. http://dx.doi.org/
10.1056/NEJM199110173251603. 3. Rauws EA, Tytgat GN. 1990. Cure of duodenal ulcer associated with
eradication of Helicobacter pylori. Lancet 335:1233–1235. http://dx.doi.org/
10.1016/0140-6736(90)91301-P. 4. Kim SS, Ruiz VE, Carroll JD, Moss SF. 2011. Helicobacter pylori in the
pathogenesis of gastric cancer and gastric lymphoma. Cancer Lett 305:
228–238. http://dx.doi.org/10.1016/j.canlet.2010.07.014. January/February 2015
Volume 3
Issue 1
e00006-15 Genome Announcements Genome Announcements
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Cycling Kinematics in Healthy Adults for Musculoskeletal Rehabilitation Guidance
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Cycling Kinematics in Healthy Adults for
Musculoskeletal Rehabilitation Guidance Haeun Yum
Ewha Womans University
Hyang Kim
Myongji Hospital
Taeyong Lee
Ewha Womans University
Moon Seok Park
Seoul National University Bundang Hospital
Seung Yeol Lee
(
kernels00@naver.com
)
Hanyang University College of Medicine Research Article License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Version of Record: A version of this preprint was published at BMC Musculoskeletal Disorders on
December 1st, 2021. See the published version at https://doi.org/10.1186/s12891-021-04905-2. Page 1/16 Page 1/16 Abstract Background: Stationary cycling is commonly used for postoperative rehabilitation of physical disabilities,
but few studies have focused on the three-dimensional (3D) kinematics of rehabilitation. This study
aimed to elucidate the three-dimensional lower limb kinematics of musculoskeletally healthy people and
the effect of sex and age on kinematics using a controlled bicycle configuration. Methods: Thirty-one healthy adults participated in the study. The stationary cycle positioning was
standardized using the LeMond method by setting the saddle height to 85.5% of the participant’s inseam. The participants maintained a pedaling rate of 10–12 km/h, and the average value of three successive
cycles of the right leg was used for analysis. The pelvis, hip, knee, and ankle joint motions during cycling
were evaluated in the sagittal, coronal, and transverse planes. Kinematic data were normalized to 0–
100% of the cycling cycle. The Kolmogorov-Smirnov test, Mann-Whitney U test, Kruskal-Wallis test, and k-
fold cross-validation were used to analyze the data. Results: In the sagittal plane, the cycling ranges of motion (ROMs) were 1.6° (pelvis), 43.9° (hip), 75.2°
(knee), and 26.9° (ankle). The coronal plane movement was observed in all joints, and the specific ROMs
were 6.6° (knee) and 5.8° (ankle). There was significant internal and external rotation of the hip (ROM:
11.6°), knee (ROM: 6.6°), and ankle (ROM: 10.3°) during cycling. There was no difference in kinematic
data of the pelvis, hip, knee, and ankle between sexes (p = 0.12 to 0.95) and among ages (p = 0.11 to
0.96) in all anatomical planes. Conclusions: The kinematic results support the assertion that cycling is highly recommended for
comprehensive musculoskeletal rehabilitation. These results may help clinicians choose a target recovery
ROM based on healthy and non-elite individuals and issue suitable guidelines to patients. Methods This prospective study was approved by the institutional board of our hospital. Informed consent was
obtained from all participants. All procedures were performed in accordance with relevant guidelines. Participants who were over 18 years of age (i.e., legal adults) were competitively included in this study. Patients diagnosed with any musculoskeletal disease, those with a history of musculoskeletal trauma
(including ligamentous injury and fracture), and those who were determined to have musculoskeletal
deformities after the physician’s physical examination were excluded from the study. To figure out the
effects of age on kinematics, the participants were divided by the age of 10; group 1: <20 years old, group
2: 21–35 years old, group 3: 36–50 years old, and group 4: >50 years old. The physical examination was
performed by a surgeon with 14 years of orthopedic experience. Background Musculoskeletal rehabilitation programs provide extensive orthopedic rehabilitation post-surgery or injury. Rehabilitation helps patients to regain muscle and joint function and to restore bone health by building
strength and restoring flexibility and mobility, which reduces pain. Bones and joints in humans weaken
with aging [1], and many countries are becoming aging societies. Thus, musculoskeletal rehabilitation is
of considerable importance. Stationary cycling is commonly recommended for individuals with a variety of disabilities, such as knee
osteoarthritis, anterior cruciate ligament (ACL) reconstruction, and total hip arthroplasty. Cycling reduces
the load on the knee joint [2, 3] and ACL [4–7]; the tibiofemoral compressive forces during cycling are
between 0.3 and 2 times the body weight, while other full weight-bearing rehabilitative exercises (e.g.,
walking, stair ascent/descent) generate forces of approximately 2–4 times the bodyweight [4, 8, 9]. The
patellofemoral compressive force [9, 10], shear stress [11], tibiofemoral shear force [12], and ACL strain [9,
10, 13, 14] are low during cycling, yet the quadriceps and hamstring muscles are strengthened as knee Page 2/16 Page 2/16 stability increases [7, 11, 15–17]. Pedaling also increases the range of motion (ROM) of the hip, knee, and
ankle joints [15, 18–20]. Many studies have investigated joint kinematics during cycling, but most have been conducted on
patients with orthopedic disabilities [11, 14, 21]. Few studies have performed kinematic research on
healthy individuals. Furthermore, when healthy study subjects were examined, they were high-level or
experienced cyclists [22–26], and the study did not focus on target ROMs for rehabilitation purposes. Additionally, most studies analyzed two-dimensional kinematic data and focused on the sagittal plane
joint kinematics during cycling [1]. The three-dimensional (3D) kinematic data from musculoskeletally
healthy individuals can serve as a clinical guide for appropriate cycling interventions, leading to more
consistent rehabilitation program results. Therefore, this study aimed to elucidate 3D kinematics of the
lower extremity joints in musculoskeletally healthy and non-elite adults during cycling, determine
kinematic differences depending on sex and age, and provide the literature on cycling rehabilitation. Acquisition of kinematic data Bicycle configuration can affect cycling performance. Thus, the positioning of each participant on the
stationary cycle was standardized with the LeMond method, which is widely used and based on the
empirical experience of a famous cyclist, Greg LeMond [27, 28]. The saddle height was measured from
the center of the bottom bracket to the top of the seat along the seat tube. The saddle height was set at
88.3% of the distance between the highest point of one’s inner thigh to the heel of one’s foot, called an
inseam (Fig. 1). This percentage is based on the average height of Westerners, and it is common to
multiply the length by 0.855 for application to Asians. Therefore, the configuration of the saddle height
was set at 85.5% of the inseam in this study. A pedaling rate of 10–12 km/h was required throughout testing. To ensure that the rate was maintained,
participants rode a bicycle ergometer (HealthWay, ROBUST S5, Korea) for several minutes before
recording the kinematics. Data collection began when the participants were acquainted with the speed
and kept a steady rate. We obtained 3D kinematic data using a Kestrel Digital System (Motion Analysis, Page 3/16 Rohnert Park, CA, USA) equipped with 10 cameras. The Helen Hayes marker set was used to place
markers on the top, back, and front of the head; both shoulders, elbows, and wrists; anterior superior iliac
spine; thigh wand; lateral knee; shank wand; lateral ankle; heel; toe; and sacrum [29]. The movements of
the pelvis, hip, knee, and ankle joints during cycling were evaluated in the sagittal, coronal, and transverse
planes. The kinematic data were normalized to 0–100% of the cycling cycle. The right pedal at 0° was
designated as 0%, and the right pedal after one 360° rotation (i.e., returned to 0°) was designated as
100%. The average value of three successive cycles was used for analysis. Statistical analyses The lower extremity kinematics for the participants’ right side were used for analysis to avoid duplication
of demographics [30]. The Kolmogorov-Smirnov test was used to verify the normality of the distribution
of continuous variables. Descriptive statistics (i.e., mean ± standard deviation) were used to summarize
the participants’ demographic and kinematic data. Comparisons between male and female groups were
conducted using the Mann-Whitney U test based on data characteristics. The Kruskal-Wallis test was
used to compare the kinematics among age groups. K-fold cross-validation was used to evaluate
machine-learning models with a limited data sample. All statistical analyses were conducted using SPSS
version 20.0 (IBM Co., Chicago, IL), and a p-value < 0.05 was considered significant. Results Thirty-one participants were finally included in this study. The mean age at the time of examination was
35.0 ± 13.8 years (range, 18.2–68.1 years) (Table 1, Supplement 1). The mean saddle height was 68.0 ±
1.7 cm (range, 64.9–72.2 cm) for men and 64.8 ± 1.1 cm (range, 63.4–66.2 cm) for women (Table 1). Page 4/16 Table 1
Patient demographics. Parameter
Value
No. subjects (Male/Female)
31 (23/8)
Age
35.0 ± 13.8 (18.2–68.1)
Height
172.1 ± 5.1 (162.9–185.0)
Male
174.1 ± 4.2 (166.8–185.0)
Female
166.5 ± 2.9 (162.9–170.0)
Inseam
78.5 ± 2.4 (74.2–84.5)
Male
79.5 ± 1.9 (75.9–84.5)
Female
75.8 ± 1.3 (74.2–77.4)
Saddle height
67.1 ± 2.1 (63.4–72.2)
Male
68.0 ± 1.7 (64.9–72.2)
Female
64.8 ± 1.1 (63.4–66.2)
Age, Height, Inseam, and Saddle height; mean ± standard deviation (range)
Age = decimal years
Saddle height = inseam x 0.855
ti
b
d i
ll th
l
(T bl 2 Fi
2) D
i
li
th
l i Table 1 Table 1
Patient demographics. Patient demographics. The joint motions were observed in all three planes (Table 2, Fig. 2). During cycling, the pelvis moved very
little in the sagittal plane. The ROMs were 43.9 ± 3.7° (hip), 75.2 ± 7.2° (knee), and 26.9 ± 10.5° (ankle). The coronal plane movement was observed in all joints; particularly, the knee ROM was 6.6 ± 2.7°, and the
ankle ROM was 5.8 ± 3.2°. The transverse plane movement was also observed in all major lower extremity
joints. Internal and external rotation occurred in the hip (11.6 ± 4.5°), ankle (10.3 ± 4.9°), and mainly in the
knee joints (6.6 ± 2.7°). coronal plane movement was observed in all joints; particularly, the knee ROM was 6.6 ± 2.7 , and the
e ROM was 5.8 ± 3.2°. The transverse plane movement was also observed in all major lower extremity
s. Internal and external rotation occurred in the hip (11.6 ± 4.5°), ankle (10.3 ± 4.9°), and mainly in the
joints (6 6 ± 2 7°) Page 5/16 Page 5/16 Page 5/16 Table 2 Table 2
Sagittal, coronal, and transverse plane kinematics of lower extremity during cycling. Rt:
Right, ROT: Rotation. Results The kinematic data of the pelvis, hip, knee, and ankle joints did not differ between the sexes (p = 0.12 to
0.95) or among ages (p = 0.11 to 0.96) in all anatomical planes. In k-fold cross-validation of the age
groups, the area under the curve was between 0.475 and 0.610. Results Range of motion (•)
Maximum value (•)
Sagittal
plane
Pelvis
1.6 ± 0.6
(0.7–3.6)
Posterior tilt
-15.0 ± 3.5
(-24.1 – -7.7)
Anterior tilt
16.6 ± 3.6
(8.9–25.3)
Hip
43.9 ± 3.7
(36.7–51.5)
Extension
-43.0 ± 5.1
(-54.4 – -34.5)
Flexion
86.9 ± 4.3
(79.7–98.0)
Knee
75.2 ± 7.2
(60.1–94.1)
Extension
-34.0 ± 9.8
(-57.2 – -14.7)
Flexion
109.3 ± 3.9
(102.7–118.5)
Ankle
26.9 ± 10.5
(10.8–47.0)
Dorsiflexion
7.6 ± 8.1
(-6.2–28.3)
Plantar flexion
19.2 ± 7.6
(1.6–31.8)
Coronal
plane
Pelvis
7.1 ± 2.5
(2.0–11.7)
Rt side up
3.5 ± 2.1
(-1.2–7.2)
Rt side down
3.6 ± 2.4
(-1.1–7.8)
Hip
5.0 ± 1.8
(1.6–10.8)
Adduction
10 ± 3.4
(5.9–21.8)
Abduction
-5.1 ± 2.9
(-13.6 – -0.9)
Knee
6.6 ± 2.7
(2.5–12.0)
Varus
1.6 ± 2.8
(-4.2–6.44) Range of motion (•)
Maximum value (•)
Valgus
5.0 ± 2.2
(0.3–9.2)
Ankle
5.8 ± 3.2
(2.1–14.2)
Inversion
1.5 ± 6.0
(-14.1–13.0)
Eversion
4.3 ± 6.5
(-10.2–20.6)
Transverse
plane
Pelvis
3.2 ± 1.9
(0.9–8.7)
Internal ROT
2.4 ± 3.1
(-4.2–8.8)
External ROT
0.8 ± 3.3
(-4.3–12.5)
Hip
11.6 ± 4.5
(3.3–23.8)
Internal ROT
6.3 ± 3.5
(-0.0–13.7)
External ROT
5.3 ± 6.1
(-5.9–20.6)
Knee
6.6 ± 2.7
(2.5–12.0)
Internal ROT
5.0 ± 2.2
(0.3–9.2)
External ROT
1.6 ± 2.8
(-4.2–6.4)
Ankle
10.3 ± 4.9
(3.9–22.0)
Internal ROT
4.4 ± 5.1
(-5.7–18.9)
External ROT
6.0 ± 4.3
(-1.0–13.2)
ematic data of the pelvis, hip, knee, and ankle joints did not differ between the sexes (p = 0.12
r among ages (p = 0.11 to 0.96) in all anatomical planes. In k-fold cross-validation of the age
the area under the curve was between 0.475 and 0.610. ussion Range of motion (•) Maximum value (•) The kinematic data of the pelvis, hip, knee, and ankle joints did not differ between the sexes (p = 0.12 to
0.95) or among ages (p = 0.11 to 0.96) in all anatomical planes. In k-fold cross-validation of the age
groups, the area under the curve was between 0.475 and 0.610. The kinematic data of the pelvis, hip, knee, and ankle joints did not differ between the sexes (p = 0.12 to
0.95) or among ages (p = 0.11 to 0.96) in all anatomical planes. In k-fold cross-validation of the age
groups, the area under the curve was between 0.475 and 0.610. Discussion Page 7/16
Cycling is one of the most effective orthopedic rehabilitation methods to recover joint ROM with less
weight load. Most studies have examined cycling in two dimensions, especially the sagittal plane [19, 31,
32], but recent studies have indicated that coronal and transverse movements also occur during cycling Page 7/16 [1, 10, 24]. Recently, 3D motion analysis is becoming critical for assessing full rehabilitation potential. Thus, this study evaluated the 3D lower limb kinematics of musculoskeletally healthy people during
stationary cycling and aimed to provide guidance for a target recovery ROM for physical rehabilitation. This study showed that considerable movement occurs in the sagittal plane and also in the coronal and
transverse planes. We also found that customizing the saddle height leads to constant joint kinematics. [1, 10, 24]. Recently, 3D motion analysis is becoming critical for assessing full rehabilitation potential. Thus, this study evaluated the 3D lower limb kinematics of musculoskeletally healthy people during
stationary cycling and aimed to provide guidance for a target recovery ROM for physical rehabilitation. This study showed that considerable movement occurs in the sagittal plane and also in the coronal and
transverse planes. We also found that customizing the saddle height leads to constant joint kinematics. The saddle position is often selected based on comfort. Improper positioning can lead to knee joint
overuse injuries [2, 31, 33, 34] and inconsistent kinematics. Numerous methods have been proposed to
determine the proper saddle height configuration [10]. We selected the LeMond method, as it is common,
reliable [33], and simple to apply, which is important because most patients use public bicycles in clinics
for rehabilitation purposes rather than private ones. We adjusted the saddle height to the length of each
participant’s inseam by multiplying the Asian-specific ratio. To our knowledge, this is the first study that
considers the cycling rehabilitation environment based on race and individuals. We found sagittal, coronal, and transverse movements in all joints during standardized ergometer cycling,
enabling comprehensive rehabilitation guidance. When the sagittal joint ROMs obtained during ergometer
cycling were compared with means of normal ROMs, the hip ROM was approximately 31%, the knee ROM
was approximately 54%, and the ankle ROM was approximately 42% of normal [7, 35–40] (Fig. 3). The
cycling kinematics of the lower limb joints were also compared to normal walking kinematics. Discussion The
normal sagittal plane ROM during a human gait cycle is on average approximately 45° in the hip (ranging
from 10° [extension] to 35° [flexion]), 55° in the knee (ranging from 5° [flexion] to 60° [flexion]), and 30° in
the ankle (ranging from 15° [dorsiflexion] to 15° [plantarflexion]) [41, 42]. This indicates that the hip and
knee joints were much more flexed during cycling and that the ankle joint motion was similar to that
during walking. The overall joint motion during pedaling may not have an advantage over that during
walking. However, the angle range where joint motion occurred during cycling was distinct from that
during walking, suggesting that pedaling has effects that cannot be obtained by walking only. Thus, in
terms of kinematics, cycling for musculoskeletal rehabilitation is highly recommended. Extra
rehabilitative exercise is necessary to restore the ROM that is not covered by cycling. Lower limb
kinematics are influenced by the saddle height [7, 10, 23]. Thus, the hip, knee, and ankle joint motions can
be adjusted by changing the saddle height. For example, more plantar flexion could be created by
increasing saddle height. Further investigation is needed to determine how cycling ROMs could be
broadened for rehabilitation. For the knee and ankle joints, significant internal and external rotation and coronal plane movements
were observed. Cycling is recommended for individuals with knee disabilities because it puts less weight
on the knee [4]. However, cycling considerably rotates the knee, which should be considered before
starting bicycle rehabilitation for injuries adversely affected by rotational motion, such as meniscus and
ankle ligament injuries. Understanding the 3D joint kinematics in healthy and normal individuals may
help clinicians plan a target recovery ROM and issue guidance to patients. Page 8/16 This study attempted to recruit all adult patients of all ages for comparison according to sex and 10-year
age groups. Pelvis, hip, knee, and ankle joint kinematics did not differ between sexes or among age
groups, indicating that customizing the saddle height per individual results in constant kinematics
regardless of sex and age. No studies have investigated the effects of sex and age on joint kinematics in
the context of rehabilitation. Thus, these results provide a good reference for planning lower limb
rehabilitation. There were a few limitations in this study. First, there were a small number of participants. Discussion This was a
pilot study in preparation for a large population study, and to overcome the small sample size, additional
k-fold cross-validation was conducted. Second, the saddle height was set considering an empirical
percentage for leg length (0.855 for Asians), and the bicycle configuration did not consider the handlebar
position. Comparative research is needed to determine if other bicycle configurations change the effect
on joint ROM or rehabilitation. Conclusions This study identified that stationary cycling generates movement in the sagittal, coronal, and transverse
planes, enabling comprehensive lower limb rehabilitation. The findings can serve as a guide for setting
the target kinematics during musculoskeletal rehabilitation with a stationary bicycle for individuals with
orthopedic disabilities. The saddle height was adjusted for each participant, producing consistent joint
motions. Given the limited number of studies on bicycle 3D movement for the general public (non-
professional athletes), further work is warranted to determine a suitable ROM for cycling rehabilitation
customizable to race and physical conditions. Abbreviations 3D: Three-dimensional ROM: Range of motion Rt: Right ROT: Rotation Consent for publication Not applicable. Authors’ contributions All authors (HY, HK, TL, MSP, and SYL) in this manuscript made significant contributions to the study
design. HY and SYL analyzed and interpreted the data, and wrote the article, and approved the final
version of the manuscript. HK and MSP acquired and analyzed the data. TL helped to draft the
manuscript and critically revised the manuscript. All authors have read and approved the final version of
the manuscript. Ethics approval Seoul National University Bundang Hospital Institutional Review Board in Seongnam, Korea approved the
study protocol. (IRB No: B-2103/673-107). Informed consent was obtained from all participants. Page 9/16 Funding Not applicable. Competing interests The authors declare that they have no competing interests. Availability of data and materials The datasets used and/or analyzed during this study are available from the corresponding author on
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During Single-Leg Assisted and Unassisted Cycling. J Strength Cond Res. 2015;29. doi:10.1519/JSC.0000000000000905. 33. Swart J, Holliday W. Cycling Biomechanics Optimization—the (R) Evolution of Bicycle Fitting. Curr
Sports Med Rep. 2019;18:490-6. doi:10.1249/JSR.0000000000000665. 33. Swart J, Holliday W. Cycling Biomechanics Optimization—the (R) Evolution of Bicycle Fitting. Curr
Sports Med Rep. 2019;18:490-6. doi:10.1249/JSR.0000000000000665. Page 12/16 Page 12/16 34. Hull ML, Jorge M. A method for biomechanical analysis of bicycle pedalling. J Biomech. 1985;18(9):631-44. doi:10.1016/0021-9290(85)90019-3. 35. American Academy of Orthopaedic S. Joint motion : method of measuring and recording. Chicago:
American Academy of Orthopaedic Surgeons; 1965. 36. Boone DC, Azen SP. Normal range of motion of joints in male subjects. J Bone Joint Surg Am. 1979;61(5):756-9. 37. Roaas A, Andersson GB. Normal range of motion of the hip, knee and ankle joints in male subjects,
30-40 years of age. Acta Orthop Scand. 1982;53(2):205-8. doi:10.3109/17453678208992202. 38. Kendall HO, Kendall FP, Wadsworth GE. Muscles, Testing and Function: Williams and Wilkins; 1971. 38. Kendall HO, Kendall FP, Wadsworth GE. Muscles, Testing and Function: W 39. Roach KE, Miles TP. doi:10.1080/17461391.2019.1635651. Normal hip and knee active range of motion: the relationship to age. Phys Ther. 1991;71(9):656-65. doi:10.1093/ptj/71.9.656. 40. Soucie JM, Wang C, Forsyth A, Funk S, Denny M, Roach KE, et al. Range of motion measurements:
reference values and a database for comparison studies. Haemophilia. 2011;17(3):500-7. doi:10.1111/j.1365-2516.2010.02399.x. 41. Pietraszewski B, Winiarski S, Jaroszczuk S. Three-dimensional human gait pattern - Reference data
for normal men. Acta Bioeng Biomech. 2012;14:9-16. doi:10.5277/abb120302. 42. Neumann DA. Kinesiology of the musculoskeletal system : foundations for physical rehabilitation. St. Louis, Mo.: Mosby/Elsevier; 2010. Figures Page 13/16 Figure 1
Measurements for the inseam (A) and saddle height (B), and the experimental setup showing a
participant cycling on the stationary bike with the Helen Hayes marker set attached (C). Figure 1 Figure 1 Measurements for the inseam (A) and saddle height (B), and the experimental setup showing a
participant cycling on the stationary bike with the Helen Hayes marker set attached (C). Page 14/16 Page 14/16 Figure 2
Hip, knee, and ankle joint angles for one complete revolution (0°–360°) of the bicycle crank for each
participant's right leg in the sagittal, coronal, and transverse planes. Figure 2 Supplement1.docx Figure 3 Comparisons among normal range of motions (ROM data reported by Kendall et al. [38, 39], Ericson et al. [7], the American Academy of Orthopaedic Surgeons [35], Boone et al. [36], Roaas et al. [37], and Soucie et
al. [40]), and joint excursion during walking (Neumann [42] and Pietraszewski et al. [41]), stair or level
walking (Ericson et al. [7]), and cycling (present study) of hip (A), knee (B), and ankle (C) joints. (degrees) Comparisons among normal range of motions (ROM data reported by Kendall et al. [38, 39], Ericson et al. [7], the American Academy of Orthopaedic Surgeons [35], Boone et al. [36], Roaas et al. [37], and Soucie et
al. [40]), and joint excursion during walking (Neumann [42] and Pietraszewski et al. [41]), stair or level
walking (Ericson et al. [7]), and cycling (present study) of hip (A), knee (B), and ankle (C) joints. (degrees) Comparisons among normal range of motions (ROM data reported by Ken Figure 2 Hip, knee, and ankle joint angles for one complete revolution (0°–360°) of the bicycle crank for each
participant's right leg in the sagittal, coronal, and transverse planes. Page 15/16 Page 15/16 Figure 3 Figure 3
Comparisons among normal range of motions (ROM data reported by Kendall et al. [38, 39], Ericson et al. [7], the American Academy of Orthopaedic Surgeons [35], Boone et al. [36], Roaas et al. [37], and Soucie e
al. [40]), and joint excursion during walking (Neumann [42] and Pietraszewski et al. [41]), stair or level
walking (Ericson et al. [7]), and cycling (present study) of hip (A), knee (B), and ankle (C) joints. (degrees)
Supplementary Files Figure 3 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Page 16/16
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Analisis Strategi Persaingan Air Mineral Dengan Menggunakan Metode Markov Chain Dan Game Theory
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Jurnal Senopati : Sustainability, Ergonomics, Optimization, and Application of Industrial Engineering
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ABSTRACT Halaman:
85 – 97
Tanggal penyerahan:
21 agustus 2022
Tanggal diterima:
29 April 2023
Tanggal terbit:
30 April 2023 Bottled water is drinking water packaged in plastic bottle. Bottled water often has
added ingredients such as minerals and vitamins that provide unique taste and
packaged in an attractive packaging. There are several brands of bottled water
currently in the market such as Aqua, Club, Nestle, and Le Mineral. This paper
presents strategy analysis using the Markov Chain method. This method can find a
competitive strategy as an effort to increase the competitiveness of the company. The four brands mentioned earlier were used in the Markov chain analysis. From
the results of the Markov Chain analysis, it was found that the average time needed
for state A (Aqua) to move to state L (Le Mineral) was 8 periods (months), and the
average time needed for state C (Club) to move to state L (Le Mineral) is two
periods (months). And for the results of Game Theory comparison of Aqua vs
Nestle, the optimal strategy for Aqua is obtained, namely Strategy X2 (Design) and
Strategy X4 (color) with a value of games (value of games) of -11 while for Nestle
the optimal strategy is obtained, namely Y2 Strategy (Design) and Y4 Strategy
(color) with a game size (value of games) of -11. For the comparison of Aqua vs Le
Mineral, the optimal strategy is obtained, namely 24 using pure strategy. Keywords: strategy, competition, markov chain, steady state ABSTRAK EMAIL EMAIL Air Mineral dalam kemasan merupakan air yang mengandung bahan-bahan larut
lain dan memberi nilai-nilai lain yang di kemas dalam bentuk ekonomis dan
menarik. Semakin berkembangnya persaingan semakin banyak pula merek Air
Mineral di pasaran. Penelitian ini membahas tentang analisis strategi menggunakan
metode Markov chain dan game theory. Metode ini dapat menemukan strategi yang
kompetitif sebagai upaya untuk meningkatkan daya saing perusahaan dan
menemukan strategi baru untuk bertahan di pasar. Terdapat empat merek yang
digunakan untuk membangun matriks matriks dan game theory.Dari hasil analisis
Markov Chain maka di temukan bahwa rata-rata waktu yang dibutuhkan untuk state
A (Aqua) berpindah ke state L (Le Mineral) adalah sebesar 8 periode (bulan), dan
rata-rata waktu yang dibutuhkan untuk state C (Club) berpindah ke state L (Le
Mineral ) adalah sebesar dua periode (bulan). Dan untuk hasil Game Theory
perbandingan Aqua vs Nestle Di peroleh strategi optimal bagi Aqua, yaitu Strategi
X2 (Desain) dan Strategi X4 (warna) dengan besar permainan (value of games)
sebesar -11 yang artinya keuntugan terbesar bagi Aqua adalah -11 sedangkan bagi
Nestle Di peroleh strategi optimal, yaitu Strategi Y2 (Desain) dan Strategi Y4
(warna) dengan besar permainan (value of games) sebesar -11 yaitu kerugian
terkecil bagi nestle adalah -11. Untuk perbandingan Aqua vs Le Mineral di peroleh
strategi optimal yaitu 24 dengan menggunakan streategi murni yang artinya
keuntungan terbesar bagi Aqua adalah 24 dan kerugian terkecil bagi nestle adalah
24. 1iisriyana@itsnupasuruan.ac.id Kata kunci : strategi, persaingan, markov chain, steady state Analisis Strategi Persaingan Air Mineral Dengan Menggunakan Metode Markov
Chain Dan Game Theory
Iis Riyana1
1Jurusan Teknik Industri, Institut Teknologi dan Sains Teknologi Nadhlatul Ulama Pasuruan, Wr. Iis Riyana1
1Jurusan Teknik Industri, Institut Teknologi dan Sains Teknologi Nadhlatul Ulama Pasuruan, Wr. Dowo, Kec. Pohjentrek, Pasuruan, y
urusan Teknik Industri, Institut Teknologi dan Sains Teknologi Nadhlatul Ulama Pasuruan, Wr. owo, Kec. Pohjentrek, Pasuruan, PENDAHULUAN Kota Pasuruan Jawa Timur merupakan daerah yang subur yang dikelilingi oleh pegunungan dengan
berbagai sumber mata air yang jernih dan melimpah. Dengan potensi pegunungan tersebut maka
sumber mata air di Kabupaten Pasuruan cukup melimpah. Potensi ini menjadi daya tarik, 85 Jurnal SENOPATI
Vol.4 No.2, April 2023 Jurnal SENOPATI
Vol.4 No.2, April 2023 e-ISSN: 2714 - 7010 khususnya bagi industry yang bergerak di bidang Air Minum Dalam Kemasan (AMDK) untuk
memanfaatkan potensi sumber air Pegunungan yang kaya dengan mineral. Perusahaan air tebanyak
di Jawa Timur terdapat di pasuruan. Perusahaan air mineral berskala besar yang berada di Pasuruan
seperti Club, Cheers, Cleo serta beberapa merk lainnya yang di hasilkan langsung dari sumber mata
air pegunungan yang berada di kabupaten Pasuruan. Dari Banyaknya perusahaan air mineral
berskala besar maupun kecil pasti akan timbul permasalahan berupa adanya peralihan konsumen air
mineral yang menyebabkan salah satu merek air mineral akan mengalami kekurangan permintaan
[1]. Oleh karena itu perlu dilakukan analisis strategi agar merek air mineral dapat bersaing untuk
mempertahankan konsumen. Penyelesaian permasalahan menggunakan teori permainan dilakukan den- gan strategi murni untuk
mencari nilai titik kesetimbangan, jika strategi murni tidak menghasilkan titik kesetimbangan maka
penyelesaian dilakukan meng- gunakan strategi campuran dengan mereduksi baris dan kolom
menggunakan prinsip dominasi dan penyelesaian pada strategi murni untuk mencari titik ke-
setimbangan. Jika titik kesetimbangan tidak diperoleh, maka nilai permainan akan dihitung
menggunakan salah satu metode alternatifnya yaitu Program Linear, Aljabar Matriks, atau Metode
Grafik. Jika teori permainan digunakan untuk mencari strategi optimal, maka dalam penelitian diperlukan
juga melihat pergerakan pelanggan dalam berpindah menggunakan suatu produk atau merek agar
dapat melakukan pen- ingkatan strategi sehingga konsumen tetap tertarik menggunakan produk
terse-but dan juga sebagai update kondisi pasar. Perhitungan mengenai perpindahan merek dapat
dilakukan menggunakan metode Markov Chain dengan menghi-tung probabilitas transisinya dalam
periode waktu tertentu. Markov Chain merupakan alat untuk memodelkan perubahan status
bersyarat dengan modelprobabilitas transisi keadaannya. Penyelesaian menggunakan Markov
Chain dengan melakukan perhitungan probabilitas pada periode tertentu pada ma- triks probabilitas
transisinya, kemudian melakukan perkalian matriks proba- bilitas waktu sebelumnya dengan
matriks transisinya hingga periode waktu yang diinginkan. Besarnya perhitung Besarnya
perhitungan Markov Chain menandakan bahwa seberapa besar minat masyarakat untuk
menggunakan produk tersebut [2]. Penelitian ini dilakukan untuk menentukan strategi bersaing bagi setiap brand air mineral
berdasarkan analisis terhadap peralihan pemilihan merek air mineral (brand switching) yang
dilakukan oleh konsumen [3]. PENDAHULUAN Kontribusi yang diharapkan adalah hasil penelitian ini dapat
dijadikan pertimbangan atau pun sebagai data pendukung dalam pengambilan keputusan yang akan
dilakukan oleh setiap Perusahaan brand air mineral Untuk menentukan strategi bersaing dalam
pangsa pasar. Pengumpulan Data Pengumpulan data dilakukan untuk mendapatkan informasi data yang dibutuhkan untuk proses
penelitian yang selanjutnya akan pakai sebagai pemecahan dari permasalahan yang telah
ditentukan. Pengumpulan data terdiri dari beberapa tahapan, yaitu: (a) Perancangan Kuesioner terbuka untuk memperoleh pendapatkonsumen, (a) Perancangan Kuesioner terbuka untuk memperoleh pendapatkonsumen, (a) Perancangan Kuesioner terbuka untuk memperoleh pendap (b) Penentuan sampel minimal dengan Persamaan Slovin, (c) Pengumpulan data, setelah menyebar kuesioner maka akan mendapat data dari responden yang
selanjutnya direkapitulasi. (c) Pengumpulan data, setelah menyebar kuesioner maka akan mendapat data dari responden yang
selanjutnya direkapitulasi. (d) membuat atribut permainan (e) merekap nilai persaingan (f) membuat matriks nilai permainan (g) menghitung maximin dan minimax (h) memodifikasi perolehan matriks (i) mencari solusi optimal menggunakan POM. Pengolahan Data. Data yang telah didapatkan kemudian diolah. Pengolahan data dilakukan untuk
mendapatkan informasi sesuai dengan apa yang dibutuhkan untuk memenuhi tujuan yang ingin
dicapai oleh peneliti. Dalam pengolahan data ini akan menggunakan metode rantai markov. METODE Metode penelitian dalam penelitian ini adalah kuantitatif deskriptif, dimana akan data akan diolah
dengan pemberian skoring, bobot maupun nilai-nilai tertentu untuk memudahkan dalam
menganalia dan menguraikan sesuai dengan indeks, nilai atau parameter-parameter tertentu. Terkait
dengan metode penelitian ini dapat dijelaaskan pada Gambar 1 di bawah ini 86 Jurnal SENOPATI
Vol.4 No.2, April 2023 e-ISSN: 2714 - 7010 e ISSN: 2714 7010
Vol.4 No.2, Ap
87
Gambar 1. Diagram Alir Metode Penelitian Gambar 1. Diagram Alir Metode Penelitian 87 87 Jurnal SENOPATI
Vol.4 No.2, April 2023 e-ISSN: 2714 - 7010 Tahap Pengumpulan dan Pengolahan Data Dalam tahap ini yaitu melakukan pengumpulan data oleh peneliti guna melengkapi data yang
diperlukan selama proses penelitian. Data yang didapat nanti akan diolah dengan menggunakan
metode-metode yang relevan untuk memecahan masalah. Penyebaran Kuesioner Pendahuluan Kuesioner adalah susunan pertanyaan yang tertulis yang disampaikan kepada responden [4]. Sebelum melakukan penyebaran kuesioner, harus melakukan pengujian terhadap kuesioner apakah
kuesioner tersebut tepat (valid) dan bersifat stabil (reliabel). Berdasarkan pertanyaan-pertanyaan
dalam kuesioner, maka uji validitas dapat menggunakan uji validitas konstruk sesuai tahapan-
tahapan yang telah dijelaskan pada bab sebelumnya. Sedangkan untuk uji reliabilitas dapat
menggunakan metode repeated measure.Langkah pertama yang dilakukan adalah dengan
melakukan penyebaran kusioner online yang disebar melalui media sosial watshapp. Setelah
pengisian kuesioner, dilakukan rekapitulasi hasil kuesioner pendahuluan tersebut apakah kriteria
responden sesuai dengan apa yang ingin diketahui oleh peneliti atau tidak, kemungkinan
penyimpangan kriteria dapat disebabkan adanya kesalah pahaman responden terhadap kuesioner. Sehingga harus dilakukan penyaringan kuesioner supaya sesuai kriteria. Berdasarkan hasil
penyaringan kuesioner, kriterian responden yang telah ditetapkan telah sesuai dengan apa yang di
harapkan oleh peneliti. =N/(1+(Nxe²))
=41.781/(1+(41.781x10%)) =107 Karena jumlah sampel yang telah di ambil sebanyak 107, maka sampel yang diambil telah
memenuhi jumlah sampel minimal yang harus di ambil (107>100). Penetuan Sampel Minimal Kuesioner yang telah di sebar haruslah memenuhi kecukupan sampel minimal yang di butuhkan. Secara statistika dinyatakan bahwa ukuran sampel yang semakin besar diharapkan akan
memberikan hasil yang semakin baik [5]. Pengukuran sampel menggunakan rumus slovin maka
dapat dihitung berapa minimal sampel yang harus diambil [6]. Hal ini bertujuan untuk mengetahui
apakah sampel yang diambil oleh penulis sudah mencukupi atau belum mencukupi [7]. Dari
penyaringan hasil kuesioner, diketahui bahwa kuesioner yang disebarkan dan kembali adalah
sebanyak 107 yang asalnya adalah 152. Diketahui populasi wilayah yang kan di teliti adalah 41.781
orang dengan tingkat kesalahan 10%. Berikut adalah perhitungan jumlah sampel minimal yang
dibutuhkan: =N/(1+(Nxe²))
=41.781/(1+(41.781x10%)) 88 Jurnal SENOPATI
Vol.4 No.2, April 2023 e-ISSN: 2714 - 7010 Pengolahan Data Uji Validitas Kuesioner Uji Validitas Kuesioner
Tabel 1 Uji Validitas
Pertanyaan ke-
Nilai Korelasi (rhitung)
Keterangan
1
0,336317
Valid
2
0,497246
Valid
3
0,201652
Valid
4
0,259905
Valid
5
0,2867
Valid
6
0,668508
Valid
7
0,573165
Valid
8
0,667463
Valid
9
0,654258
Valid
10
0,597059
Valid
Dari table di atas maka dapat di simpulkan bahwa semua data bersifat valid. Dari table di atas maka dapat di simpulkan bahwa semua data bersifat valid. Tabel 2 Uji Realibilitas
Variabel
Ralpha
Rkritis
Kriteria
Minat dan daya Tarik Konsumen
6.6012
0.600
Reliabel Dari table di atas maka dapat di simpulkan variabel minat dan daya Tarik konsumen setelah di uji
realibilitas menggunakan aplikasi SPSS bersifat Reliabel. Analisa Rantai Markov
Tabel 3 Perpindahan Konsumen
D
Merek
Ke merek
Konsumen
akhir
a
Aqua
Club
Nestle
Lea Mineral
r
Aqua
14
13
0
2
29
i
Club
0
23
0
33
56
M
Nestle
0
0
12
0
12
e
Le Mineral
0
0
0
10
10
r
Konsumen Awal
14
16
12
45
107
k Analisa Rantai Markov Analisa Rantai Markov Dari data pola perpindahan konsumem merek pada Tabel 3 diatas didapatkan i = A, C, N, L ; j =
A,C, N, L ( Ades, Club, Nestle dan Lea Mineral ) jumlah pelanggan berpindah dari merk I ke state j
disimbolkan dengan Xij. Serta probabilitas transisi dari state I ke state j disimbolkan dengan pij. Maka nilai Pij=
[8] Sehingga dapat dihitung probabilitas perpindahan jumlah pelanggan
pada masing-masing merek Air Mineral yang dapat dilihat pada Tabel 4 di bawah ini: Tabel 4 Probabilitas Perpindahan Konsumen
Da
Merek
Ke merek Tabel 4 Probabilitas Perpindahan Konsumen
Da
Merek
Ke merek
Ri
Aqua
Club
Nestle
Le Mineral
M
Aqua
0,4828
0,4483
0,0000
0,0690
er
Club
0,0000
0,7931
0,0000
1,1379
k
Nestle
0,0000
0,0000
0,4138
0,0000
Le mineral
0,0000
0,0000
0,0000
0,3448 Tabel 4 Probabilitas Perpindahan Konsumen 89 Jurnal SENOPATI
e-ISSN: 2714 - 7010
Vol.4 No.2, April 2023 Jurnal SENOPATI
Vol.4 No.2, April 2023 e-ISSN: 2714 - 7010 Selanjutnya akan dibentuk sebuah matriks probabilitas transisi awal untuk dapat bergerak dari satu
state ke state yang berikutnya. Pengolahan Data Nilai-nilai pada matriks probabilitas transisi awal diambil dari nilai-
nilai yang ada pada Tabel 4 proporsi perpindahan pelanggan dan matriks probabilitas transisi awal
adalah sebagai berikut: Tabel 5 Probabilitas Transisi Awal
T=
0,7368
0,6842
0,0000
0,1053
0,0000
0,4107
0,0000
0,5893
0,0000
0,0000
1,0000
0,000
0,0000
0
0,0000
1 Setelah berbentuk matriks probabilitas awal, kemudian di lakukan perhitungan matriks probabilitas
untuk periode-periode selanjutnya [9]. Berikut adalah penyajian penentuan matrik probabbilitas
transisi untuk periode pertama yang menunjukkan probabilitas loyalitas konsumen pada masing-
masing merek Air Mineral. Dalam Kemasan setelah bulan pertama : Dalam Kemasan setelah bulan pertama : Tabel 6 Probabilitas Transisi Periode Pertama
T23=
0,7368
0,6842
0,0000
0,1053
0,0000
0,4107
0,0000
0,5893
0,0000
0,0000
1,0000
0,0000
0,0000
0
0,0000
1
T=
0,7368
0,6842
0,0000
0,1053
0,0000
0,4107
0,0000
0,5893
0,0000
0,0000
1,0000
0,0000
0,0000
0
0,0000
1
T1=
0,54
0,79
0,00
0,59
0,00
0,17
0,00
0,83
0,00
0,00
1,00
0,00
0,00
0,00
0,00
1
Perhitungan seperti di atas dilakukan hingga nilai nilai pada matriks bersifat konstan. Nilai-nilai
matriks yang bersifat konstan dinamakan Steady State Probability [10]. Berdasarkan hasil
perhitungan, kondisi steady state didapatkan pada periode ke 23 berikut adalah matriks
probabilitassteady state. Perhitungan seperti di atas dilakukan hingga nilai nilai pada matriks bersifat konstan. Nilai-nilai
matriks yang bersifat konstan dinamakan Steady State Probability [10]. Berdasarkan hasil
perhitungan, kondisi steady state didapatkan pada periode ke 23 berikut adalah matriks
probabilitassteady state. Tabel 7 Hasil Perhitungan Steady State
T23=
0,00
0,00
0,00
2,997
0,00
0,00
0,00
1,00
0,00
0,00
1,00
0,00
0,00
0,00
0,00
1,00 Pada matriks diatas, warna hijau melambangkan besarnya peluang konsumen akan loyal pada suatu
merek Air Mineral dalam jangka waktu yang panjang. Kemudian dapat di sajikan sebuah table
steady state probability berdasarkan nilai terbesar hingga nilai terkecil. Analisa Absorbing Markov Chain Dengan memasukkan nilai 𝐍 dan 𝐈, maka Rata-rata waktu yang dibutuhkan untuk state A dan C berpindah ke state L dapat dihitung
dengan menggunakan persamaan (𝐈-𝐍)-1𝟏. Dengan memasukkan nilai 𝐍 dan 𝐈, maka Rata-rata waktu yang dibutuhkan untuk state A dan C berpindah ke state L dapat dihitung
dengan menggunakan persamaan (𝐈-𝐍)-1𝟏. Dengan memasukkan nilai 𝐍 dan 𝐈, maka I -N =
-
(I-N)-1=
=
(1-N)-11= I -N =
-
(I-N)-1=
=
(1-N)-11= I -N =
-
(I-N)-1=
=
(1-N)-11= Hasil ini menunjukkan bahwa rata-rata waktu yang dibutuhkan untuk state A berpindah ke
state L adalah sebesar 8 periode, dan rata-rata waktu yang dibutuhkan untuk state C berpindah ke
state L adalah sebesar 2 periode. Analisa Absorbing Markov Chain Analisa Absorbing Markov Chain 1. Bentuk standar dari absorbing markov chain 1. Bentuk standar dari absorbing markov chain Bentuk standar dari absorbing markov chain adalah sebagai berikut: Bentuk standar dari absorbing markov chain adalah sebagai berikut: 90 Jurnal SENOPATI
e-ISSN: 2714 - 7010
Vol.4 No.2, April 2023
Tabel 8 Bentuk Standar Absorbing
Non absorbing
Absorbing
P=
Absorbing
N
A
Non absorbing
0
1
Dengan menyusun matriks transisi sesuai dengan bentuk standar, maka:
Tabel 9 Bentuk Standar Absorbing
P=
A
C
N
L
A
0,7268
0,6842
0
0,11
C
0
0,4107
0
0,59
N
0
0
1
0
L
0
0
0
1
Dengan :
Tabel 9 analisa absorbing
N=
0,73
0,68
A=
0
0,11
O=
0
0
I=
1
0
0,00
0,41
0
0,59
0
0
0
1
Rata-rata waktu yang dibutuhkan untuk state A dan C berpindah ke state L dapat dihitung
dengan menggunakan persamaan (𝐈-𝐍)-1𝟏. Dengan memasukkan nilai 𝐍 dan 𝐈, maka Jurnal SENOPATI
e-ISSN: 2714 - 7010
Vol.4 No.2, April 2023
Tabel 8 Bentuk Standar Absorbing
Non absorbing
Absorbing
P=
Absorbing
N
A
Non absorbing
0
1
Dengan menyusun matriks transisi sesuai dengan bentuk standar, maka:
Tabel 9 Bentuk Standar Absorbing
P=
A
C
N
L
A
0,7268
0,6842
0
0,11
C
0
0,4107
0
0,59
N
0
0
1
0
L
0
0
0
1
Dengan :
Tabel 9 analisa absorbing
N=
0,73
0,68
A=
0
0,11
O=
0
0
I=
1
0
0,00
0,41
0
0,59
0
0
0
1
Rata-rata waktu yang dibutuhkan untuk state A dan C berpindah ke state L dapat dihitung
dengan menggunakan persamaan (𝐈-𝐍)-1𝟏. Dengan memasukkan nilai 𝐍 dan 𝐈, maka Jurnal SENOPATI
Vol.4 No.2, April 2023 e-ISSN: 2714 - 7010 Tabel 8 Bentuk Standar Absorbing
Non absorbing
Absorbing
P=
Absorbing
N
A
Non absorbing
0
1
Dengan menyusun matriks transisi sesuai dengan bentuk standar, maka: Dengan menyusun matriks transisi sesuai dengan bentuk standar, maka: Tabel 9 Bentuk Standar Absorbing
P=
A
C
N
L
A
0,7268
0,6842
0
0,11
C
0
0,4107
0
0,59
N
0
0
1
0
L
0
0
0
1
Dengan : Dengan : g
Tabel 9 analisa absorbing
N=
0,73
0,68
A=
0
0,11
O=
0
0
I=
1
0
0,00
0,41
0
0,59
0
0
0
1 Rata-rata waktu yang dibutuhkan untuk state A dan C berpindah ke state L dapat dihitung
dengan menggunakan persamaan (𝐈-𝐍)-1𝟏. Uji Reliabilitas Nilai suatu kuisioner dianggap reliabel apabila memberikan nilai α > 0.60. Dari hasil uji reliabilitas
dengan bantuan software SPSS diperoleh nilai α = 0.709> 0.60, yang artinya setiap strategi yang
digunakan pada kuisioner dinyatakan reliabel Nilai suatu kuisioner dianggap reliabel apabila memberikan nilai α > 0.60. Dari hasil uji reliabilitas
dengan bantuan software SPSS diperoleh nilai α = 0.709> 0.60, yang artinya setiap strategi yang
digunakan pada kuisioner dinyatakan reliabel Analisis Game Theory Analisis Game Theory di lakukan dengan cara pengumpulan data berupa penilaian responden
terhadap atribut Air Mineral. Berikut atribut yang di gunakan yang akan di jabarkan pada tabel Tabel 10 atribut dan kode
KODE
ATRIBUT
A1
Rasa
A2
Desain
A3
Harga
A4
Warna
A5
Promosi Probabilitas Absorpsi Probabilitas absorpsi dari state A dan C ke state L dapat dihitung dengan menggunakan persamaan
(𝐈 - 𝐍)-1𝐀. Dengan memasukkan nilai (𝐈 - 𝐍)-1 yang sudah dihitung sebelumnya dan 𝐀, maka: (1-N)-1A= Berdasarkan perhitungan di atas dapat di lihat bahwa merek Air Mineral yang mendapatkan nilai
Probabilitas Absorpsi yang paling tinggi adalah Le Mineral yang artinya bahwa dalam masa yang
akan dating konsumen Aqua dan Club mempunyai kemungkinan terserap ke Le Mineral. Berdasarkan perhitungan di atas dapat di lihat bahwa merek Air Mineral yang mendapatkan nilai
Probabilitas Absorpsi yang paling tinggi adalah Le Mineral yang artinya bahwa dalam masa yang
akan dating konsumen Aqua dan Club mempunyai kemungkinan terserap ke Le Mineral. Setelah mengetahui hasil dari Analisa Probabiltas Absorpsi, dapat di lihat bahwa dalam
masa yang akan datang konsumen dari produk Air Mineral Aqua dan Club akan terserap ke Le
Mineral dan Nestle artinya ada strategi yang harus di perbaiki di dalam Air Mineral Aqua dan
Club. Sehingga di perlukan strategi yang optimal untuk dapat membuat Aqua dan Club dapat
mengalahkan persaingan di masa mendatang terutama dengan Nestle dan Le Mineral. Adapun metode yang di gunakan yaitu Game Theory di karenakan Metode Game Theory
biasanya di gunakan sebagai salah satu cara mencari strategi yang paling optimal dalam 2 produk,
dengan mengetahui strategi yang optimal maka di harapkan Aqua khususnya bisa meningkatkan
strategi yang kurang optimal menjadi strategi yang lebih optimal, dengan tidak mengabaikan ciri 91 Jurnal SENOPATI
Vol.4 No.2, April 2023 Jurnal SENOPATI
Vol.4 No.2, April 2023 e-ISSN: 2714 - 7010 khas dari Aqua sendiri. Dengan di temukannya streategi yang optimal di harapkan bisa di terapkan
di perusahaan untuk mempertahankan perusahaan Aqua di masa yang akan datang. Analisis Game Theory Uji Validitas Uji validitas dalam kuisioner dengan n= 41, derajat kebebasan (df) = n -2 = 97 dan tingkat
signifikansi 5% maka diperoleh 𝒓𝒕𝒂𝒃𝒆𝒍=0,367. Kriteria pengambilan keputusan jika 𝒓𝒉𝒊𝒕𝒖𝒏𝒈
lebih besar dari 𝒓𝒕𝒂𝒃𝒆𝒍 maka butir pertanyaan dinyatakan valid. Adapun hasil validitas data
kuisioner secara lengkap diberikan pada Tabel. A. Uji validitas Perbandingan Aqua dan Nestle A. Uji validitas Perbandingan Aqua dan Nestle
Tabel 11 uji validitas perbandingan aqua dan nestle
No
Atribut
rhitung
rtabel
Keterangan
1. Rasa
0.5529
0.19
Valid
2. Desain
0.6073
0.19
Valid
3. Harga
0.7880
0.19
Valid
4. Warna
0.3535
0.19
Valid
5. Promosi
0.5931
0.19
Valid B. Uji Validitas perbandingan Aqua dan Le Mineral B. Uji Validitas perbandingan Aqua dan Le Mineral B. Uji Validitas perbandingan Aqua dan Le Mineral
Tabel 12 uji validitas perbandingan aqua dan Le Mineral
No
Atribut
rhitung
rtabel
1. Rasa
0,5697
0,19
2. Desain
0,5937
0,19
3. Harga
0,7986
0,19
4. Warna
0,3217
0,19
5. Promosi
0,5616
0,19 Pengolahan Data Game Theory Data pada penelitian ini diperoleh dari hasil kuisioner yang berisi tentang perbandingan setiap
atribut yang ada. Variabel yang digunakan oleh dibedakan menjadi variabel X untuk Aqua dan
variabel Y untuk Nestle B untuk aqua C untuk Le Mineral. Berikut diberikan atribut pada game
theory yang digunakan sebagai variabel pada Tabel 92 Jurnal SENOPATI
Vol.4 No.2, April 2023 e-ISSN: 2714 - 7010 Tabel 13 variabel Xaqua dan Ynestle Tabel 13 variabel Xaqua dan Ynestle
Atribut
pada
permainan
Variable yang di gunakan
Variable yang di gunakan
Aqua
Nestle
Aqua
Le mineral
Rasa
X1
Y1
B1
C1
Desain
X2
Y2
B2
C2
Harga
X3
Y3
B3
C3
Warna
X4
Y4
B4
C4
Promosi
X5
Y5
B5
C5 Dari atribut di atas dapat digunakan untuk membentuk nilai persaingan antara Aqua dan
Nestle, aqua dan le minerlal. Matriks nilai perolehan Aqua dan Nestle digunakan langkah pertama
untuk strategi murni. Bagi pemain baris menggunakan aturan miximin dan untuk pemain kolom
menggunakan aturan minimax. Tabel 14 Hasil Nilai Teori Permainan untuk Strategi Murni
Y1
Y2
Y3
Y4
Y5
Minimax
X1
-16
-24
23
-26
-9
-26
X2
-6
0
-24
-32
-20
-32
X3
-22
20
8
13
-32
-32
X4
-18
18
12
14
14
-18 Maximin
X5
-26
-22
-26
-17
22
-26
Maximun
-6 minimax
20
23
14
22 Dari matriks permainan air mineral Aqua dan Nestle pada tabel 14 di atas dapat dilihat bahwa nilai
maksimin tidak sama dengan nilai minimaks yang artinya titik pelana atau sadle point tidak dicapai
dan bukan merupakan strategi optimum, maka permainan tidak dapat diselesaikan dengan
menggunakan strategi murni. Dimana nilai minimaks nya adalah -6 dan nilai maksimin adalah -18. Langkah selanjutnya yang akan dilakukan adalah dengan menggunakan program linier dengan
metode simplek. untuk menjamin nilai permainan (v) bernilai positif, maka semua elemen matriks
pembayaran di tambahkan dengan suatu nilai dengan harga mutlak dari elemen yang terkecil. Untuk semua elemen matriks nilai pemain Air Mineral Aqua vs Nestle di tambahkan dengan k=32. Akan di jabarkan pada tabel di bawah ini. Tabel 15 Matriks perolehan modifikasi permainan Air Mineral Aqua dan Nestle
P11
Y1
Y2
Y3
Y4
Y5
P1
X1
16
8
55
6
23
X2
26
32
8
0
12
X3
10
52
40
45
0
X4
14
50
44
46
46
X5
6
10
6
15
54 Untuk Pemain Baris karena pemain baris adalah maximizing player, maka tujuannya adalah memaksimumkan V, atau
sama dengan meminimumkan Maka dapat dirumuskan kedalam bentuk program linier untuk
pemain baris sebagai berikut: meminimumkan Z = 93 Jurnal SENOPATI
e-ISSN: 2714 - 7010
Vol.4 No.2, April 2023
16x1 + 26x2 + 18x3 + 14x4 +6x5 ≥ 1
8x1 + 32x2 + 48x3 + 44x4 +6x5 ≥ 1
55x1 + 8x2 + 48x3 + 44x4 + 6x5 ≥ 1
6x1 + 0x2+ 53x3 + 46x4 + 15x5 ≥ 1
23x1 + 12x2 + 8x3 + 46x4 + 54x5 ≥ 1
X1,X2,X3,X4,X5 ≥ 0
Persoalan di atas kemudian di selesaikan dengan menggunakan program POM PQM5.0 dengan
tabel awal sebagai berikut : Jurnal SENOPATI
Vol.4 No.2, April 2023 e-ISSN: 2714 - 7010 Persoalan di atas kemudian di selesaikan dengan menggunakan program POM PQM5.0 dengan
tabel awal sebagai berikut : Persoalan di atas kemudian di selesaikan dengan menggunakan program POM PQM5.0 dengan
tabel awal sebagai berikut : Tabel 16 Matriks perolehan modifikasi permainan air mineral Aqua dan Nestle
Maximize
Y1
Y2
Y3
Y4
Y5
RHS
1
1
1
1
1
Constraint 1
16
8
55
6
23
>=
1
Constraint 2
26
32
8
0
12
>=
1
Constraint 3
10
52
40
45
0
>=
1
Constraint 4
14
50
44
46
46
>=
1
Constraint 5
6
10
6
15
54
>=
1
Setelah di operasikan pada program QM 5.0 maka di peroleh hasil optimal sebagai berikut : Setelah di operasikan pada program QM 5.0 maka di peroleh hasil optimal sebagai berikut : X1
X2
X3
X4
X5
1
1
1
1
1
Constraint 1
16
26
10
14
6
>=
1
0,0385
Constraint 2
8
32
52
50
10
>=
1
0
Constraint 3
55
8
40
44
6
>=
1
0
Constraint 4
6
0
45
46
15
>=
1
0,01
Constraint 5
23
12
0
46
54
>=
1
0
Solution
0
0,0268
0
0,0217
0
0,0485
minimize
RHS
Dual
Tabel 17 solusi optimal permainan air mineral Aqua dan Nestle Tabel 17 solusi optimal permainan air mineral Aqua dan Nestle Karena elemen-elemen matriks perolehan pada permainandi atas telah di tambahkan dengan K=32,
maka nilai pemainannya menjadi V = 21-32=-11. Di peroleh strategi optimal bagi Aqua, yaitu Strategi X2 (Desain) dan Strategi X4 (warna) dengan
besar permainan (value of games) sebesar -11. Untuk Pemain kolom (Air Mineral Nestle) Karena
pemain
baris
adalah
minimizing
player,
maka
tujuannya
adalah
meminimumkan V, atau sama dengan memaksimumkan . Maka dapat dirumuskan kedalam
bentuk program linier untuk pemain baris sebagai berikut: 94 Jurnal SENOPATI
Vol.4 No.2, April 2023 e-ISSN: 2714 - 7010 persoalan di atas kemudian di selesaikan dengan menggunakan program POM PQM5.0 dengan
tabel awal sebagai berikut : g
Tabel 18 matriks perolehan modifikasi permainan air mineral Aqua dan Nestle Y1
Y2
Y3
Y4
Y5
1
1
1
1
1
Constraint 1
16
8
55
6
23
>=
1
Constraint 2
26
32
8
0
12
>=
1
Constraint 3
10
52
40
45
0
>=
1
Constraint 4
14
50
44
46
46
>=
1
Constraint 5
6
10
6
15
54
>=
1
maximze
RHS
Setelah di operasikan pada program QM 5.0 maka di peroleh hasil optimal sebagai berikut : Setelah di operasikan pada program QM 5.0 maka di peroleh hasil optimal sebagai berikut : Setelah di operasikan pada program QM 5.0 maka di peroleh hasil optimal sebagai berikut : Tabel 19 solusi optimal permainan air mineral Aqua dan Nestle
Y1
Y2
Y3
Y4
Y5
1
1
1
1
1
Constraint 1
16
8
55
6
23
>=
1
0
Constraint 2
26
32
8
0
12
>=
1
0,0268
Constraint 3
10
52
40
45
0
>=
1
0
Constraint 4
14
50
44
46
46
>=
1
0,0217
Constraint 5
6
10
6
15
54
>=
1
0
Solution
0,0385
0
0
0,01
0
0,0485
maximze
RHS
Dual Tabel 19 solusi optimal permainan air mineral Aqua dan Nestle tabel 19 di atas di peroleh solusi optimal, yaitu: Karena elemen-elemen matriks perolehan pada permainandi atas telah di tambahkan dengan K=32,
maka nilai pemainannya menjadi V = 21-32=-11. Di peroleh strategi optimal bagi Nestle, yaitu Strategi Y2 (Desain) dan Strategi Y4 warna dengan
besar permainan (value of games) sebesar -11. Berdasarkan Dengan nilai perolehan pada tabel 4.19 95 Jurnal SENOPATI
Vol.4 No.2, April 2023 e-ISSN: 2714 - 7010 yaitu perbandingan Aqua vs Le Mineral dicoba langkah pertama dengan menggunakan strategi
murni. Bagi pemain baris akan menggunakan aturan maximin dan pemain kolom akan
menggunakan aturan minimax yang akan di jabarkan di tabel 20. KESIMPULAN Dari penelitian ini dapat diperoleh hasil kesimpulan, bahwa dari Analisis Strategi Persaingan Air
Mineral dengan Metode Markov Chain didapatkan bahwa merek Air Mineral yang mendapatkan
nilai Probabilitas Absorpsi yang paling tinggi adalah Le Mineral yang artinya bahwa dalam masa
yang akan datang konsumen Aqua dan Club mempunyai kemungkinan terserap ke Le Mineral. Hasil Analisis Strategi dengan Metode Game Theory untuk perbandingan Aqua vs Nestle Di
peroleh strategi optimal bagi Aqua, yaitu Strategi X2 (Desain) dan Strategi X4 (warna) dengan besar
permainan (value of games) sebesar -11 sedangkan bagi Nestle Di peroleh strategi optimal, yaitu
Strategi Y2 (Desain) dan Strategi Y4 (warna) dengan besar permainan (value of games) sebesar -11. Untuk perbandingan Aqua vs Le Mineral di peroleh strategi optimal yaitu 24 dengan menggunakan
streategi murni. Dari penelitian ini dapat diperoleh hasil kesimpulan, bahwa dari Analisis Strategi Persaingan Air
Mineral dengan Metode Markov Chain didapatkan bahwa merek Air Mineral yang mendapatkan
nilai Probabilitas Absorpsi yang paling tinggi adalah Le Mineral yang artinya bahwa dalam masa
yang akan datang konsumen Aqua dan Club mempunyai kemungkinan terserap ke Le Mineral. Hasil Analisis Strategi dengan Metode Game Theory untuk perbandingan Aqua vs Nestle Di
peroleh strategi optimal bagi Aqua, yaitu Strategi X2 (Desain) dan Strategi X4 (warna) dengan besar
permainan (value of games) sebesar -11 sedangkan bagi Nestle Di peroleh strategi optimal, yaitu Untuk Pemain kolom (Air Mineral Nestle) P11
P1
24
maximin
24
minimax
24
28
25
33
62
18
12
-12
12
4
14
1
24
-10
-14
-12
10
3
14
Y1
Y2
Y3
-32
-10
9
X5
11
-34
-34
-14
-32
-12
13
Y5
X1
X2
X3
X4
Y4
28
33
25
62
Tabel 20 matriks nilai perolehan Aqua dan Le Mieral
Tabel 20 di ketahui nilai minimaks sudah sama dengan nilai maksimin sehingga sadle point sudah
tercapai. Dengan menggunakan strategi murni di peroleh nilai keuntungan maksimal bagi Aqua dan
kerugian minimum bagi Le mineral adalah sebesar 24. Tabel 20 di ketahui nilai minimaks sudah sama dengan nilai maksimin sehingga sadle point sudah
tercapai. Dengan menggunakan strategi murni di peroleh nilai keuntungan maksimal bagi Aqua dan
kerugian minimum bagi Le mineral adalah sebesar 24. DAFTAR PUSTAKA [1]
F. F. Sugiarto, “Analisa Strategi Bersaing PT. Surya Cipta Mandiri,” Agora, vol. 4, no. 2,
pp. 250–258, 2016, [Online]. Available:
http://publication.petra.ac.id/index.php/manajemen-bisnis/issue/view/215 [2]
Y. Inda Pertiwi et al., “ANALISIS RANTAI MARKOV UNTUK MENGETAHUI
PELUANG PERPINDAHAN MEREK KARTU INTERNET GSM PRABAYAR (Studi
Kasus: Program Studi Matematika Fakultas Matematika dan Ilmu Pengetahuan Alam),” pp. 1–11, 2014. [3]
I. Y. Mayasari and R. Indriyani, “Analisis Strategi Bersaing pada PT.Citra Surya Pacific
Agora, vol. 4, no. 2, pp. 188–196, 2016. [4]
A. Boujnoui, A. Zaaloul, and A. Haqiq, “Mathematical model based on game theory and
Markov chains for analysing the transmission cost in SA-ZD mechanism,” Int. J. Comput. Inf. Syst. Ind. Manag. Appl., vol. 10, pp. 197–207, 2018. f
y
g
pp
pp
[5]
N. Salkind, “Random Sampling,” Encycl. Res. Des., pp. 42–57, 2012, doi:
10.4135/9781412961288.n364. [6]
Y. I. Pertiwi, “ANALISIS RANTAI MARKOV UNTUK MENGETAHUI PELUANG 96 Jurnal SENOPATI
Vol.4 No.2, April 2023 e-ISSN: 2714 - 7010 PERPINDAHAN MEREK KARTU INTERNET GSM PRABAYAR ( Studi Kasus :
Program Studi Matematika Fakultas Matematika dan Ilmu Pengetahuan Alam )
Yuli.inda14@gmail.com,” pp. 1–11, 2014. [7]
R. Widi, “Uji Validitas Dan Reliabilitas Dalam Penelitian Epidemiologi Kedokteran Gigi,”
Stomatognatic, vol. 8, no. 1, pp. 27–34, 2011. [8]
F. N. Masuku, Y. A. R. Langi, C. Mongi, and R. Markov, “Analisis Rantai Markov Untuk
Memprediksi Perpindahan Konsumen Maskapai Penerbangan Rute Manado-Jakarta
Analysis of Markov Chain To Predict Consumer Movement of Airline Route Manado-
Jakarta,” pp. 1–5, 2020. [9]
M. Gugutu, D. Hatidja, and Y. A. R. Langi, “Kecenderungan Penggunaan Merek Kartu
Seluler Pra Bayar GSM Menggunakan Analisis Biplot (Studi Kasus Mahasiswa Fakultas
Pertanian Unsrat Manado),” J. MIPA, vol. 2, no. 1, p. 23, 2013, doi:
10.35799/jm.2.1.2013.746. j
[10]
J. F. Andry, “Implementasi Penerapan Markov Chain Pada Database Marketing Studi Kasus
Pelanggan E-Commerce,” vol. 5, no. 1, pp. 94–108, 2015. 97 97
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https://openalex.org/W3108165450
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Biological effectiveness of organic and inorganic products for the management of Maconellicoccus hirsutus green in soursop crop
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Food Science and Technology
| 2,021
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cc-by
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1 Introduction semi-tropical regions of the world. M. hirsutus was first found
in Mexico in 1999, in the city of Mexicali, Baja California, later
in February 2004 in the municipality of Bahía de Banderas,
Nayarit, and Puerto Vallarta, Jalisco (Hernández et al., 2013). Soursop (Annona muricata L.) is the most important species
in the Annonaceae family. The main soursop producer in the
world is Mexico with a production of 29,228 t, distributed in
the states of Nayarit, Colima, Michoacán, Guerrero, Veracruz,
Puebla, Tabasco, Jalisco and Campeche, with an area of 3,693 ha
(Servicio de Información Agroalimentaria y Pesquera, 2018). M. hirsutus is a pest of economic importance not only for
the damage it causes to the plant, but also for being a regulated
pest. In Mexico, there are regulations at internal verification
points for the mobilization of host products of M. hirsutus
(Hernández et al., 2013). However, the main form of dispersion
is through the mobilization of infested plant material (México,
2014). The global economic losses due to M. hirsutus in the
Caribbean region during the period 1995-2008 were approximately
USD $ 18.3 million. For the Caribbean subregion, losses were
approximately 138 million dollars (Rivero, 2007). With respect
to the soursop area cultivated in Mexico, there was a sustained
increase until 2006 (two years after reporting the presence of
M. hirsutus in Nayarit, subsequently, there was a 26% reduction
in production until 2009, this decrease was mainly associated
presence and effects of M. hirsutus (Servicio de Información
Agroalimentaria y Pesquera, 2010). Recent advances has been made in the study of the soursop
pulp which in research has been made by Amariz et al. (2018) in
the use of soursop fibrous pulp as a byproducts which has been
proven to have chemical properties and bioactive components that
can potentially be use in food industry. Studies, by Nascimento et al. (2020) revealed that the physicochemical properties of frozen
soursop pulps has shown to be a good source of essential elements
to human, but was low in manganese and zinc. Findings by
Guimarães et al. (2019) revealed that the use of HIUS technology
induced both negative (degradation of the ascorbic acid, decrease
of some minerals and production of many volatile compounds)
and beneficial (an increase of phenolic content, improvement
of the antioxidant and anti-hypertensive activity, reduction of
undesired minerals) changes in the nutritional profile of the
soursop whey beverage. Abstract The pink mealybug Maconellicoccus hirsutus Green is a pest of economic importance in various crops around the world. The objective was to determine the biological effectiveness of organic and inorganic products for the management of M. hirsutus in
the soursop crop. The experiment was carried out in the communities of “Lima de Abajo” and “El Capomo”, in the municipality of
Compostela, Nayarit, Mexico, during the period from September to November 2018 and from July to August 2019. The treatments
were Ara3, Inter-a-ch-c, Evolution, a complex of plant extracts (Capsicum chinense + Allium sativum + Allium cepa + Artemisia
absinthium), Mos Blanc® (Imidacloprid), farmers’ control (Detergent Blanca Nieves® + Sodium Hypochlorite Cloralex®) and the
absolute control (Water). The nymphs and adult females of M. hirsutus were counted after each application, in young shoots. The results showed that in “Lima de Abajo”, Ara 3 registered the highest percentage of suppression after 28 days. In “El Capomo”,
Imidacloprid recorded the greatest suppression after 28 days of application. In both communities, the treatments Ara 3, Evolution
and Imidacloprid registered the least number of living individuals. The results indicated that Ara 3 and Evolution were the most
promising alternatives in the management against M. hirsutus in the crop. Keywords: Nayarit; suppression; Ara 3; evolution; pink mealybug. Practical Application: Management of mealybug population using organic and inorganic produc Received 20 Jan., 2020
Accepted 23 June, 2020
1Posgrado en Ciencias Biológico Agropecuarias, Universidad Autónoma de Nayarit, Xalisco, Nayarit, Carrertera Tepic-Compostela, México.
*Corresponding author: therolaestwick@gmail.com ISSN 0101-2061 (Print)
ISSN 1678-457X (Online) ISSN 0101-2061 (Print)
ISSN 1678-457X (Online) Food Science and Technology OI:
D
https://doi.org/10.1590/fst.01820 Biological effectiveness of organic and inorganic products for the management of
Maconellicoccus hirsutus green in soursop crop Therola CALIFA-ESTWICK1*
, Agustín ROBLES-BERMÚDEZ1, Octavio Jhonathan-Cambero CAMPOS1,
Gregorio LUNA-ESQUIVEL1, Néstor ISIORDIA-AQUINO1 Therola CALIFA-ESTWICK1*
, Agustín ROBLES-BERMÚDEZ1, Octavio Jhonathan-Cambero CAMPOS1,
Gregorio LUNA-ESQUIVEL1, Néstor ISIORDIA-AQUINO1 Accepted 23 June, 2020
1Posgrado en Ciencias Biológico Agropecuarias, Universidad Autónoma de Nayarit, Xalisco, Nayarit, Carrertera Tepic-Compostela, México.
*Corresponding author: therolaestwick@gmail.com 1 Introduction However this crop has been affected
by various phytosanitary problems, of which Maconellicoccus
hirsutus Green is one of the main pests (Hernández et al., 2013;
Cham et al., 2019). M. hirsutus is an exotic pest native to Southeast
Asia whose distribution is cosmopolitan from the tropical and The pink mealybug affects all the phenological stages of the
crop. The presence of this pest favours the development of sooty
mould; due to its complex relationship and interdependence
with other pests such as fruit flies and the symbiotic relationship 343 Food Sci. Technol, Campinas, 41(2): 343-348, Apr.-June 2021 Organic inorganic management Maconellicoccus hirsutus green that this pest establishes with ants to protect itself from their
predators (Hernández et al., 2013). During the maturation of
the female mealybug, it produces a waxy layer on the surface,
This waxy layer is one of the factors that reduces the action of
the chemical products, for this reason it is necessary to apply
chemical products in certain instars where waxy layer is not
present (Rivero, 2007; Kondo et al., 2010). The cultural control is also another method used which
consists of the pruning or elimination of hosts that are positive
for M. hirsutus in the areas under phytosanitary control. This includes the elimination of weed residues, cleaning of
ridges, water distribution channels and affected agricultural
production areas (Servicio Nacional de Sanidad, Inocuidad y
Calidad Agroalimentaria, 2019). It is also convenient to apply a
soap or oil solution before pruning foliage or fruits, in order to
immobilize the immature stages and thus minimize the dispersion
of M. hirsutus (Servicio Nacional de Sanidad, Inocuidad y Calidad
Agroalimentaria, 2019). Therefore, the search for less harmful
chemical alternatives in pest control is also a viable strategy,
for this reason, the objective of this research was to determine
the biological effectiveness of different organic and inorganic
products for the management of M. hirsutus with the possibility
of it being included as a sustainable pest management strategy
of soursop in Nayarit, Mexico. Another factor that constitutes a limitation in the production
of fruit trees in the world is the ecological imbalance caused
by improper practices of insect pest management. Annually,
2.5 million tons of pesticides are applied worldwide. Of the total
pesticides applied, 20 to 30% are insecticides (Orozco et al., 1998). 2 Materials and methods Two experiments were carried out on three year old soursop
orchards in a vegetative stage, with trees 5 to 8 m high in the
municipality of Compostela, Nayarit; The first one located in
the community of Lima de Abajo located at 21 ° 06`02 ”N and
105 ° 12`45” W during September to November 2018, and the
second in the community of “El Capomo” located at 21 ° 21`09 ”N
and 104 ° 05`57” W during July to August 2019.The samples
obtained from “Lima de Abajo” were hermetically transferred to
the Agricultural Parasitology Laboratory of the Multidisciplinary
Center for Scientific Research (CEMIC 03) of the Autonomous
University of Nayarit. In the community of ‘El Capomo’, data
collection was carried out directly in the field. Insect specimens
were preserved in 70% alcohol for identification with the help of
stereoscopic and compound microscopes, both of the VELAB®
and Labomed® brand, as well as Miller’s taxonomic keys (1999). Confirmation of the species was carried out by specialists
from the Entomology and Phytopathology Laboratory of the
University of Nuevo León (UANL).The products evaluated
were INTER-A-CH-C (Allium sativum, Capsicum frutescens,
Cinnamomum zyelanicum) (International Química de Copper
SA de CV), Ara3 (Ricinus communis, Azadirachta indica,
Argemone mexicana) (Proan, Mexico), Evolution (Allium sativum,
Quillaja saponaria) (Terra Nova, Mexico), detergent (Blanca
Nieves®) (linear anionic surfactant, silicate, carbonate, sulfate and
tripolyphosphate) + Cloralex® (sodium hypochlorite) (Alen de
México), imidacloprid (Sifatec, Mexico), and a combination of
Habanero Chile (Capsicum chinense) + garlic (Allium sativum)
+ onion (Allium cepa) + wormwood (Artemisia absinthium). Extract formulation for ‘Lima de Abajo’: 100 g of C. chinense,
100 g of A. cepa, 100 g of A. sativum, 150 g of leaves and flowers
of A. absinthium, 2 liters of water, 20 g of detergent (Blanca
nieves®), dissolved in 10 liters of water.Formula adjusted for
‘El Capomo’: 200 g of C. chinense, 200 g of A. cepa, 200 g of
A. sativum, 100 g of leaves and flowers of A. absinthium, 2 liters
of water, 20 g of detergent (Blanca nieves®), dissolved in 10 liters
of water.Chile habanero, wormwood, garlic and onion were
poured into a blender (Osterizer®) to obtain only the liquid,
the pulp was subsequently discarded. The detergent treatment
(Blanca Nieves®) + chlorine (Cloralex®) included in this research In various crops, up to six applications per year are made
without effective control (Hernández et al., 2008). 1 Introduction The greatest amount of pesticides applied in crops of high
economic value such as fruit trees, only an insignificant amount
of the chemical (less than 0.1%) manages to reach the target pest
(Pimentel & Levitan, 1986). This means that more than 99.9% of
the pesticide applied is lost in the environment, where it can cause
adverse effects to beneficial fauna, soil, water and atmosphere
pollution (Pimentel et al., 1993). Despite the indiscriminate use
of pesticides, effective control over this pest is not achieved or
because the insecticide applications were neither timely nor
adequate (Pimentel et al., 1993).The management strategies of
M. hirsutus consist mainly of chemical, biological and cultural
control. The most commonly used method to combat pest
insects, in Mexico is chemical control; however, there are no
authorized pesticides for use in soursop (Comisión Federal para
la Protección contra Riesgos Sanitarios, 2018). The chemical
control of M. hirsutus is based on the use of products such as
Horta 25 (diazinon); Dimetri 400 (dimethoate) and Siroco
(cypermethrin), Cyrux 20%, paraffinic oil, citroline, with
detergent to reduce the damages attributed to fruits infested by
M. hirsutus when populations are at low levels (Kondo et al., 2010;
Hernández et al., 2013). 3.1 Evaluation of organic and inorganic products 2018 The effectiveness of the seven treatments was evaluated against
the mealybug species morphologically identified as M. hirsutus. The mealybug host used in this study was the soursop orchard
in open field conditions. According to the results (Table 2), an
initial population was registered in the experimental area, which
ranged between (11.4) and (15.25) M. hirsutus per young shoots,
without presenting statistical differences prior to the application
of the treatments. At 7 and 14 days after application, statistical
differences were observed between treatments. The variables evaluated corresponded to the average
mealybugs in each treatment before and after the application:
The average population of M. hirsutus present was determined by
the quantification of nymphs and adult females, where four young
shoots of five cm in length per tree were collected. The formula
used to calculate the percentage of effectiveness after the application
of the treatments was that described by Henderson & Tilton
(1955): % of effectiveness = 100 × (1 - (Ca * T d) / (Cd * T a),
where: Td = number of M. hirsutus after application Ta = number
of M. hirsutus before application Cd = number of M. hirsutus
in the control after application Ca = number of M. hirsutus in
the control before application. The design of the experiment
was completely randomized with five repetitions, where each
repetition was a tree and from each tree four terminal (young)
shoots of five cm in length was evaluated. The data were analyzed
by means of the analysis of variance with the general linear
model to determine the differences between the treatments and
later, the comparison of the mean by means of the Tukey test
(P < 0.05). All statistical analyzes were performed with Statistix
version 10 software. The lowest population of M. hirsutus was recorded in the
farmers control with a value of (5.05) and (0.25) M. hirsutus
per young shoots, which was significantly lower than other
treatments. The maximum population of M. hirsutus was
observed in T4 (Chile Habanero + Wormwood + Onion + Garlic)
(13.25) and (10.05) at 7 and 14 days respectively. At 21 days
after application, significant differences were observed between
treatments, the lowest population of M. hirsutus was recorded
in Ara 3 (2.45) and the highest population was observed in the
absolute control (10.05). 2 Materials and methods As a result
of the intensive use of insecticides, the number of them capable
of exercising satisfactory control is reduced every day, mainly
due to the development of resistance (Riley & King, 1994;
Servín et al., 2007). An important part of chemical management
is insecticide resistance management (Food and Agriculture
Organization, 2012). However, various producers in Mexico
use pesticides in the hope of reducing the impact of pests on
their crops; information is lacking as to whether or not these
chemicals are actually efficient, and the negative impact it has
on nature (Hernández et al., 2008). The use of pesticides for the control of agricultural pests, is
not the only option to achieve success in production, there are
alternatives that do not affect the balance of populations, nor
induce the development of resistance neither displace species. Biological control is one such method. In Mexico, an integrated
pest management program was created with emphasis on biological
control with the release of the parasitoid Anagyrus kamali
Moursi; a total of 7,024,000 individuals were released in 2011,
generating a 98% reduction of the mealybug population at that
period (Servicio Nacional de Sanidad, Inocuidad y Calidad
Agroalimentaria, 2014). 344 Food Sci. Technol, Campinas, 41(2): 343-348, Apr.-June 2021 Califa-Estwick et al. 3 Results and discussion as a famers control, is a product commonly used by the farmer. The mortality in the treatment water was used as a control for
this calculation. The treatments evaluated were the products
or mixtures of products described in (Table 1). Which were
previously mixed in a volume of three liters of water, which was
later applied between 8 and 9 in the morning evenly covering
foliage and fruits. Food Sci. Technol, Campinas, 41(2): 343-348, Apr.-June 2021
345/348 345 TRT-Treatment; BA-Before application; DAA-Days After application. The means separated by the same letter between columns mean that there is no significant difference between
treatments according to the Tukey test at (P > 0.05). 3.1 Evaluation of organic and inorganic products 2018 Percentage of effectiveness after the application of products for the control of M. hirsutus were between 6.14 and 99.39% in the
community of “Lima de Abajo”. Figure 1. Percentage of effectiveness after the application of products for the control of M. hirsutus were between 6.14 and 99.39% in the
community of “Lima de Abajo”. after application against P. marginatus in papaya and differ
from the results by Samman et al. (2016), who reported that
Imidacloprid obtained (84.21%) mortality against M. hirsutus
72 hours after application. application was reduced with a significant difference between
treatment 1 and 2. The lowest average population was recorded in Ara 3, with
0.15 mealybug per young shoot. The products in decreasing order
of effectiveness were T1> T7> T6> T3> T4> T5> T2. At 14, 21
and 28 days no statistical differences were observed between
treatments (Table 3) although a decrease in M. hirsutus was
observed during the three weeks. The highest mean reduction
of M. hirsutus at 14, 21 and 28 days was Ara 3 (0.15), Evolution
(0.2) and Imidacloprid (0.05) respectively. Based on the results,
Ara 3 and Evolution can be considered one of the best options
as an alternative to insecticides. The data taken after 14 days of application showed that the
farmers control caused the greatest reduction (97.67%) followed
by Ara3, INTER-A-CH-C, Evolution, Imidacloprid 60.34, 49.19,
48.87 and 47.73% respectively however the combination treatment
(Chile Habanero + Garlic + onion + wormwood) had no effect
on the population of M. hirsutus. Castillo et al. (2018), reported
that Imidacloprid was the most effective treatment with a control
of (33%) against whitefly in melon cultivation. Regarding the overall efficacy of the 21-day treatments,
the data revealed that Imidacloprid, Ara 3, farmers control
and INTER-A-CH-C maintain the highest reduction 78.03,
75.38, 68.96 and 65.44% respectively, followed by Evolution and
(Habanero Chile + garlic + onion + wormwood) with (19.62%)
and (1.6%). Similar results by El-Zahi et al. (2016) indicated
that imidacloprid effectively controlled the cotton mealybug,
registering an average decrease of (89.2%), in populations
compared to the untreated control. Figure 2 Reveals that all products in this trial, except the
absolute control, showed a level of effectiveness between 7.6
and 97.88%. Ara 3, Imidaclorpid and Evolution showed the
most control compared to INTER-A.CH-C, which was least
effective during the four weeks of applications. 3.1 Evaluation of organic and inorganic products 2018 At 28 days after application, the highest average was obtained
in T5 without significant differences with T2 and with significant
differences with respect to the other treatments. The information
suggests that at 21 and 28 days after the application Ara 3 was
significantly superior in the suppression of the population of
M. hirsutus in the soursop orchard. Table 1. Products and doses used for the management of M. hirsutus. Trat
Products
Dose
T1
Ara 3
10 mL/L
T2
INTER-A-CH-C
10 mL/L
T3
Evolution
15 mL/L
T4
Chile habanero-Onion-Wormwood-Garlic
100 mL/L
T5
Absolute control (water)
3L
T6
Farmers control (Blanca Nieves®) +Cloralex®
2.5 mL/L
T7
Imidacloprid
2.5 mL/L Table 1. Products and doses used for the management of M. hirsutus. The results in Figure 1, indicated that the absolute control
obtained an efficiency of 0% compared to the other treatments. Data after 7 days of application, revealed that the farmers control
significantly reduced the amount of M. hirsutus (56.24%) followed
by INTER-A-CH-C (26.18%), Ara3 (14.14%), Evolution (14.04%). The lowest reduction was caused by Imidacloprid (6.14%)
followed by (Chile habanero + Garlic + onion + wormwood)
that had no effect on the population of M. hirsutus. These results
differ from those found by Gowda et al. (2013), who revealed
that Imidacloprid recorded a mortality of (65.76%), 7 days Table 2. Effectiveness of products before and after the application in the “Lima de Abajo” community. TRT
Products
Average mealybug population/treatment
BA
DAA
T0
T+7
T+14
T+21
T+28
T1
Ara3
11.4 a
9ab
3.85cd
2.45b
0.05b
T2
INTER-A-CH-C
15.25 a
10.35 a
6.6abc
4.6ab
3.8ab
T3
Evolution
12.4 a
9.8ab
5.4bcd
8.7ab
0.45b
T4
Chile habanero-Onion-Wormwood-Garlic
12.35 a
13.25 a
11 a
10.6a
1.4b
T5
Absolute control (water)
11.8 a
10.85 a
10.05ab
10.3a
8.5 a
T6
Farmers control (Blanca Nieves® mas Cloralex®)
12.55 a
5.05b
0.25d
3.4b
0.7b
T7
Mos blanc (Imidacloprid)
14.6 a
12.6 a
6.5abc
2.8b
0.55b
TRT-Treatment; BA-Before application; DAA-Days After application. The means separated by the same letter between columns mean that there is no significant difference between
treatments according to the Tukey test at (P > 0.05). Table 2. Effectiveness of products before and after the application in the “Lima de Abajo” community. of products before and after the application in the “Lima de Abajo” community. Average mealybug population/treatment Food Sci. Technol, Campinas, 41(2): 343-348, Apr.-June 2021 345 Organic inorganic management Maconellicoccus hirsutus green Figure 1. 3.1 Evaluation of organic and inorganic products 2018 The increase in
effectiveness for (Chile habanero + garlic + onion + wormwood)
can be attributed to the increase in the extract concentration used
in this trial. With the adjusted dosage of garlic, Chile habanero,
wormwood and onion, you can reach a level of effectiveness of
M. hirsutus similar to other products in this trial. It can be seen that all the products used showed an efficiency
greater than 65% at 28 days of application, the products that
obtained the most efficiency were Ara 3 (99.39%), Evolution
(94.96%), Imidacloprid (94.77%), regional control (92.26%),
(Habanero Chile + Garlic + Onion + Wormwood) (84.26%)
and INTER-A-CH-C (65.4%). The treatments with active ingredients based on Allium
sativum, Capsicum chinense and Capsicum frutescens work as a
repellent and inhibit insect ingestion, in the same way Vindas
(2011) explained that Chile extract, rich in capsicin, affects the
digestion system of insects. In this regard Vásquez (2000) mentions
that the garlic smell changes the natural smell that each plant
produces, which deceives insects because it causes an alteration
and confusion due to the thiosulfates that garlic contains. Food Sci. Technol, Campinas, 41(2): 343-348, Apr.-June 2021 3.2 Evaluation of organic and inorganic products 2019 Table 3 shows the results of the average population of
M.hirsutus. The initial mean population was 4.8 to 8.25 mealybug
per young shoot / treatment, which showed no significant
differences. The average population of M. hirsutus at 7 days after It was discovered that Ara 3 is the most effective treatment
after day 7 of application with maximum suppression (96.23%)
followed by Imidacloprid, Evolution, regional control, (Chile Food Sci. Technol, Campinas, 41(2): 343-348, Apr.-June 2021 346 Califa-Estwick et al. Table 3. Effectiveness of products before and after application, in the community of “El Capomo”. Table 3. Effectiveness of products before and after application, in the community of “El Capomo”. TRT
Products
Average mealybug population/treatment
BA
DAA
T0
T+7
T+14
T+21
T+28
T1
Ara3
4.8 a
0.15b
0.15 a
0.4 a
0.1 a
T2
INTER-A-CH-C
5.8 a
4.45 a
2.8 a
2.25 a
0.65a
T3
Evolution
8.25 a
1.8ab
2 a
0.2 a
0.15a
T4
Chile habanero-Onion-Wormwood-Garlic
6.5 a
2.15ab
1.8 a
0.21 a
0.65a
T5
Absolute control (water)
3.55 a
2.95ab
3.1 a
2.3 a
1.3a
T6
Farmers control (Blanca Nieves® mas Cloralex®)
5.7 a
1.7ab
2.1 a
0.8 a
1.15a
T7
Mos blanc (Imidacloprid)
6.45 a
1.1ab
0.85 a
1 a
0.05a
TRT-Treatment; BA-Before application; DAA-Days After application. The means separated by the same letter between columns mean that there is no significant difference between
treatments according to the Tukey test at (P > 0.05). Average mealybug population/treatment TRT-Treatment; BA-Before application; DAA-Days After application. The means separated by the same letter between columns mean that there is no significant difference between
treatments according to the Tukey test at (P > 0.05). Figure 2. Percentage of effectiveness after the application of products for the control of M. hirsutus were between 7.6 and 97.88 % in the community
of “El Capomo”. Figure 2. Percentage of effectiveness after the application of products for the control of M. hirsutus were between 7.6 and 97.88 % in the community
of “El Capomo” iveness after the application of products for the control of M. hirsutus were between 7.6 and 97.88 % in the commun Figure 2. Percentage of effectiveness after the application of products for the control of M. hirsutus were between 7.6 and 97.88 % in the community
of “El Capomo”. those recorded by Romero (2017) revealed that garlic and onion
extract showed 96.45% efficacy in Rambután against mealybug. 3.2 Evaluation of organic and inorganic products 2019 habanero + Garlic + onion + wormwood) and INTER-A-CH-C
with 79.47, 73.74, 64, 60.19 and 7.6%, respectively. However, Domínguez (2013) mentioned that the combination
of garlic, Chile habanero, onion and aloe as an organic extract
obtained control of up to 90% against M. hirsutus in tulip plants
with the help of cultural control (pruning). After day 14, the
results showed a mortality rate of 96.42, 84.91, 72.24, 68.29,
57.81 and 44.72% for Ara 3, Imidacloprid, Evolution, (Chile
habanero + garlic + onion + wormwood), farmers control and
INTER-A-CH- C respectively. In this trial, it can be seen that all the products used have
an efficacy of more than 44.9% after 28 days of application,
Imidacloprid (97.88%), Evolution (95.04%) and Ara 3 (94.31%)
showed an efficacy greater than 94% compared to (Chile habanero
+ garlic + onion + wormwood) (72.69%), INTER-A-CH-C
(69.39%) and farmer control (44.9%), which was less than 72%. Food Sci. Technol, Campinas, 41(2): 343-348, Apr.-June 2021 References Amariz, A., Lima, M. A. C., & Alves, R. E. (2018). Quality and antioxidant
potential of byproducts from refining of fruit pulp. Food Science
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O. A. (2018). Evaluación de tres extractos naturales contra Bemisia
tabaci en el cultivo del melón, puntarenas, Costa Rica. Agronomía
Costarricense, 42(2), 93-106. http://dx.doi.org/10.15517/rac.v42i2.33781. Orozco, S. M., López, P. J., Farías, L. J., & Arenas, V. M. (1998). Sustentabilidad en el manejo de plagas y enfermedades de cultivos
agrícolas. Revista Mexicana de Fitopatología, 16(1), 46-54. Cham, A. K., Luna-Esquivel, G., Robles-Bermúdez, A., Ríos-Velasco, C.,
Coronado-Blanco, J. M., & Cambero-Campos, O. J. (2019). Insects
associated with the soursop (Annona muricata L.) Crop in Nayarit,
Mexico. The Florida Entomologist, 102(2), 359-365. http://dx.doi. org/10.1653/024.102.0211. Pérez, S. J., Ángel-Ríos, D. M., Arteaga, D. A., Hernández, C. E., & Damián,
N. A. (2013). Hongos entomopatogenos y extractos vegetales contra
escama blanca (Aulacaspis tubercularis NEWSTEAD), en cultivo de
mango en San Luis la Loma, municipio de Tecpan de Galeana, gro. México. Entomología Mexicana, 12(1), 452-455. Retrieved from
http://www.entomologia.socmexent.org/revista/2013/CB/452-455.pdf Comisión Federal para la Protección contra Riesgos Sanitarios –
COFEPRIS. (2018). Consulta de Registros Sanitarios de Plaguicidas,
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amounts reaching pests. BioScience, 36(2), 86-91. Pimentel, D., Mc Laughlin, L., Zepp, A., Lakitan, B., Kraus, T., Kleinman,
P., Vancini, F., Roach, W. J., Graap, E., Keeton, W. S., & Selig, G. (1993). Environment and economic effects of reducing pesticide
use in agriculture. Agriculture, Ecosystems & Environment, 46(1-4),
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(Maconellicoccus hirsutus Green) en tulipán (Hibiscus) mediante
un extracto orgánico (pp. 33-43). México, Quintana Roo: Instituto
tecnológico de la zona maya. El-Zahi, S., Aref, S. A. E.-S., & Korish, S. K. M. (2016). The cotton mealybug,
Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae) as a
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Plant Protection Research, 56(2), 113-115. http://dx.doi.org/10.1515/
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hirsutus (GREEN), Un peligro potencial para la agricultura cubana. Rev. Protección, 22(3), 167-175. Food and Agriculture Organization – FAO. (2012). References Código internacional
de conducta para la distribución y utilización de plaguicidas. Directrices
sobre la prevención y manejo de la resistencia a los plaguicidas. Rome:
FAO. Retrieved from http://www.fao.org/fileadmin/templates/agphome/
documents/Pests_Pesticides/Code/Annotated_Guidelines_SP.pdf Romero, P. M. J. (2017). Efecto de productos químicos y orgánicos para el
control de cochinilla harinosa (Dysmicoccus brevipes, Pseudococcidae)
en rambután, nuevo progreso, San Marcos (pp. 29-30). Guatemala:
Universidad San Rafael Landivar. Gowda, G. B., Kumar, L. V., Jagadish, K. S., Kandakoor, S. B., & Rani,
A. T. (2013). Efficacy of insecticides against papaya mealybug,
Paracoccus marginatus Williams and Granara de Willink (Hemiptera:
Pseudococcidae). Current Biotica, 7(3), 161-173. Samman, F., Mubashar, H., Muhammad, F. M., Nadia, N., Noor, A., & Zaheer,
A. (2016). Field efficacy of some insecticides against hibiscus
mealybug, Maconellicoccus hirsutus (Hemiptera: Pseudococcidae). Journal of Entomology and Zoology Studies, 4(1), 240-244. Guimarães, J. T., Silva, E. K., Ranadheera, C. S., Moraes, J., Raices, R. S. L., Silva, M. C., Ferreira, M. S., Freitas, M. Q., Meireles, M. A. A., & Cruz, A. G. (2019). Effect of high-intensity ultrasound on the
nutritional profile and volatile compounds of a prebiotic soursop
whey beverage. Ultrasonics Sonochemistry, 55, 157-164. http://dx.doi. org/10.1016/j.ultsonch.2019.02.025. PMid:30853535. Servicio de Información Agroalimentaria y Pesquera – SIAP. (2010). Anuario Estadístico de la Producción Agrícola. Retrieved from http://
www.siap.gob.mx/cierre-de-la-produccion-agricola-porestado/ Servicio de Información Agroalimentaria y Pesquera – SIAP. (2018). Anuario Estadístico de la Producción Agrícola. Retrieved from http://
www.siap.gob.mx/cierre-de-la-produccion-agricola-porestado/ Henderson, C. F., & Tilton, E. W. (1955). Test with acaricides against
the brow wheat mite. Journal of Economic Entomology, 48(2), 157-
161. http://dx.doi.org/10.1093/jee/48.2.157. Servicio Nacional de Sanidad, Inocuidad y Calidad Agroalimentaria –
SENASICA. (2014). Laboratorio regional de producción de agentes
de control biológico. México: SENASICA. Retrieved from http://
www.senasica.gob.mx/ Hernández, F. L. M., Bautista, M. M., Carrillo, S. J. L., Sánchez, A. H.,
Urías, L. M. A., & Salas, A. M. D. (2008). Control del barrenador
de las semillas, Bephratelloides cubensis Ashmead (Hymenoptera:
Eurytomidae) en guanábana Annona muricata L. (Annonales:
Annonaceae). Acta Zoológica Mexicana, 24(1), 199-206. http://
dx.doi.org/10.21829/azm.2008.241631. Servicio Nacional de Sanidad, Inocuidad y Calidad Agroalimentaria –
SENASICA. (2019). Cochinilla rosada del hibisco (Maconellicoccus
hirsutus) Green, 1908 (Ficha técnica, No. 6, pp. 22). México: SENASICA. Servín, R., García, H. J. L., & Troyo, D. E. (2007). Buenas prácticas en el
manejo plagas para una agricultura, ganadería y producción forrajera
sostenible en zonas áridas (pp. 85). La Paz, México: Editorial Centro
de Investigaciones Biológicas del Noroeste. Hernández, F. L. M., Gómez, J. Organic inorganic management Maconellicoccus hirsutus green Organic inorganic management Maconellicoccus hirsutus green Nascimento, M. M., Jesus, R. M., Santos, H. M., Silva, A. L. S. Jr.,
Campos, N. M. C. O., Silva, E. G. P., & Lôbo, I. P. (2020). Quality
pattern evaluation of frozen soursop pulps: an assessment based on
chemical composition and chemometric analysis. Food Science and
Technology (Campinas), 40(2), 508-516. http://dx.doi.org/10.1590/
fst.04919. 4 Conclusions The results obtained by Pérez et al. (2013) revealed that
Ricinus communis extracts resulted in 63% mortality of A. tuberculars and 41.5% after the first application in mango. After
21 days, the results revealed that Evolution (96.26%) and (Chile
habanero + Garlic + onion + wormwood) (95.25%) had their
highest reduction peak compared to Ara 3, regional control,
Imidacloprid and INTER-A- CH-C with efficiency of 87.14, 78.34,
76.07 and 40.12% respectively. These results are consistent with As an ecological alternative, Ara 3 (R. communis, A. indica,
A. mexicana) and Evolution (Allium sativum, Quillaja saponaria)
were the most effective products to suppress the incidence of
M. hirsutus on soursop trees in both location. In the communities of “Lima de Abajo” and “El Capomo” Ara
3, Evolution and Imidaclorpid recorded the highest percentage of
suppression and showed the lowest number of living individuals
of M. hirsutus, 28 days after application. 347 Food Sci. Technol, Campinas, 41(2): 343-348, Apr.-June 2021 References R., & Andrés, A. J. (2013). Importancia,
plagas insectiles y enfermedades fungosas del cultivo de guanábano
(pp. 1-33). Nayarit, México: Campo Experimental Santiago Ixcuintla. Kondo, D. T., López-Bermudez, R., & Quintero, E. M. (2010). Manejo
integrado de insectos escama (Hemiptera: Coccoidea) con énfasis
en control biológico. Novedades Técnicas, 7-14. Vásquez, A. O. L. (2000). Manejo de cochinilla (Dysmicoccus brevipes)
en el cultivo de piña orgánica en la zona del Lago de Yojoa, Honduras. México. Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y
Alimentación — SAGARPA. (2014). Manual Técnico-Operativo del
manejo de la Cochinilla Rosada del Hibisco Maconellicoccus hirsutus
(Green) (pp. 36). México: SAGARPA. Vindas, A. M. (2011). Evaluación de productos naturales para el control
de la cochinilla harinosa, (Dysmicoccus brevipes Cockerell) (Hemiptera:
Pseudococcidae), en el fruto de pina, San Carlos (Tesis de maestría). Universidad de Costa Rica, San Carlos, Costa Rica. Food Sci. Technol, Campinas, 41(2): 343-348, Apr.-June 2021 348
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A life course approach to injury prevention: a "lens and telescope" conceptual model
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© 2011 Hosking et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited. Background Our understanding of injury, its causes and opportunities
for prevention are informed by paradigms drawn from a
range of disciplines, including epidemiology, biomecha-
nics, ergonomics and the behavioral and social sciences
[1,2]. As many injuries follow acute events and are consid-
ered to be relatively sudden in onset [3], it is tempting to
focus on the short-term and proximal influences on injury
(e.g., speeding as a risk factor for a motor vehicle crash, or
the time elapsed to availability of definitive trauma care as
a risk factor for injury-related deaths). While such factors
are undeniably important, risks of injury are also influ-
enced by living conditions, such as the urban environment,
and access to money and other resources [4]. There are a
host of distal factors that influence these domains, not all
of which are inherently obvious or actively considered as The life course approach is notable for its longer-term
temporal perspective, and is an increasingly influential fra-
mework in a range of areas of health, especially preventa-
tive medicine [5-7]. In this paper, we examine the models
and concepts that have most strongly influenced the field
of injury, drawing on seminal articles, book chapters and
other prominent resources, and describe the extent to
which life course concepts have been addressed in existing
approaches to injury. We then summarize the develop-
ment of and main concepts inherent in the life course
approach, and discuss how this approach can be applied
to the field of injury. Drawing on this exploration, we offer
a model that integrates the two perspectives to address the
burden of injury across the life course. Jamie Hosking1†, Shanthi Ameratunga1*†, Susan Morton2 and Danilo Blank3 Jamie Hosking1†, Shanthi Ameratunga1*†, Susan Morton2 and Danilo Blank3 Jamie Hosking1†, Shanthi Ameratunga1*†, Susan Morton2 and Danilo Blank3 Abstract Background: Although life course epidemiology is increasingly employed to conceptualize the determinants of
health, the implications of this approach for strategies to reduce the burden of injuries have received little
recognition to date. Methods: The authors reviewed core injury concepts and the principles of the life course approach. Based on this
understanding, a conceptual model was developed, to provide a holistic view of the mechanisms that underlie the
accumulation of injury risk and their consequences over the life course. Results: A “lens and telescope” model is proposed that particularly draws on (a) the extended temporal dimension
inherent in the life course approach, with links between exposures and outcomes that span many years, or even
generations, and (b) an ecological perspective, according to which the contexts in which individuals live are critical,
as are changes in those contexts over time. Conclusions: By explicitly examining longer-term, intergenerational and ecological perspectives, life course
concepts can inform and strengthen traditional approaches to injury prevention and control that have a strong
focus on proximal factors. The model proposed also serves as a tool to identify intervention strategies that have
co-benefits for other areas of health. amenable to intervention in many injury prevention
strategies. * Correspondence: s.ameratunga@auckland.ac.nz
† Contributed equally
1Section of Epidemiology and Biostatistics, School of Population Health,
University of Auckland, Auckland Mail Centre, Auckland 1142, New Zealand
Full list of author information is available at the end of the article Hosking et al. BMC Public Health 2011, 11:695
http://www.biomedcentral.com/1471-2458/11/695 Hosking et al. BMC Public Health 2011, 11:695
http://www.biomedcentral.com/1471-2458/11/695 Open Access Common injury concepts Within the range of potential inter-
ventions, Haddon advocated that preference should be
given to “passive” injury prevention strategies - those that
protect the individual without action on the part of that
individual - over “active” measures requiring individual
action [18]. The interactions between people and broader notions
of ecology and environment are also fundamental
aspects of the life course approach [28,29]. We review
the evolution of the latter as a prelude to considering its
integration and application to the field of injury. The life course approach Life course epidemiology has been defined as “the study
of long term effects on later health or disease risk of phy-
sical or social exposures during gestation, childhood,
adolescence, young adulthood and later adult life” [30, p. 778]. In addition to examining biological, behavioral and
psychosocial pathways to disease that operate across the
life course of an individual, the approach invites a consid-
eration of how risks and attributes are transmitted across
generations [30]. These concepts are not new or specific
to epidemiology, and have been promoted for decades in
other disciplines such as psychology, sociology, demogra-
phy, anthropology and biology [30]. Evidence of correla-
tions between early life factors and adult health was
reported over 70 years ago [31], and the link between
early childhood factors and adult health was a matter of
concern at least as far back as the first years of the 20th
century [32,33]. Runyan introduced a third dimension to the Haddon
matrix to aid the decision-making process, introducing cri-
teria such as effectiveness, cost, equity and feasibility [2]. Her approach specifically integrated injury concepts into
Bronfenbrenner’s social-ecologic model, demonstrating
opportunities to intervene across multiple levels of the
social environment [20]. Rivara provided a framework
describing the process by which basic etiologic research
can be translated into improved injury outcomes via the
development, testing and implementation of injury inter-
ventions [21]. Common injury concepts Although a range of different conceptual approaches to
injury have been proposed [8], a relatively small number
of models and concepts remain central in most injury
textbooks [1,9-16] and in current leading teaching * Correspondence: s.ameratunga@auckland.ac.nz
† Contributed equally
1Section of Epidemiology and Biostatistics, School of Population Health,
University of Auckland, Auckland Mail Centre, Auckland 1142, New Zealand
Full list of author information is available at the end of the article Hosking et al. BMC Public Health 2011, 11:695
http://www.biomedcentral.com/1471-2458/11/695 Page 2 of 8 Page 2 of 8 resources, such as the TEACH-VIP resource developed
by the World Health Organization [17]. We summarize
these common concepts here. enforcement (e.g., laws mandating use of child-resistant
packaging for hazardous medicines) [1]. The environment
(physical and social) is sometimes identified as a distinct
category, as in the Haddon matrix. William Haddon’s seminal contributions to conceptualiz-
ing causes and measures to prevent injuries included the
matrix that combined the epidemiological triad of host,
agent and environment with a temporal dimension cover-
ing pre-event, event and post-event phases [18]. In doing
so, he drew on John Gordon’s earlier work, which described
how the causative factors in injury can be categorized
according to the three components of the epidemiological
triad, and which also described the environment as having
physical, biological and social dimensions [10]. An example
of how the Haddon matrix [18] can be used to identify
potential injury determinants and interventions is shown in
Table 1. Theories of behavior change have been applied to a
number of injury prevention strategies, typically interven-
tions targeting the “host”, rather than the “vehicle” or
“environment” [22,23]. While not their primary focus,
several of these theories explicitly acknowledge the role
of environmental barriers and facilitators in behavior
change [22]. As apparent from the above, with increasing
awareness and emphasis on the environmental context,
current concepts in injury prevention promote considera-
tion of “ecological” approaches, in which all parts of a
model “influence each other as part of a connected sys-
tem” [24, p. 557]. The application of such thinking is
increasingly evident in fields such as violence prevention
[25] and community safety promotion [26,27]. Complementing this matrix, Haddon proposed ten
types of injury countermeasures which comprise a tem-
porally ordered sequence of approaches designed to con-
trol, modify and interrupt the process of energy transfer
from the hazard causing injury to the individual(s) that
can be affected [19]. Applicability of life course concepts to injury
prevention Extending concepts regarding “determinants of injury”
Compared to existing injury models, a life course
approach explicitly extends the temporal dimension of
relevance when considering determinants of injury. While the “pre-event” phase in the Haddon matrix is
typically (although not exclusively) applied to a time per-
iod that is relatively proximal to the time of the injury
event, a life course approach views the relevant determi-
nants as having effects that accrue over a lifetime, with
potentially important effects across generations. Interge-
nerational effects have been demonstrated in the agricul-
tural setting, where parental injury has been associated
with a higher risk of subsequent injury to children [43]. This could be related to intergenerational associations in
injury risk behaviors, or to the sharing of hazardous
environments across generations. There is a need for
research that more clearly identifies the mechanisms that
mediate or counter the transmission of these risks in dif-
ferent settings. This initially challenged the predominant paradigm of
chronic disease causation, which tended to focus on proxi-
mal determinants and risk factors in adults. The presence
of atherosclerotic changes early in life revealed that focus-
ing on risk factors in adulthood (such as smoking and
blood pressure), while important, would offer only a par-
tial solution to preventing cardiovascular disease [6]. g
A life course approach to health and disease largely
developed to integrate these two polarized views of disease
causation - the focus on distal perinatal risk factors, and
the focus on proximal risk factors in adults - by applying a
more sophisticated perspective that acknowledges that
relevant exposures occur throughout the entire life span. Also fundamental to the modern life course approach is a
recognition of the importance of the environment and
ecological context [5]. Nancy Krieger’s eco-social model
shares some of the key principles involved by explicitly
acknowledging the importance of the conditions in which
people are born, live, work and retire [37]. In other words,
“changing individuals need to be studied in a changing
world” [[30], p. 781]. g
Similarly, while socioeconomic status is established as
an important determinant of injury rates [44], the rele-
vant periods of influence can occur across the life
course. As demonstrated for chronic diseases, socioeco-
nomic trajectories across the life course and intergenera-
tional correlations in factors such as income, social class
and employment status can mediate important social
inequities in health outcomes [4,45,46]. The life course approach Regardless of the model, the strategies used to prevent
injuries are often discussed in terms of the “three Es”:
education (e.g., instructing parents to keep medicines out
of children’s reach); engineering and design of specific
agents, products and the physical environment (e.g.,
designing child-resistant medicine packaging); and A life course approach in epidemiology was developed
largely as a response to the “fetal origins of adult Table 1 Use of the Haddon matrix to consider targets for interventions to reduce the burden of car crash injuries
Host/person
Agent/vehicle
Environment (physical and social)
Pre-event
Driver skills and experience
Speeding
Roadworthiness of vehicles
Visibility of vehicles
Good brakes
Graduated driver licensing systems
Traffic speed limits
Event
Seatbelt use
Presence of seatbelts, airbags and other vehicle safety features
Median barriers
Laws mandating seatbelt use
Post-event
First aid knowledge
Presence of co-morbidities
Features that make access easier for emergency services
Features that avoid post-crash explosions
Emergency services
Treatment and rehabilitation services Table 1 Use of the Haddon matrix to consider targets for interventions to reduce the burden of car crash injuries
Host/person
Agent/vehicle
Environment (physical and social)
Pre-event
Driver skills and experience
Speeding
Roadworthiness of vehicles
Visibility of vehicles
Good brakes
Graduated driver licensing systems
Traffic speed limits
Event
Seatbelt use
Presence of seatbelts, airbags and other vehicle safety features
Median barriers
Laws mandating seatbelt use
Post-event
First aid knowledge
Presence of co-morbidities
Features that make access easier for emergency services
Features that avoid post-crash explosions
Emergency services
Treatment and rehabilitation services Hosking et al. BMC Public Health 2011, 11:695
http://www.biomedcentral.com/1471-2458/11/695 Page 3 of 8 disease” hypothesis in which size at birth (usually mea-
sured by birth weight) shows a consistent graded asso-
ciation with cardiovascular risk factors and disease
outcomes later in life [34]. When this hypothesis was
initially proposed, it was suggested that this association
was mediated through some form of biological program-
ming that occurs in early life, particularly during intrau-
terine development. The concept (referred to more
recently as the “developmental origins of health and dis-
ease” [35,36]) drew attention to a body of research sup-
porting the notion that several chronic diseases that
become apparent in adulthood are linked to patterns of
early life growth (both prenatal and postnatal). dimension has not been represented. In the next section,
we analyze the relevance of these two main concepts to
injury, providing some specific examples that illustrate
the concepts. Applicability of life course concepts to injury
prevention Furthermore,
these inequalities must be considered in the context of
multiple levels of influence, including the individual and
the neighborhood levels [37]. A life course approach
highlights the need to address the social inequalities in
injury as well as overall injury rates. While the implica-
tions at national and regional levels are obvious as
attested to by many studies and reviews investigating
socioeconomic inequalities in injury, the issues involved
are particularly salient at the global level where hazar-
dous environments and inequities in access to resources
of many kinds place people living in low- and middle-
income countries at much greater risk of injury com-
pared with those in higher-income countries [47]. For
example, while childhood drowning in swimming pools
have drawn much-needed policy attention in many
high-income countries, rates of drowning are not only
higher by several orders of magnitude in countries such
as Bangladesh and China, but also demand attention to
much more challenging contextual issues, such as the
risks from flooding of homes and play areas [47]. Intergenerational influences are also considered
actively in a life course approach. For example, research
in this domain has identified that child birth size is pre-
dicted not only by the characteristics of the mother (e.g. mother’s birth size, adult height and parity) but also by
characteristics of the grandmother (e.g. grandmother’s
adult height and parity) [38]. While researchers have previously considered the
implications of the life course approach for topics such
as suicide [39], child behavior [40], child abuse [41] and
combat [42], this approach is not explicitly incorporated
in current injury prevention frameworks. Two main concepts are represented in the modern life
course approach: an extended temporal dimension, with
links between exposures and outcomes that span many
years, or even generations; and an ecological perspective,
according to which the contexts in which individuals live
are critical, as are changes in those contexts over time. Although some injury control frameworks have incorpo-
rated ecological considerations, the extended temporal Hosking et al. BMC Public Health 2011, 11:695
http://www.biomedcentral.com/1471-2458/11/695 Hosking et al. BMC Public Health 2011, 11:695
http://www.biomedcentral.com/1471-2458/11/695 Page 4 of 8 in life, and physically active children are more likely to
become physically active adults [57]. Physical activity has
also been shown to be an important component of falls
prevention in the elderly [58]. The ecology of injury
h
l
l d Building on the features noted above, injury prevention
strategies with effects that can reach across the life
course can be particularly powerful. For example, one
home visiting intervention was found not only to reduce
child abuse and neglect [52], but also to reduce injury
risk factors for children in the intervention group when
they were older, in the form of problem drinking in ado-
lescence [53]. Several other social and health outcomes
were also improved [54]. This suggests a need to be
mindful both of the potential duration of intervention
effects, and also the multiple outcomes that may stem
from a single intervention. While educational interven-
tions often target proximal injury risk factors, education
is also a component of the more distal concept of socioe-
conomic status, suggesting that some interventions may
result in both proximal and distal effects. Home visiting
interventions, which can address multiple injury risk fac-
tors for several different people in a family, also reflect
the importance of employing an ecological perspective
that recognizes that people “influence each other as part
of a connected system” [24, p. 557]. However, more stu-
dies are needed to demonstrate the long-term effects of
injury prevention interventions [55]. The ecological dimension of the life course approach is
an important feature of the proposed model that is, to
some extent, distinct from the traditional injury preven-
tion frameworks that consider “host”, “vehicle” and
“environment”, or the “three Es” as specific concepts. Ecological models present the world as a set of inter-
linked ecosystems whose parts reciprocally influence one
another, so both “host” and “vehicle” are constituent
parts of these ecosystems. This is exemplified by the
dynamic interaction between people, vehicles and the
physical environment in a busy road. The “safety in num-
bers” concept, according to which injury risk for indivi-
dual cyclists reduces as cyclist numbers increase [63],
further illustrates the interdependence within the ecology
of the road. Broader ecological effects also occur through the con-
tribution of transport emissions to climate change, with
consequent increases in injury risk factors related to
increasingly frequent extreme weather events and other
natural disasters [64]. Conversely, the public health
response to climate change offers substantial opportu-
nities for injury prevention [65]. In the chronic disease sphere, the discovery of athero-
sclerotic changes as early as the second decade of life led
to calls for the “lifelong prevention of atherosclerosis”
[6, p. 1129]. Applicability of life course concepts to injury
prevention With the neighborhood
environment being a recognized determinant of physical
activity at all ages [59], exposure to activity-friendly envir-
onments early in life could thus be an important environ-
mental determinant of falls in later life. In some situations,
the hazardous exposure may interact with other age-
related factors to increase the risk of injury over the life
course. For example, exposure to high levels of occupa-
tional noise affects the cumulative risk of significant hear-
ing impairment, which may manifest later in life due to
the additive effects of age-related hearing loss [60]. Com-
munity-level factors that can influence drinking initiation
among youth [61] can also increase the risk of subsequent
alcohol-related unintentional injury in adulthood [62]. These findings indicate the significant benefits that could
result from comprehensive injury prevention strategies
that consider intervention opportunities both early in life
and in later years, with particular awareness of the physical
and social environments. There are many examples revealing intergenerational
correlations in injury risk. Being physically punished as a
child is not only associated with being the victim of spou-
sal abuse later in life (an effect across the life course of an
individual), it is also associated with abusing, as an adult,
one’s own child [48]. This abuse, in turn, has lifelong
health implications for the victim [41]. The alcohol con-
sumption of parents is correlated with their adolescent
children’s alcohol consumption [49], and early onset of
alcohol drinking (for example, aged under 14 years) not
only predicts problem drinking later in life, but also pre-
dicts early onset of alcohol drinking in that person’s chil-
dren [50]. Furthermore, while the utility of labeling
individuals as “accident-prone” is debatable [51], there is
little doubt that the characteristics of the social and phy-
sical environments that people live in directly influence
their risk of injury. A life course perspective emphasizes
the need to see “injury proneness” as representing not
fixed, intrinsic personal characteristics, but rather, an
opportunity to create safer environments for populations
identified as being at higher long-term risk of injury. The ecology of injury
h
l
l d Taking a lifelong approach to injury preven-
tion can be considered in the same light. For example,
while mortality from falls occurs overwhelmingly in the
elderly [56], risk factors for falls accumulate throughout
the life course. Trajectories in physical activity start early Injury interventions should take account of, and capita-
lize on, these interactions. For example, low-speed zones
and traffic calming interventions that enhance the safety
of vulnerable road users [63,66] could, in theory, catalyze
and mutually reinforce a “safety in numbers” effect for
pedestrians and cyclists. Over time, if more people walk
and cycle, the safety in numbers effect could increase,
further encouraging pedestrians and cyclists, while also Hosking et al. BMC Public Health 2011, 11:695
http://www.biomedcentral.com/1471-2458/11/695 Page 5 of 8 Symbolically, the tool takes the form of a telescope and
lens - representing its extended time dimension, and its
ecological and intergenerational focus. The “lens” contains,
at its centre, the host (the person at risk of injury) and the
vehicle (through which the transfer of energy occurs, such
as a motor vehicle in road traffic injury, or a firearm in the
case of gunshot wounds). The host and vehicle are located
within and influenced by the social and physical environ-
ments, ranging from the home environment to the local
community and the global context. This combines the
commonly used concepts in the Haddon matrix with the
broader levels of environmental influence described by
many ecological models [26-28,37,70,71]. These environ-
mental factors are not only of fundamental importance in
the causation of injury, but also in the level of functioning
and disability after injury, as illustrated by the Interna-
tional Classification of Functioning, Disability and Health
[72]. We represent the boundaries between the ecological
layers in the tool with dashed lines in recognition of the
dynamic interactions between all components of the
“lens”. galvanizing environmental policy actions that promote
and support active modes of travel. pp
A key feature of interventions such as traffic calming
that improve the safety of the road environment for
pedestrians and cyclists is their potential to improve
opportunities for more active lifestyles that do not inher-
ently increase the risk of serious injuries. Integrating
injury prevention with physical activity promotion goals,
instead of pursuing each in isolation, can help to manage
pre-existing conflicts between these domains of health. The ecology of injury
h
l
l d For example, concerns about the safety of child pedes-
trians are likely to have contributed to the decline in
walking to school in recent years [67]. While being driven
to school reduces that child’s pedestrian injury risk, it
increases the risks from physical inactivity, indicating a
need for alternative solutions that improve safety and
physical activity simultaneously. An ecological approach
addresses these tensions explicitly by designing and
implementing interventions to maximize co-benefits
across multiple domains of health. For example, it has
been argued that reducing global fossil fuel dependence
could simultaneously reduce the large global problems of
road traffic injury, obesity and climate change, by transi-
tioning from dependence on motor vehicles to increased
use of active transport [68]. The evaluation of interven-
tions also needs to address the multiple outcomes that
may stem from interventions, as well as the interdepen-
dence between different spheres from the home setting
to the city level [69]. By fitting this “lens” on the end of the “telescope”, a life
course dimension is added to the tool. As people move
through the different stages of the life course, their perso-
nal attributes evolve, as do the dynamic social and physi-
cal environments with which they interact. All of these
factors influence the accumulation of injury risk for indi-
viduals, as well as for their families and future genera-
tions. Accordingly, each of these factors constitutes a
potential target for injury prevention interventions. A life course model for injury prevention An example of the application of the model is provided
by the topic of climate change, referred to earlier. The glo-
bal physical environment (e.g. climate) and macro-social
environment (e.g. climate policy) are interdependent. Both
influence host factors (e.g. travel behavior), which itself
influences road traffic injury risk. Travel behavior also
affects greenhouse gas emissions, which contribute to
future climate change that will be experienced by the host
in later stages of life, as well as by future generations. The preceding section demonstrates that life course and
ecological influences on injury are conceptually plausible
and supported by empirical evidence. In this section we
propose a model that integrates both injury prevention
and life course concepts. As noted earlier, the Haddon matrix remains the most
prominent injury conceptual model, with a central place
in textbooks and current leading teaching resources. As
well as a temporal dimension, the Haddon matrix identi-
fies host, agent and environmental factors (both social and
physical) that contribute to injury. The two main concepts
represented in the modern life course approach are an
extended temporal dimension and an ecological perspec-
tive. Figure 1 presents a schematic view of a model that
integrates the salient concepts of the Haddon matrix and
the life course approach to inform injury prevention. Author details
1 Author details
1Section of Epidemiology and Biostatistics, School of Population Health,
University of Auckland, Auckland Mail Centre, Auckland 1142, New Zealand. 2Centre for Longitudinal Research, School of Population Health, University of
Auckland, Auckland Mail Centre, Auckland 1142, New Zealand. Conclusions 3Departamento de Pediatria, Faculdade de Medicina, Universidade Federal
do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil. final catalysts in pathways that result in injury. Rather, we
suggest that explicitly integrating a life course approach
helps identify strategies that actively address broader social
and ecological determinants as well as achieve co-benefits
across multiple health domains for many generations. p
g
The authors declare that they have no competing interests. Acknowledgements
No external sources of funding were received for this research. Authors’ contributions
d
d f d h JH and SA drafted the manuscript. All authors contributed to the study
conception and analysis, reviewing and revising the article, and approved
the final manuscript. Acknowledgements
No external sources of funding were received for this research. Received: 17 May 2011 Accepted: 8 September 2011
Published: 8 September 2011 Conclusions While injuries are typically attributed to discrete events,
these are often strongly associated with social and ecologi-
cal influences including risks accumulated throughout the
life course. Some injury risk factors, such as those relating
to alcohol use, exposure to violence and socioeconomic
status, are also transmissible between generations. Acknowledging the importance of a longer-term perspec-
tive on injury - along the lines of a “chronic disease” - we
propose a “lens and telescope” model that integrates tradi-
tional injury prevention and life course approaches. In
doing so, we do not deny the vital significance of addres-
sing the immediate events surrounding an injury, or the In comparison with the Haddon matrix (Table 1), this
tool emphasizes broader ecological influences and life
course and intergenerational determinants of injury. Furthermore, the tool can be used to consider potential
long-term effects that can arise both from injuries and
from interventions. Hosking et al. BMC Public Health 2011, 11:695
http://www.biomedcentral.com/1471-2458/11/695 Page 6 of 8 SOCIAL
ENVIRONMENT
PHYSICAL
ENVIRONMENT
The Lens
Regional and national
macrosocial factors
Regional and national
physical environment
Friends and family
Home
Community
Neighbourhood
Host
Agent / vehicle
Global macrosocial factors
Global physical environment
The Telescope
Look forwards for the outcomes associated with an intervention,
a determinant or an injury in a given stage of life
Next
generation
Fetal
development
Adolescence
Adulthood
Older age
Previous
generation
Childhood
Look backwards for preventing an injury in a given stage of life
Figure 1 Ecological model of injury across the life course: the “lens and telescope”. The Telescope
Look forwards for the outcomes associated with an intervention,
a determinant or an injury in a given stage of life
Next
generation
Fetal
development
Adolescence
Adulthood
Older age
Previous
generation
Childhood
Look backwards for preventing an injury in a given stage of life Look backwards for preventing an injury in a given stage of life The Telescope Look forwards for the outcomes associated with an intervention,
a determinant or an injury in a given stage of life Look forwards for the outcomes associated with an intervention,
a determinant or an injury in a given stage of life ENVIRONMENT The Lens Figure 1 Ecological model of injury across the life course: the “lens and telescope”. 3Departamento de Pediatria, Faculdade de Medicina, Universidade Federal
do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil. Author details
1Section of Epidemiology and Biostatistics, School of Population Health,
University of Auckland, Auckland Mail Centre, Auckland 1142, New Zealand.
2Centre for Longitudinal Research, School of Population Health, University of
Auckland, Auckland Mail Centre, Auckland 1142, New Zealand. Competing interests p
g
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disability and health: ICF: the international classification of functioning,
disability and health Geneva: World Health Organization; 2002. Pre-publication history The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-2458/11/695/prepub The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-2458/11/695/prepub doi:10.1186/1471-2458-11-695
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Trait Aggressiveness Is Not Related to Structural Connectivity between Orbitofrontal Cortex and Amygdala
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Introduction in the brain. Measuring the diffusion of water within each voxel,
different parameters can be estimated with DTI. Fractional
anisotropy (FA) reflects the coherence of diffusion in a given
voxel. Mean diffusivity (MD), on the other hand, is inversely
related to the quality of white matter connections [8]. Inhibitory control of amygdala activity by prefrontal structures
is a crucial aspect of emotion regulation and dysfunction in this
network is associated with anxiety-related disorders [1] as well as
aggression [2]. Altered functional connectivity between the
prefrontal cortex, especially the medial orbitofrontal cortex
(mOFC), and the amygdala has been shown to be related to
aggressive tendencies in pathologically aggressive patients and
healthy samples [3,4]. Accordingly, integrity and strength of
structural connections between medial prefrontal cortex and
amygdala might equally be related to interindividual variability
in aggression. q
y
Several studies using DTI have shown a relationship between
alterations in prefrontal-subcortical anatomical connections or
prefrontal white matter volume and aggression-related psychopa-
thology. Psychopathic men show reduced FA-values in the right
UF, and the number of streamlines in the bilateral UF is negatively
related to antisocial behavior in psychopaths [9,10]. In adults with
antisocial personality disorder, FA-values were found to be
decreased in a range of brain areas including the right UF. MD-
values were increased in the right UF, among other regions [11]. Schizophrenic men presented with a negative relationship between
inferior frontal FA-values and motor impulsivity, and with a
positive relationship between inferior frontal diffusivity and
aggression scores [12]. Reciprocal connections between prefrontal cortex and amyg-
dala exist anatomically via the uncinate fascicle (UF), which
connects prefrontal cortex and the anterior temporal lobe [5,6]. Research in rhesus monkeys suggests that anatomical connections
between prefrontal cortex and amygdala are highly reciprocal, i.e. areas of prefrontal cortex receiving strong input from the
amygdala also possess a high density of efferent fibers projecting
back to the respective amygdala region [7]. While evidence exists
that the UF is associated with the transmission of socio-emotional
information [6], research in this domain and especially concerning
specific functions such as control of aggressive impulses is still
scarce and inconclusive. In adolescents (mean age = 18.4 years) with conduct disorder
(CD), who present with aggressive and antisocial behavior,
increased FA-values were found in the UF [13]. Trait Aggressiveness Is Not Related to Structural
Connectivity between Orbitofrontal Cortex and
Amygdala Frederike Beyer, Thomas F. Mu¨ nte, Juliana Wiechert, Marcus Heldmann, Ulrike M. Kra¨mer*
Dept. of Neurology, University of Lu¨beck, Lu¨beck, Germany Frederike Beyer, Thomas F. Mu¨ nte, Juliana Wiechert, Marcus Heldmann, Ulrike M. Kra¨mer*
Dept. of Neurology, University of Lu¨beck, Lu¨beck, Germany Abstract * Email: umkraemer@gmail.com Competing Interests: The authors have declared that no competing interests exist. * Email: umkraemer@gmail.com June 2014 | Volume 9 | Issue 6 | e101105 Abstract Studies in both pathological and healthy samples have suggested altered functional connectivity between orbitofrontal
cortex (OFC) and amygdala as a possible cause of anger and aggression. In patient populations presenting with pathological
aggression, there is also evidence for changes in structural connectivity between OFC and amygdala. In healthy samples,
however, the relationship between white matter integrity and aggression has not been studied to date. Here, we
investigated the relationship between trait aggressiveness and structural OFC-amygdala connectivity in a large sample
(n = 93) of healthy young men. Using diffusion tensor imaging, we measured the distribution of fractional anisotropy and
mean diffusivity along the uncinate fascicle bilaterally. We found no differences in either measure between participants high
and low in physical aggressiveness, or between those high and low in trait anger. Our results therefore argue against a
direct relationship between structural OFC-amygdala connectivity and normal-range trait aggressiveness. Citation: Beyer F, Mu¨nte TF, Wiechert J, Heldmann M, Kra¨mer UM (2014) Trait Aggressiveness Is Not Related to Structural Connectivity between Orbitofrontal
Cortex and Amygdala. PLoS ONE 9(6): e101105. doi:10.1371/journal.pone.0101105 Editor: Allan Siegel, University of Medicine & Dentistry of NJ - New Jersey Medical School, United States of America Editor: Allan Siegel, University of Medicine & Dentistry of NJ - New Jersey Medical School, United States of America Received March 13, 2014; Accepted May 22, 2014; Published June 30, 2014 Received March 13, 2014; Accepted May 22, 2014; Published June 30, 2014 Copyright: 2014 Beyer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. authors confirm that all data underlying the findings are fully available without restriction. Data are available from the NITRC at: http://
mdti/. ta Availability: The authors confirm that all data underlying the findings are fully available without restriction. Data are available fro
ww.nitrc.org/projects/ymdti/. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. Data are available
www.nitrc.org/projects/ymdti/. Funding: This study was supported by the German Research Foundation (KR3691/5-1) and through intramural funding by the University of Lu¨beck (E36-2011). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. PLOS ONE | www.plosone.org Participants 99 healthy male volunteers were recruited for this study. All
participants were free of psychiatric and neurological disorders
(self-report). Since differences between men and women have been
shown both for white matter properties [16,17,18] and physical
aggression scores [19], we limited our study to male participants. Of the investigated subjects, 5 were excluded from data analysis
due to artifacts in their DTI-images or anatomical abnormalities. Data from one participant was excluded because the AFQ
tractography failed due to a low number of original tracts
identified in the whole brain tractography step (see below for
detailed information on the tractography). Thus, data of 93
participants was included in the analysis. Mean age of the
participants was 23.2 years (SD = 2.7) and all but 7 were right
handed. For a subsample of 34 participants, we used behavioral data
from a version of the original TAP by Taylor: The opponent
started with low punishment levels (1–2) and ended with the
highest selection, being 8 (mean = 4.0), shuffled in 4 blocks (means
= 1.5/3.5/5.5/7.5, ranges = 1–2, 3–4, 5–6, 7–8). Each participant
played 24 trials against the confederate and won 50% of them. As
behavioral aggression measure for each participant, we used the
mean punishment selection across all trials. For a second subsample of 33 participants, we used behavioral
data from a version of the TAP adapted for functional MRI and
including video sequences showing the ostensible opponent. The
video sequences were presented at the beginning of each decision
phase and showed the opponent bearing a neutral or angry facial
expression. The TAP consisted of 60 trials which were divided into
three blocks, and the opponent selected medium to high
punishments (3–8, mean = 4.8). The fMRI results of this study
will be presented elsewhere [22]. For the present purpose, we only
considered the mean punishment selection across all trials as
behavioral measure of aggression. A-priori estimations of required sample size were difficult, as
there is little evidence for the effect sizes to be expected in healthy
samples. With n = 93, a = .005 (to account for multiple compar-
isons) and b = .8, the minimum effect size detectable is r = .72. This is considerably smaller than the effects in the above-cited
studies comparing pathological and normal populations, which
showed effect sizes between 1.25 and 2.5 [9,11,13]. Behavioral Aggression measure As an additional aggression measure, we used behavioral data
derived from the Taylor Aggression Paradigm (TAP) [21] in two
subsamples of our subjects. The TAP is set up as a competitive
reaction time task in which the participant competes against an
ostensible opponent in multiple rounds. In each round of the TAP,
the participant must press a button as quickly as possible in
response to a visual cue and try to be faster than his opponent. The
loser of each round is punished with an aversive stimulus, the
intensity of which is determined by the winner. Both outcome of
the reaction time task and the opponent’s punishment selection are
under control of the experimenter. Thus, in the TAP, the
punishment selected by the opponent serves as a method for
provocation and the punishment selected by the participant serves
as a measure of aggression. Evidently, the reviewed literature is based on patient popula-
tions presenting with complex syndromes, precluding any definite
conclusions concerning the importance of the UF for the control of
anger or aggression. If information transmitted by this fiber tract is
important for social information processing involved in the
development of non-aggressive behavioral strategies and emotion-
al regulation, this might also be reflected in healthy samples. In this study, we investigated the relationship between measures
of diffusivity in the UF and trait aggressiveness in healthy young
men. If strength of the UF indeed is of specific importance for
control of anger and aggression, a negative relationship between
anger and aggression scores and FA-values would be expected,
with a reverse relationship between MD-values and aggression
scores. We implemented the TAP such that at the beginning of each
round, the participant selected a punishment level for his
opponent. This was followed by the reaction time task and an
outcome phase, during which the participant was informed
whether he had won or lost and which punishment level his
opponent had selected. As a punishment stimulus, we used an
aversive noise that could be adjusted in terms of loudness on a
scale from 1–8. In lost trials, the noise was played in the respective
loudness at the end of the outcome phase. The outcome of the
reaction time task, and also the opponent’s selections, were
controlled by the computer. The mean punishment level selected
by the participant was used as a measure of aggressive behavior. Orbitofrontal-Amygdala Connectivity in Aggression (mean age 14.3 years), impairments in structural amygdala-
orbitofrontal connectivity in CD patients may develop during the
maturation
of
white
matter
across
adolescence
and
early
adulthood. Participants The study was approved by the Ethics Committee of the
University of Lu¨beck and performed according to the Declaration
of Helsinki. Written informed consent was obtained from all
participants. For both subsamples, the behavioral aggression measure used
reflects the participants’ propensity to aggress in situations that
include some form of provocation. All participants were confront-
ed with an opponent who at least in some trials selected high
punishments. However, there were also differences both in the
mean punishment level participants received and the general setup
of the task (in subsample 2, the task was adapted for fMRI and
thus longer, and included video sequences). Thus, while the two
samples could not be investigated as one group, both behavioral
measures reflect reactive aggressive behavior in a competitive
interactive context. Trait Aggression measure As a measure of trait aggression, participants filled out a
German version of the Buss and Perry Aggression Questionnaire
(AQ) [20], which comprises four subscales: anger, physical
aggression, verbal aggression and hostility. Since we hypothesized
that
deficits
in
prefrontal-amygdala
connectivity
should
be
specifically associated with emotional dysregulation and aggressive
behavior, we focused our analysis on the anger and physical
aggression subscales. The physical aggression scale contains items
such as ‘‘If somebody hits me, I hit back’’ or ‘‘I have become so
mad that I have broken things.’’ The anger scale contains items
such as ‘‘I flare up quickly but I get over it quickly’’ or ‘‘Some of
my friends think I’m a hothead.’’ Answers in the AQ are given on
a 5-point Likert scale, with high scores representing high
aggressiveness [19]. Introduction As white matter
increases into early adulthood [14], the authors interpret this
finding as a reflection of premature maturation of this fiber tract in
adolescents with CD. However, another study investigating youths
with conduct disorder and psychopathic traits found no difference
in FA-values in the UF compared to healthy controls [15]. The
authors argue that since the investigated sample was rather young Diffusion weighted tensor imaging (DTI) is a non-invasive
imaging method allowing for in-vivo measurement of fiber tracts PLOS ONE | www.plosone.org June 2014 | Volume 9 | Issue 6 | e101105 1 June 2014 | Volume 9 | Issue 6 | e101105 June 2014 | Volume 9 | Issue 6 | e101105 UF properties and physical aggression Participants with a physical aggression score of 1.63 and lower
were classified as low aggressive, resulting in a 46:47 ratio of low:
high aggressive participants (mean aggression scores 1.36 and
2.32; SD = 0.7). Low and high aggressive participants did not differ significantly
in their FA- or MD-values for the UF in either hemisphere. With a
more lenient significance threshold of p,.05 uncorrected, we
observed higher FA-values for the low aggressive group bilaterally
in the anterior part of the UF, close to the prefrontal ROI. In the
left UF, group differences were observed for nodes 76–95, in the
right UF for nodes 89–95, as well as nodes 97, 99 and 100. This
trend for statistical differences might point to ‘‘true’’ personality
effects which are diminished by the rather rough approach of a
median-split. To ensure that we did not miss a true effect, we
further investigated the distribution of FA-values on a descriptive
level. A true personality effect on FA values should be clearly
observable in participants with extreme trait scores. For the node
with the greatest difference between groups (node 83 in the left
UF; means
= 0.547 and 0.511), we therefore plotted the
distribution of FA-values for the low and high aggressive groups
together with the FA-values of the nine participants with the
highest and lowest aggression scores. As can be seen in figure 1C,
there was no consistent trend of highest aggression being
associated with lowest FA-values or vice versa, supporting the
assumption that the observed group differences in FA-values
constitute a chance finding. Using this method, Yeatman et al. [24] were able to show
systematic variations in FA-values along fiber tracts that were
consistent between subjects. They found that developmental
changes of FA-values vary along fiber tracts, underlining the
importance of investigating separate nodes rather than mean-FA-
values for entire tracts. Furthermore, they demonstrated the
validity of this method by replicating previous findings of
correlations between reading skills and FA-values in children. For each subject, we extracted the FA- and MD-values for the
left and right UF. The UF is defined by a seed ROI in the anterior
temporal lobe and an end-ROI close to the OFC. We then used
SPSS to analyze the relationship between DTI-values and AQ
scores. Since the scores on the anger and physical aggression scales
were not normally distributed (Kolmogorov-Smirnov-test p,
.001), we did not perform correlational analyses on the data. UF properties and physical aggression Instead, we performed a median split based on the physical
aggression subscale and compared FA- and MD-values between
high and low aggressive participants using independent sample t-
tests for each of the 100 nodes of the UF in each hemisphere. We
then followed the same procedure for the anger subscale. Data analysis Along the left UF, mean FA-values ranged from 0.31–0.56 (SD
0.039–0.066). As can be seen in figure 1A, mean FA-values
increased along the tract from the ROI in the temporal lobe
towards the prefrontal ROI. Figure 1B shows the distribution of
FA-values along the left UF in a representative subject. These
results were similar for the right UF (means 0.32–0.55; SD 0.032–
0.050) and are consistent with previous findings concerning the
distribution of FA-values along the UF [24]. We used the open-source software FSL 5.0 (http://neuro. debian.net/pkgs/fsl-5.0.html#binary-pkg-fsl-5-0) [23] for eddy
correction and extraction of diffusion tensors from the original
diffusion weighted images. We also used FSL’s brain extraction
tool to skull-strip DTI (probability-cutoff 0.3) and anatomical
images (cutoff 0.5). For further analysis, we used the Automated Fiber Quantifica-
tion
(AFQ)
toolbox
(http://white.stanford.edu/newlm/index. php/AFQ). This is an open-source matlab-based DTI analysis
tool built to automatically identify the 20 major fiber tracts in
individual DTI-images in their native space. Details on the
methods implemented by the AFQ-toolbox can be found in [24]. Briefly, analysis with AFQ consists of the following processing
steps: first, the eddy-corrected, skull-stripped DTI-images were
imported into AFQ format and aligned with the respective skull-
stripped T1-weighted image. Then, deterministic fiber-tracking
was performed across the whole brain and subsequently, for each
of the 20 target tracts tract segmentation was performed based on
two waypoint-regions of interest (ROIs). These tracts were then
refined and cleaned by removing aberrant fibers. Further analysis
focused on the portion of the tract between the two waypoint
ROIs. This section was divided into 100 equally spaced nodes,
such that FA- and MD-values, or other values of interest, could be
investigated for each node separately rather than averaged across
the whole tract. Mean MD-values along the left UF ranged from 0.75–0.89 (SD
0.033–0.072). Along the right UF, mean MD-values ranged from
0.74–0.83 (SD 0.032–0.043). The median of the physical aggression scores was 1.67, with
quartiles 1.38 and 2.11 (mean score = 1.85). The median of the
anger scores was 2.0, with quartiles 1.57 and 2.5 (mean score
= 2.16). Orbitofrontal-Amygdala Connectivity in Aggression 2 mm; in-plane voxel size 262 mm; single-shot EPI sequence; TR
= 7852 ms; TE = 60 ms; flip angle 90u; FOV = 224 mm; matrix
= 1126112. High resolution structural images were obtained
applying a T1-weighted 3D turbo gradient echo sequence with
SENSE (FOV = 240 mm; matrix = 2406240; 180 sagittal slices
of 1 mm thickness). We accepted group differences as significant if they survived a p-
value cutoff of 0.05, false-discovery-rate (FDR-) corrected. For the
UF, we performed FDR-correction separately for the left and right
tract. For the exploratory whole-brain analysis, we corrected for all
calculated tests (18 tracts with 100 nodes each). 2 mm; in-plane voxel size 262 mm; single-shot EPI sequence; TR
= 7852 ms; TE = 60 ms; flip angle 90u; FOV = 224 mm; matrix
= 1126112. High resolution structural images were obtained
applying a T1-weighted 3D turbo gradient echo sequence with
SENSE (FOV = 240 mm; matrix = 2406240; 180 sagittal slices
of 1 mm thickness). DTI data acquisition Images were recorded on a Philips Achieva 3-T scanner (Philips
Healthcare, the Netherlands) with a standard 8-channel head coil. Diffusion weighted images (1 image without diffusion gradients
and 32 images with diffusion gradients applied) were recorded
with the following specifications: 70 axial slices; slice thickness PLOS ONE | www.plosone.org June 2014 | Volume 9 | Issue 6 | e101105 2 Orbitofrontal-Amygdala Connectivity in Aggression UF properties and anger The critical value for the median split was 2.0. Since 41
participants had an anger score below and 42 participants had an
anger score greater than this, we excluded participants with an
exact score of 2.0 (n = 10) in order to yield comparable group sizes
(mean scores = 1.56 and 2.75; SD = 0.71).The groups high and
low in trait anger did not differ in their FA- or MD-values across
the UF in either hemisphere. As an exploratory approach, we also compared FA-values
between high and low aggressive participants for the remaining 18
fiber tracts. Note that as not all tracts can be detected reliably in all
participants, these comparisons were based on slightly different
group sizes (range: 86–93). For the behavioral aggression data, we correlated mean
aggression scores from the TAP with FA-values for the left and
right UF. This was done separately for the two groups for which
we had data from different versions of the TAP. For the remaining 18 fiber tracts, we also did not observe any
significant effects for trait physical aggression or trait anger. June 2014 | Volume 9 | Issue 6 | e101105 PLOS ONE | www.plosone.org 3 Orbitofrontal-Amygdala Connectivity in Aggression Figure 1. Descriptive results. The distribution of mean FA-values along the left uncinate fascicle (UF) between the tract defining regions of
interest (ROIs) is shown (A). Standard deviations for each of the 100 nodes are shown in blue. B shows the distribution of FA-values along the left UF
for a representative subject. Tract-defining ROIs are depicted in red. C shows mean FA-values for the low and high aggressive groups as defined by
the median split based on physical aggression scores for node 83 of the left uncinate fascicle. Single subject FA-values are depicted for the nine
participants with the lowest and highest aggression scores. Data points encircled in black represent two cases. doi:10.1371/journal.pone.0101105.g001 Figure 1. Descriptive results. The distribution of mean FA-values along the left uncinate fascicle (UF) between the tract defining regions of
interest (ROIs) is shown (A). Standard deviations for each of the 100 nodes are shown in blue. B shows the distribution of FA-values along the left UF
for a representative subject. Tract-defining ROIs are depicted in red. UF properties and anger C shows mean FA-values for the low and high aggressive groups as defined by
the median split based on physical aggression scores for node 83 of the left uncinate fascicle. Single subject FA-values are depicted for the nine
participants with the lowest and highest aggression scores. Data points encircled in black represent two cases. doi:10.1371/journal.pone.0101105.g001 UF properties and aggressive behavior values in adolescents around 18 years [13] and decreased FA-
values in adults [11]. As white matter maturation continues into
early adulthood [14] it is possible that differences in the speed of
white matter maturation within our sample eliminated effects of
trait aggressiveness. Nevertheless, our results argue against a direct
relationship between trait aggressiveness and integrity of the UF in
healthy male participants. Such a relationship should be reflected
in lower UF maturation in high aggressive individuals, which we
did not observe. For the behavioral variant of the TAP, mean punishment
selection was 3.80 (SD = 1.33; n = 34). For the fMRI-variant of
the TAP, we observed a mean punishment selection of 4.3 (SD
= 0.86; n = 33). In support of our results for the AQ, we found no significant
correlations between aggressive behavior and FA-values in the left
or right UF. June 2014 | Volume 9 | Issue 6 | e101105 References 1. Kim MJ, Loucks RA, Palmer AL, Brown AC, Solomon KM, et al. (2011) The
structural and functional connectivity of the amygdala: from normal emotion to
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16. Koolschijn PC, Crone EA (2013) Sex differences and structural bra 3. Passamonti L, Rowe JB, Ewbank M, Hampshire A, Keane J, et al. (2008)
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20. Herzberg PY (2003) Faktrostruktur, Gu¨tekriterien und Konstruktvalidita¨t der
deutschen U¨ bersetzung des Aggressionsfragebogens von Buss und Perry. Zeitschrift fu¨r Differentielle und Diagnostische Psychologie 24: 311–323. temporal cortex in the human brain. J Comp Neurol 115: 333–3 6. Von Der Heide RJ, Skipper LM, Klobusicky E, Olson IR (2013) Dissecting the
uncinate fasciculus: disorders, controversies and a hypothesis. Brain 136: 1692–
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provocation and the tendency to inhibit aggression. Journal of Personality 35:
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reactivity to angry facial expression in a social interaction correlates with
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tensor imaging. Frontiers in Neuroscience 7: 31. 23. Smith SM, Jenkinson M, Woolrich MW, Beckmann CF, Behrens TE, et al. (2004) Advances in functional and structural MR image analysis and
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connections on the road to psychopathy. Molecular Psychiatry 14: 946–953,
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profiles of white matter properties: automating fiber-tract quantification. PLoS
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connectivity in psychopathy. Journal of Neuroscience 31: 17348–17357. 25. Montag C, Reuter M, Weber B, Markett S, Schoene-Bake JC (2012) Individual
differences in trait anxiety are associated with white matter tract integrity in the
left temporal lobe in healthy males but not females. Neuroscience 217: 77–83. 11. Sundram F, Deeley Q, Sarkar S, Daly E, Latham R, et al. (2012) White matter
microstructural abnormalities in the frontal lobe of adults with antisocial
personality disorder. Cortex 48: 216–229. p
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12. Hoptman MJ, Volavka J, Johnson G, Weiss E, Bilder RM, et al. (2002) Frontal
white matter microstructure, aggression, and impulsivity in men with
schizophrenia: a preliminary study. Biological Psychiatry 52: 9–14. p
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26. Xu J, Kober H, Carroll KM, Rounsaville BJ, Pearlson GD, et al. (2012) White
matter integrity and behavioral activation in healthy subjects. Human Brain
Mapping 33: 994–1002. 13. Passamonti L, Fairchild G, Fornito A, Goodyer IM, Nimmo-Smith I, et al. (2012) Abnormal anatomical connectivity between the amygdala and orbito-
frontal cortex in conduct disorder. PLoS One 7: e48789. 27. Sisti HM, Geurts M, Gooijers J, Heitger MH, Caeyenberghs K, et al. (2012)
Microstructural organization of corpus callosum projections to prefrontal cortex
predicts bimanual motor learning. Learning and Memory 19: 351–357. 14. Bava S, Thayer R, Jacobus J, Ward M, Jernigan TL, et al. (2010) Longitudinal
characterization of white matter maturation during adolescence. We thank Susanne Schellbach for help during data acquisition. We thank Susanne Schellbach for help during data acquisition. Conclusions While
pathological
aggression
has
been
associated
with
decreased structural connectivity between amygdala and OFC,
this does not seem to be the case in healthy men. According to our
results, between-subject variability in trait physical aggression and
anger in young men is unrelated to fractional anisotropy along the
uncinate fascicle connecting anterior temporal and prefrontal
areas. Author Contributions Conceived and designed the experiments: FB TFM UMK. Performed the
experiments: FB JW. Analyzed the data: FB JW MH. Contributed to the
writing of the manuscript: FB TFM JW MH UMK. Conceived and designed the experiments: FB TFM UMK. Performed the
experiments: FB JW. Analyzed the data: FB JW MH. Contributed to the
writing of the manuscript: FB TFM JW MH UMK. Limitations It has to be stressed that our participants were mostly college
students and as such a rather homogenous sample. Physical
aggression and anger scores were not normally distributed with a
bias towards low scores, precluding the conductance of correlation
analyses. We cannot assume, therefore, that our participants
reflected the entire range of trait aggressiveness that one might
observe in the general population. In a more representative
sample, we would expect more extreme values in aggression
scores, which then might be related to properties of the UF. In his
validation study for the German version of the AQ, Herzberg [20]
found higher mean values for the physical aggression and anger
scales than we did (2.41 and 2.64 respectively, vs. 1.85 and 2.16 in
our study). Notably, the sample in this validation study was not
limited to university students. Thus, our results argue against a
relationship between UF integrity and aggressiveness in presum-
ably high-functioning and self-controlled young men, but they
cannot be generalized to all men of this age group. Future studies
should investigate samples recruited from a broader population in
order to allow for correlational analyses to account for the whole
spectrum of variance in white matter properties and aggression
scores. Alternatively, specifically targeting extreme groups in- Orbitofrontal-Amygdala Connectivity in Aggression Orbitofrontal-Amygdala Connectivity in Aggression volved in socially accepted aggressive behavior, such as high-
contact sports might increase effect sizes in group comparisons. Another possible limitation of this study is that across the entire
sample, we only used a self-report questionnaire as measure of
aggression. However, further investigating the relationship be-
tween FA-values and aggressive behavior in the TAP for sub-
samples, we could confirm the null-findings. Thus it seems unlikely
that our results are due to a bias in self-report. aggression [4], and also healthy participants high in approach
motivation [3]. Accordingly, investigating functional connectivity
in healthy samples might reveal more subtle differences between
low and high aggressive individuals. volved in socially accepted aggressive behavior, such as high-
contact sports might increase effect sizes in group comparisons. Another possible limitation of this study is that across the entire
sample, we only used a self-report questionnaire as measure of
aggression. However, further investigating the relationship be-
tween FA-values and aggressive behavior in the TAP for sub-
samples, we could confirm the null-findings. Thus it seems unlikely
that our results are due to a bias in self-report. June 2014 | Volume 9 | Issue 6 | e101105 Discussion Other studies in healthy participants have found significant
correlations between FA-values and trait anxiety [25], behavioral
approach motivation [26] and motor learning [27]. Thus, we do
not believe that our results are based on a general lack of
variability in DTI-measures in healthy samples or insensitivity of
such measures to personality traits. This study revealed that in healthy participants, integrity of the
bilateral UF is not related to trait anger or physical aggressiveness. Thus, it appears that deficits in structural prefrontal-amygdala
connectivity in aggressive individuals [9,10] are specific to
pathological populations such as criminal offenders with psychop-
athy or adolescents and adults with conduct disorder. Regarding aggressive behavior, however, Finger et al. [15] in
their study on younger adolescents concluded that functional
connectivity between OFC and amygdala is a more sensitive
measure of maladaptive development than structural connectivity. Decreased functional connectivity between amygdala and pre-
frontal cortex in response to negative social stimuli has been
observed in adolescents with CD [28], adults with impulsive The large sample size of our study makes it unlikely that we
missed a true effect due to lack of power. It is important to note,
however, that we only investigated men in the age range of 18–30
years, with a mean age of 23. Studies investigating changes in the
UF in pathologically aggressive groups found no difference in FA-
values in children around 14 years of age [15], increased FA- PLOS ONE | www.plosone.org June 2014 | Volume 9 | Issue 6 | e101105 4 References Brain Research
1327: 38–46. 28. Decety J, Michalska KJ, Akitsuki Y, Lahey BB (2009) Atypical empathic
responses in adolescents with aggressive conduct disorder: a functional MRI
investigation. Biological Psychology 80: 203–211. 5 June 2014 | Volume 9 | Issue 6 | e101105 June 2014 | Volume 9 | Issue 6 | e101105 PLOS ONE | www.plosone.org
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|
English
| null |
Molecular Simulations of Carbohydrates with a Fucose-Binding Burkholderia ambifaria Lectin Suggest Modulation by Surface Residues Outside the Fucose-Binding Pocket
|
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To cite this version: Tamir Dingjan, Anne Imberty, Serge Pérez, Elizabeth Yuriev, Paul A. Ramsland. Molecular Sim-
ulations of Carbohydrates with a Fucose-Binding Burkholderia ambifaria Lectin Suggest Modula-
tion by Surface Residues Outside the Fucose-Binding Pocket. Frontiers in Pharmacology, 2017, 8,
10.3389/fphar.2017.00393. hal-02377915 HAL Id: hal-02377915
https://hal.science/hal-02377915v1
Submitted on 18 Jan 2021 L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
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lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers. ORIGINAL RESEARCH
published: 21 June 2017
doi: 10.3389/fphar.2017.00393 Edited by: Edited by:
Leonardo G. Ferreira,
University of São Paulo, Brazil 1 Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, VIC, Australia, 2 Centre
de Recherches sur les Macromolécules Végétales, Centre National de la Recherche Scientifique UPR5301, Université
Grenoble Alpes, Grenoble, France, 3 Département de Pharmacochimie Moléculaire, Centre National de la Recherche
Scientifique, UMR5063, Université Grenoble Alpes, Grenoble, France, 4 School of Science, RMIT University, Melbourne, VIC,
Australia, 5 Department of Surgery Austin Health, University of Melbourne, Melbourne, VIC, Australia, 6 Department of
Immunology, Central Clinical School, Monash University, Melbourne, VIC, Australia, 7 Burnet Institute, Melbourne, VIC,
Australia Reviewed by:
Hugo Verli,
Universidade Federal do Rio Grande
do Sul, Brazil
David Antony Morton-Blake,
Trinity College, Dublin, Ireland
*Correspondence:
Elizabeth Yuriev
elizabeth.yuriev@monash.edu
Paul A Ramsland
paul.ramsland@rmit.edu.au Reviewed by:
Hugo Verli,
Universidade Federal do Rio Grande
do Sul, Brazil
David Antony Morton-Blake,
Trinity College, Dublin, Ireland *Correspondence:
Elizabeth Yuriev
elizabeth.yuriev@monash.edu
Paul A Ramsland
paul.ramsland@rmit.edu.au Burkholderia ambifaria is an opportunistic respiratory pathogen belonging to the
Burkholderia cepacia complex, a collection of species responsible for the rapidly fatal
cepacia syndrome in cystic fibrosis patients. A fucose-binding lectin identified in the
B. ambifaria genome, BambL, is able to adhere to lung tissue, and may play a role in
respiratory infection. X-ray crystallography has revealed the bound complex structures
for four fucosylated human blood group epitopes (blood group B, H type 1, H type
2, and Lex determinants). The present study employed computational approaches,
including docking and molecular dynamics (MD), to extend the structural analysis of
BambL-oligosaccharide complexes to include four additional blood group saccharides
(A, Lea, Leb, and Ley) and a library of blood-group-related carbohydrates. Carbohydrate
recognition is dominated by interactions with fucose via a hydrogen-bonding network
involving Arg15, Glu26, Ala38, and Trp79 and a stacking interaction with Trp74. Additional hydrogen bonds to non-fucose residues are formed with Asp30, Tyr35, Thr36,
and Trp74. BambL recognition is dominated by interactions with fucose, but also features
interactions with other parts of the ligands that may modulate specificity or affinity. The detailed computational characterization of the BambL carbohydrate-binding site
provides guidelines for the future design of lectin inhibitors. Specialty section:
This article was submitted to
Experimental Pharmacology and Drug
Discovery,
a section of the journal
Frontiers in Pharmacology
Received: 12 March 2017
Accepted: 06 June 2017
Published: 21 June 2017 Keywords: blood group determinants, Burkholderia ambifaria, docking, fucose, molecular dynamics Tamir Dingjan 1, Anne Imberty 2, Serge Pérez 3, Elizabeth Yuriev 1* and
Paul A. Ramsland 4, 5, 6, 7* Edited by:
Leonardo G. Ferreira,
University of São Paulo, Brazil INTRODUCTION of the role of each functional group in the natively recognized
carbohydrate (Ernst and Magnani, 2009). Cystic fibrosis morbidity is mostly due to respiratory infection
by opportunistic pathogens (Lyczak et al., 2002; O’Sullivan
and Freedman, 2009; Ciofu et al., 2013; Caverly et al.,
2015). Burkholderia cepacia is one of the most dangerous
pathogens isolated from cystic fibrosis patients; 20% of infected
individuals succumb to a rapidly fatal pneumonia termed
“cepacia syndrome” (Zahariadis et al., 2003; Blackburn et al.,
2004; Lynch, 2009). Isolated B. cepacia strains have been
classified into a steadily increasing number of species, referred
to collectively as the B. cepacia complex (currently consisting
of 20 species Vandamme et al., 1997; De Smet et al., 2015;
Martinucci et al., 2016). Most members of the complex are
resistant to multiple clinically used antibiotics, making the search
for new therapeutics more urgent (Zhou et al., 2007; Loutet and
Valvano, 2011; Podnecky et al., 2015). Burkholderia ambifaria,
a member of the B. cepacia complex, has been isolated from
both clinical and environmental samples (Coenye et al., 2001). In addition to infecting human respiratory tissue, B. ambifaria
can colonize plant rhizospheres, where it promotes growth and
protects against invading fungi (Li et al., 2002; Lee et al., 2006;
Parra-Cota et al., 2014). The crystallographic structure of BambL has been solved,
revealing a six-bladed β-propeller fold formed by three separate
protomers (Audfray et al., 2012). Each subunit contains a
single carbohydrate-binding site; upon oligomerization, three
additional binding sites are formed at the interfaces between
protomers, for a total of six binding sites in the β-propeller
fold. The intra- and inter-protomeric sites have similar
architectures and (for most blood group carbohydrates) similar
binding properties. For this reason, the present work addresses
interactions within the intra-protomeric site only. Crystal
structures of BambL have also been obtained bound to multiple
fucosylated human blood group tetrasaccharides: H type 1, H
type 2, B type 2, and Lex (PDB IDs: 3ZW2, 3ZZV, 3ZWE, and
3ZW1; Audfray et al., 2012; Topin et al., 2013; Figure 1). In
each case, the carbohydrate is bound via a buried fucose residue,
which participates in a network of hydrogen bonds within a
tight fucose-binding pocket. Blood group carbohydrate binding
specificity has also been determined by glycan array and affinity
quantified by titration microcalorimetry: strongest affinity is for
H type 2 tetrasaccharide (KD 7.5 µM) and Ley pentasaccharide
(KD 11.1 µM; Audfray et al., 2012). INTRODUCTION This binding preference
indicates that BambL is more selective for blood and tissue
carbohydrate determinants containing the type 2 epitope Fucα1-
2Galβ1-4GlcNAc. Several of the blood group and tissue antigens
recognized by BambL have not been structurally characterized
in complex with the lectin (e.g., Ley, Leb, and A). Additionally,
while existing crystal structures describe static recognition, the
dynamic behavior of BambL complexes has not been described. The relative contributions of individual binding interactions to
saccharide recognition is also unknown. Extending the structural
analysis of BambL-blood group complexes to probe these aspects
of recognition will enhance understanding of carbohydrate
recognition and facilitate inhibitor design. )
Previously, a carbohydrate-binding protein (named “BambL”)
was identified in the B. ambifaria genome; binding studies using
human tissues suggest it may play a role in infection (Audfray
et al., 2012). Opportunistic bacteria often adhere to tissues by
binding to host carbohydrates using carbohydrate-recognizing
proteins (lectins) displayed at the bacterial surface (Bavington
and Page, 2005; Imberty and Varrot, 2008; Pieters, 2011; Audfray
et al., 2013). Among the many carbohydrates present on
human cells, fucose-bearing blood group determinants are often
recognized by bacterial lectins (Lindén et al., 2008; Anstee,
2010; Holmner et al., 2010). In the cystic fibrosis respiratory
epithelium, cell-surface carbohydrates, present on glycolipids,
N-glycoproteins, and mucins, are more fucosylated than in
healthy tissue (Rhim et al., 2001; Venkatakrishnan et al., 2015). This increased fucosylation may promote adhesion by fucose-
recognizing pathogens (Stoykova and Scanlin, 2008; Audfray
et al., 2013). Known cystic fibrosis pathogens Pseudomonas
aeruginosa, Burkholderia cenocepacia and Aspergillus fumigatus,
all have lectins that bind to fucosylated human blood group
carbohydrates (Mitchell et al., 2002; Imberty et al., 2004; Sulak
et al., 2010, 2011; Houser et al., 2013, 2015). Significantly, the P. aeruginosa lectins are strongly associated with respiratory tissue
damage and bacterial load in a mouse model of lung injury,
and treatment with monosaccharides, able to specifically inhibit
lectin binding, reduces infection (Chemani et al., 2009). Similar
effects have been reported in a human P. aeruginosa infection
case study (von Bismarck et al., 2001) suggesting that interfering
with lectin-carbohydrate interactions may offer a new frontier in
anti-infective treatment (Sharon, 2006; Pera and Peters, 2014). Lectin inhibitor design begins with a thorough understanding The goal of this computational study was to characterize
BambL-saccharide binding modes and to inform future in silico
or structure-based design of inhibitors for this bacterial lectin. Abbreviations: BambL, Burkholderia ambifaria lectin; H1, H type 1; H2, H type
2; Lea, Lewis a; Leb, Lewis b; Lex, Lewis x; Ley, Lewis y; MD, Molecular Dynamics;
PDB, Protein Data Bank; vdW, van der Waals; RMSD, root mean square deviation Citation: Dingjan T, Imberty A, Pérez S, Yuriev E
and Ramsland PA (2017) Molecular
Simulations of Carbohydrates with a
Fucose-Binding Burkholderia
ambifaria Lectin Suggest Modulation
by Surface Residues Outside the
Fucose-Binding Pocket. Front. Pharmacol. 8:393. doi: 10.3389/fphar.2017.00393 June 2017 | Volume 8 | Article 393 Frontiers in Pharmacology | www.frontiersin.org 1 Molecular Simulations of BambL-Carbohydrate Recognition Dingjan et al. INTRODUCTION We were interested in identifying lectin residues that are critical
for ligand recognition and thus could be used as constraints
in prospective virtual screening. In particular, we investigated
whether the BambL binding site is restricted to recognizing
fucose or is capable of engaging non-fucose saccharides using
additional interactions. We first used docking and site mapping
to study binding modes in complexes featuring A, B, O (H), and
Lewis fucosylated carbohydrates and a library of blood-group-
related saccharides. The dynamic behavior of these systems
was then explored by molecular dynamics (MD) simulations. The recognition of fucose-containing saccharides by BambL is
accomplished by a hydrogen-bonding network between fucose
and Arg15, Glu26, Trp79, and to a lesser extent Ala38. A
hydrophobic contact is made between the fucose non-polar face
and the Trp79 imidazole. Additional hydrogen bonds outside the
fucose-binding pocket to Asp30, Thr36, Trp74, and Tyr35 are
formed in complex with multiple blood group and blood-group-
related saccharides. Residues involved in these interactions
are consistently engaged by blood-group-related saccharides, June 2017 | Volume 8 | Article 393 Frontiers in Pharmacology | www.frontiersin.org 2 Molecular Simulations of BambL-Carbohydrate Recognition Dingjan et al. gjan et al. Molecular Simulations of BambL-Carbohydrate Recognition
IGURE 1 | BambL subunit shown with blood group and tissue antigen saccharides (A, B, H, Lea, Leb, Lex, and Ley) used for simulation. BambL structure from PDB
D: 3ZZV, with the intra-protomeric binding site and ligand shown. b, Lex, and Ley) used for simulation. BambL structure from PDB FIGURE 1 | BambL subunit shown with blood group and tissue antigen saccharides (A, B, H, Lea, Leb, Lex, and Ley) used for simulation. BambL structure from PDB
ID: 3ZZV, with the intra-protomeric binding site and ligand shown. FIGURE 1 | BambL subunit shown with blood group and tissue antigen saccharides (A, B, H, Lea, Leb, Lex, and Ley) used for simulation. BambL structure from PDB
ID: 3ZZV, with the intra-protomeric binding site and ligand shown. Frontiers in Pharmacology | www.frontiersin.org June 2017 | Volume 8 | Article 393 3 Molecular Simulations of BambL-Carbohydrate Recognition Dingjan et al. suggesting they may be valuable interaction targets for BambL
inhibitors. suggesting they may be valuable interaction targets for BambL
inhibitors. Galβ1-3GlcNAc ϕ > 0◦& 180◦< ψ < −60◦. Thus, we have
used energy maps to post-filter docked poses as a means of
retaining reasonable conformations. INTRODUCTION These energy maps have
been commonly used to evaluate carbohydrate conformations
obtained from simulations and experimental work [for example
Jackson et al. (2014) and Tempel et al. (2002)]. Hydrogen bonds
and contacts were tallied using MDAnalysis (Michaud-Agrawal
et al., 2011; distance = 3.0 Å, angle = 120). MATERIALS AND METHODS A
single
BambL
subunit
containing
an
intra-protomeric
(Audfray et al., 2012) binding site was used in the below
computational studies. Docking Docking experiments were performed using the docking
program Glide 6.8 (Friesner et al., 2004, 2006; Halgren
et al., 2004; Schrödinger, 2014a) available within the molecular
modeling package Maestro (Schrödinger, 2014a,b). The BambL
crystallographic complexes were downloaded from the Protein
Data Bank, PDB (Berman et al., 2000), and the protein
structures prepared using the Protein Preparation Wizard tool
(Madhavi Sastry et al., 2013; Schrödinger, 2014b). During this
step, structural details required for the docking calculation
were specified. Double bond orders were applied for backbone
carbonyl and aromatic side chain moieties, hydrogen atoms
were added to the structure, water molecules removed, and
disulfide bonds created between cysteine side chain sulfur atoms
in close proximity. Missing atoms and side chains were added
based on the protein’s primary sequence using the Prime tool
(Schrödinger, 2014c). To remove steric clashes between added
hydrogen atoms, a minimization step was then conducted on
hydrogen atoms only, using the OPLS2005 forcefield (Banks
et al., 2005). A receptor grid was generated using default settings,
with the binding site box centered on the crystallographic ligand. Ligands were docked into the receptor grid using Standard
Precision mode with default settings. All carbohydrate atoms
were treated flexibly during docking, including all glycosidic
linkages and exocyclic groups. The lowest-energy docked poses
were retained for MD simulation. Docked poses were filtered
by glycosidic dihedral angle to exclude unfavorable high energy
carbohydrate conformations. Cutoffvalues for dihedral filtering
were chosen for each glycosidic linkage based on isoenergy
contours previously calculated with the MM3 force field from
Imberty et al. (1995). Conformations with dihedrals in the
following ranges were removed from the analysis: Fucα1-2Gal
ϕ < −130◦& 180◦< ψ < 360◦; GalNAcα1-3Gal ϕ > 240◦; Site Mapping pp
g
All BambL-blood group carbohydrate complexes were examined
using LigPlot (Wallace et al., 1995; Laskowski and Swindells,
2011). Only poses that passed the glycosidic torsion filter
requirements (see above), were used for site mapping, following a
previously developed method (Yuriev et al., 2001; Agostino et al.,
2009b, 2011, 2013; Dingjan et al., 2015a). In brief, each individual
hydrogen bond made by a particular BambL residue was counted
toward the hydrogen-bond tally. Non-polar vdW interactions
between a specific BambL residue and a carbohydrate residue
were counted as a single interaction toward the tally. The tallies
were normalized to percentages of the total number of hydrogen
bond or vdW interactions. Site maps were generated using
residue inclusion cutoffvalues for lectin-carbohydrate complexes
of 90% for hydrogen bonds, 0% for vdW interactions (Agostino
et al., 2013). Site map images were rendered using PyMOL
(Schrödinger, 2014d). Low energy blood-group and blood-group-related carbohydrate
structures were generated and simulation parameters produced
using the GLYCAM web portal (Woods, 2005; Kirschner et al.,
2008). The A and B determinants were modeled as trisaccharides
for comparison to previous binding data for the soluble type
A determinant (Audfray et al., 2012). The H type 1, H type
2, Lea and Lex determinants were modeled as tetrasaccharides
for consistency to previously determined binding data (Audfray
et al., 2012) and the Leb and Ley determinants were modeled
as tetrasaccharides to encompass the entire epitope. The library
of blood-group-related structures is shown in Supplementary
Figure 1. Frontiers in Pharmacology | www.frontiersin.org Generation of BambL-Blood Group
Complexes by Docking Complexes by Docking
To decide which of the crystallographic BambL receptor
structures to use in this study, we compared complex structures
predicted
by
re-docking
with
respective
crystallographic
complexes. The results of these cognate and cross-docking
experiments
are
shown
in
Table 1,
Figure 2. The
Lex
tetrasaccharide was poorly docked (RMSD > 2 Å) into all
BambL structures. However, all four lectin structures afforded
approximately equal performance when used as a receptor for
the other three carbohydrate ligands: overall RMSD values of
1.09–2.62 and 0.14–0.56 Å for the buried fucose (Fucα1-2Gal)
were observed. The crystallographic BambL structure from the
PDB ID: 3ZZV complex was used as the receptor structure for
site mapping and MD with all carbohydrates shown in Figure 1. In a second step, all blood group saccharides were docked in
BambL (PDB ID: 3ZZV) and the top docked poses were analyzed
for structural features relevant to recognition (Table 2). In all
cases except Lex, the majority of binding interactions were made
via a single buried fucose residue (Figure 3). The difucosylated
Leb and Ley possess two fucose residues (Fucα1-2Gal and Fucα1-
4GlcNAc in Leb or Fucα1-3GlcNAc in Ley) and therefore may
occupy the fucose-binding pocket in two ways. Of the docked Leb To decide which of the crystallographic BambL receptor
structures to use in this study, we compared complex structures
predicted
by
re-docking
with
respective
crystallographic
complexes. The results of these cognate and cross-docking
experiments
are
shown
in
Table 1,
Figure 2. The
Lex
tetrasaccharide was poorly docked (RMSD > 2 Å) into all
BambL structures. However, all four lectin structures afforded
approximately equal performance when used as a receptor for
the other three carbohydrate ligands: overall RMSD values of
1.09–2.62 and 0.14–0.56 Å for the buried fucose (Fucα1-2Gal)
were observed. The crystallographic BambL structure from the
PDB ID: 3ZZV complex was used as the receptor structure for
site mapping and MD with all carbohydrates shown in Figure 1. FIGURE 2 | Blood group carbohydrates docked into the BambL binding site
of PDB ID: 3ZZV (orange), compared to their respective experimentally
determined poses (green). The PDB IDs for experimental poses are indicated. For clarity, all carbohydrates are shown as the non-reducing-end trisaccharide
without hydrogen atoms. poses produced here, only the Fucα1-2Gal residue was predicted
in the binding pocket. Frontiers in Pharmacology | www.frontiersin.org Analysis of MD Simulations cValues shown in bold indicate cognate docking experiments. Hydrogen bonds in MD simulations were analyzed using the
Baker-Hubbard method implemented in the MDTraj (McGibbon
et al., 2015) software library. An occupancy value was assigned
to each hydrogen bond by calculating the percentage of
simulation frames in which the bond was present. Glycosidic
dihedral angles were measured using MDTraj and compared
to calculated isoenergy contours (see above). Carbohydrate
ring conformations were analyzed using Best Four-Member
Plane method from GLYCAM (Makeneni et al., 2014). CH-π
interactions were represented by measuring a shortest distance
from either of the fucose atoms C3, C4, C5, or C6 to atoms
of the indole ring of Trp74. Atom labeling corresponds to the
conventions of the PDB exchange dictionary (Berman et al.,
2003). FIGURE 2 | Blood group carbohydrates docked into the BambL binding site
of PDB ID: 3ZZV (orange), compared to their respective experimentally
determined poses (green). The PDB IDs for experimental poses are indicated. For clarity, all carbohydrates are shown as the non-reducing-end trisaccharide
without hydrogen atoms. Molecular Dynamics RMSD values compare the ligand portion common between
the docked and crystallographic ligand; RMSD values in brackets compare the fucose
portion of the docked ligand to the fucose portion of the crystallographic ligand. bCross-docking performed using the ligands used in site mapping and molecular
dynamics (Figure 1). Cognate docking performed using the ligand length present in the
crystallographic complex. cValues shown in bold indicate cognate docking experiments For all MD simulations in the NPT ensemble, temperature
was kept constant using the velocity rescaling thermostat coupled
with a time constant of 0.1 ps. Pressure was held constant at
1 bar using the Parrinello-Rahman barometer, coupled with
a time constant of 2 ps. Equations of motion were integrated
using a leap-frog integrator with a 2 fs timestep. Long-range
electrostatics were evaluated using the Particle Mesh Ewald
method. Cutoffvalues for Coulomb and vdW interactions were
set to 1.0 nm. Complexes with blood group carbohydrate ligands
were simulated in triplicate, complexes with blood-group-
related carbohydrate ligands were simulated in singlicate. Each
replicate was commenced using randomized velocities, resulting
in independent simulations with different initial velocities. RMSD of top docked pose to crystal structure (Å)a crystallographic complex. cValues shown in bold indicate cognate docking experiments. Molecular Dynamics y
MD
simulations
were
performed
using
Gromacs
5.0.4
(Berendsen et al., 1995; Van Der Spoel et al., 2005; Hess et al.,
2008; Pronk et al., 2013). Proteins were parameterized using
the AMBER99SB-ILDN (Lindorff-Larsen et al., 2010) forcefield. Carbohydrate topologies were generated using the GLYCAM06
(Kirschner et al., 2008) force field via the glycam.org web portal. The resulting AMBER-formatted topology was converted to
GROMACS format using the “acpype” tool (Sousa da Silva and
Vranken, 2012). The correctly formatted carbohydrate topology
was then combined with the protein topology to describe
the entire protein-carbohydrate system. Protein-carbohydrate
docked complexes were placed in a rhombic dodecahedral box
with a 10 Å minimum distance between solute and box wall,
and subsequently solvated using the TIP3P water model. To
maintain electrostatic neutrality, Na+ and Cl−counterions
were added by the genion module. To remove steric clashes
between nearby atoms, the system contents were minimized
using the steepest descent algorithm (maximum steps: 50,000). The positions and velocities of the solvent molecules and ions
were then equilibrated at constant volume and temperature
(NVT ensemble) using three restraint settings: with all protein
heavy atoms restrained for 100 ps, then with only backbone
atoms restrained for 100 ps (both at 10 K), followed by a 100 ps
equilibration without restraints at 300 K. Finally, the pressure
of the system was equilibrated for 300 ps without restraints
at constant atmospheric pressure (NPT ensemble) at 310 K. During all equilibration steps, positional restraints were applied
to protein residues using LINCS (Hess, 2007). The coordinates Frontiers in Pharmacology | www.frontiersin.org June 2017 | Volume 8 | Article 393 4 Molecular Simulations of BambL-Carbohydrate Recognition Dingjan et al. from the final equilibration step were used to begin production
simulation, which was conducted for 400 ns. TABLE 1 | Top scoring docked pose characterization for BambL-blood group
saccharide complexes. saccharide complexes. RMSD of top docked pose to crystal structure (Å)a
Ligand
3ZWE (1.75 Å)
3ZW2 (1.60 Å)
3ZZV (1.68 Å)
3ZW1 (1.60 Å)
Bb
1.68 (0.42)c
2.04 (0.25)
2.13 (0.36)
2.62 (0.28)
H1
1.80 (0.47)
1.09 (0.29)c
1.47 (0.14)
2.02 (0.27)
H2b
1.90 (0.39)
2.42 (0.39)
1.56 (0.25)c
1.52 (0.56)
Lex
9.47 (7.95)
4.61 (0.58)
6.94 (10.31)
7.01 (0.32)c
aThe experimental resolution of each crystallographic BambL complex is shown in
brackets beneath the PDB ID. Generation of BambL-Blood Group
Complexes by Docking bDihedral angles defined as: ϕ, O5-C1-O1-Cx; ψ, C1-O1-Cx-Cx+1. cExcluding hydrogen bonds involving the buried fucose residue. et al., 1995) energy maps (see Supplementary Figures 2, 3). An exception is the Fucα1-2Gal linkage, which is positioned in
between minima in the H type 1, H type 2, Leb and Ley top poses. In the A and B trisaccharide complexes, the Fucα1-2Gal linkage
adopted the lowest energy conformation. These results agree with
earlier BambL-blood group docking by Topin et al. (2013) in
which top docked pose glycosidic linkages also occupied a range
of energetic minima. Apart from interactions with the buried fucose residue,
additional hydrogen bonds are made between non-fucose
residues and amino acids in the four β-turn loops surrounding
the fucose-binding pocket (Table 2). The most frequently
participating residue, Asp30, interacts with non-fucose portions
of multiple saccharides (B, H1, Lea, Leb, and Ley). The imidazole
side-chain of Trp74 (which stacks against the buried fucose)
also donates a hydrogen bond to non-fucose residues in several
cases. In each case, the hydrogen bond is accepted by atoms in
a similar location: two residues away from the buried fucose,
at the GlcNAc 6-position (Leb, Ley), Gal/GalNAc 3-position (A,
B), or GlcNAc 2-position (H1). The presence of hydrogen bonds
between non-fucose portions and loop residues suggests that
BambL recognition may not rely solely on interactions with a
single buried fucose. Generation of BambL-Blood Group
Complexes by Docking As for the docked Ley poses, all of the top
20 ranked poses positioned the Fucα1-2Gal residue in the pocket,
with the exception of poses at rank 5 and 6 that predicted the
Fucα1-3GlcNAc residue in the fucose binding pocket. In a second step, all blood group saccharides were docked in
BambL (PDB ID: 3ZZV) and the top docked poses were analyzed
for structural features relevant to recognition (Table 2). In all
cases except Lex, the majority of binding interactions were made
via a single buried fucose residue (Figure 3). The difucosylated
Leb and Ley possess two fucose residues (Fucα1-2Gal and Fucα1-
4GlcNAc in Leb or Fucα1-3GlcNAc in Ley) and therefore may
occupy the fucose-binding pocket in two ways. Of the docked Leb As expected, recognition of the buried fucose (Fucα1-2Gal)
was governed by a conserved hydrogen-bonding network and a
single hydrophobic stacking interaction (Supplementary Table 1). Rather than interacting via a buried fucose, the Lex top docked
pose was placed “back-to-front” with the reducing end galactose
in the fucose-binding pocket, and the fucose directed away from
the protein. Frontiers in Pharmacology | www.frontiersin.org June 2017 | Volume 8 | Article 393 5 Molecular Simulations of BambL-Carbohydrate Recognition Dingjan et al. TABLE 2 | Top scoring docked pose characterization for BambL-blood group saccharide complexes. Ligand
Fucose RMSD (Å)a
Glycosidic dihedral anglesb
Hydrogen bondsc
Docking score (kcal/mol)
ϕ
ψ
ϕ
ψ
ϕ
ψ
A tri
0.53
Fucα1-2Gal
GalNAcα1-3Gal
GalNAc2-H6O...Asp30-OD1
−5.848
−106.8
55.9
81.3
76.8
GalNAc2-O4...Trp8-HE1
B tri
0.24
Fucα1-2Gal
Galα1-3Gal
Gla3-H6O...Asp30-OD1
−5.890
−107.9
63.9
49.5
45.8
Gla3-O3...Trp74-HE1
H1
0.43
Fucα1-2Gal
Galβ1-3GlcNAc
GlcNAcβ1-3Gal
GlcNAc2-H4O...Asp30-OD2
−6.879
−116.2
−129.6
−42.2
146.6
−106.1
159.2
GlcNAc2-O2N...Trp74-HE1
H2
0.38
Fucα1-2Gal
Galβ1-4GlcNAc
GlcNAcβ1-3Gal
Gal1-H3O...Tyr35-O
−6.597
−106.0
−88.0
−42.2
−91.8
−81.8
101.0
Gal1-H6O...Asp77-OD2
GlcNAc2-H6O...Gly76-O
Lea
0.30
Fucα1-4GlcNAc
Galβ1-3GlcNAc
GlcNAcβ1-3Gal
Gal4-H2O...Asp30-OD2
−5.706
−138.3
−147.3
−64.8
138.7
−68.1
96.6
Lex
10.22
Fucα1-3GlcNAc
Galβ1-4GlcNAc
GlcNAcβ1-3Gal
Gal1-O5...Ala38-H
−5.786
−76.4
151.5
−72.6
−112.7
−66.5
89.6
Gal1-O6...Trp79-HE1
Gal1-H6O...Glu26-OE1
Gal1-O4...Arg15-HH21
Gal1-H4O...Tyr35-OH
Fuc4-H2O...Asp30-OD2
Fuc4-H3O...Asp30-O
Gal3-H2O...Ser55-O
Gal3-O6...Thr11-HG1
Gal3-H6O...Ser13-OG
Leb
0.38
Fucα1-2Gal
Fucα1-4GlcNAc
Galβ1-3GlcNAc
Fuc2-H2O...Asp30-OD1
−7.065
−83.6
−98.8
−70.3
96.5
−39.5
166.6
Fuc2-O3...Trp8-HE1
GlcNAc1-O6...Trp74-HE1
GlcNAc1-H6O...Val57-O
GlcNAc1-HO1...Gly76-O
Ley
0.33
Fucα1-2Gal
Fucα1-3GlcNAc
Galβ1-4GlcNAc
Fuc2-H2O...Asp30-OD2
−7.007
−105.3
−138.3
−83.8
−51.3
−56.0
−103.7
GlcNAc1-H2N...Asp30-OD1
GlcNAc1-O1...Trp8-HE1
GlcNAc1-O6...Trp74-HE1
aCalculated for buried fucose residue heavy atoms between crystallographic saccharide (PDB ID: 3ZZV) and docked ligand. bDihedral angles defined as: ϕ, O5-C1-O1-Cx; ψ, C1-O1-Cx-Cx+1. Leb Ley aCalculated for buried fucose residue heavy atoms between crystallographic saccharide (PDB ID: 3ZZV) and docked ligand. Frontiers in Pharmacology | www.frontiersin.org Molecular Dynamics Simulations of
BambL-Blood Group Complexes To investigate the dynamic behavior of BambL-blood group
complexes, the lowest-energy poses generated by docking were
simulated in explicit solvent. For difucosylated Leb and Ley, the
lowest-energy poses with the Fucα1-2Gal residue in the fucose-
binding pocket were used. The poorly docked Lex complex was
also simulated, but quickly dissociated from the protein or was
unstable in the binding site (see Supplementary Figure 4). To
probe the dynamic behavior of the Lex binding interactions, the
crystallographic complex was used instead (PDB ID: 3ZW1). y
g
p
p
During MD simulations, all fucose-anchored blood group
saccharides (A, B, H type 1, H type 2, Lea, Leb, Ley) remained
bound to BambL without dissociation for the entire duration
(400 ns). Structural fluctuations in ligand RMSD were below 2 Å
in all bound complexes, reflecting relatively small changes in
ligand positions and geometries during the MD simulations (see
Supplementary Figure 5). Carbohydrate ring conformations were
found to generally adopt one of the two chair conformations
(1C4 or 4C1), while the GlcNAc rings in the H type 2, Lea, and
Lex exhibited some variation (see Supplementary Figure 6). A
similar hydrogen-bonding pattern was observed across all blood
group simulations (Figures 5, 6), featuring interactions between
the buried fucose residue and the fucose-binding pocket: Glu26
acidic group to O3 and O4 hydroxyl protons, Arg15 guanidinium
to O4 and O5 oxygen atoms, and Trp79 indole to O3 oxygen
atom. These hydrogen bonds were highly occupied (between
60 and 90% of simulation frames), with the exception of the
Glu26 hydrogen bonds in the Leb complex (50–60%). The high
occupancy of these hydrogen bonds indicates the dominant role
played by fucose in BambL-carbohydrate binding. FIGURE 3 | Binding site interactions involving fucose in BambL-blood group
saccharide docked poses. Hydrogen bonds shown as yellow dashes,
hydrophobic interactions shown as teal dashes. Non-polar hydrogens omitted
for clarity. FIGURE 4 | Site maps of a BambL subunit showing binding site residues
involved in docked pose interactions. Residues involved in 5% or fewer
interactions are colored white; residues involved in 20% or greater interactions
are colored red (for hydrogen bonding) or blue (for van der Waals). Residues
with intermediate involvement are shaded according to the color scale. In addition to the above interactions, a low-occupancy (up
to 30% of simulation frames) hydrogen bond was observed
between the Ala38 backbone amide proton and the buried
fucose 2-position hydroxyl oxygen atom. Site Mapping of BambL-Blood Group
Complexes Site mapping reveals binding site residues that are frequently
involved in interactions throughout an ensemble of docked
poses. Site maps for BambL-blood group complexes are shown
in Figure 4. These maps are based on docking results for all
carbohydrates shown in Figure 1. The BambL site maps agree Glycosidic dihedral angles in top docked poses lie close to
global or secondary minima in previously calculated (Imberty June 2017 | Volume 8 | Article 393 Frontiers in Pharmacology | www.frontiersin.org 6 Molecular Simulations of BambL-Carbohydrate Recognition Dingjan et al. fucose pocket, in close agreement with crystallographic bound
complexes. Site maps also revealed new interactions not seen in
crystal structures, identifying hydrogen bonding to Asp30 (7.1%)
and vdW interaction with Tyr35 (11.1%) as regularly occurring
across all docked poses. FIGURE 3 | Binding site interactions involving fucose in BambL-blood group
saccharide docked poses. Hydrogen bonds shown as yellow dashes,
hydrophobic interactions shown as teal dashes. Non-polar hydrogens omitted
for clarity. Molecular Dynamics Simulations of
BambL-Blood Group Complexes In contrast to the
highly occupied hydrogen bonds, this interaction engages a
backbone proton rather than a side-chain; combined with the
low occupancy, this suggests a less significant contribution
by this hydrogen bond to carbohydrate binding. Alongside
hydrogen-bonding interactions, stacking of the fucose C3-C4-
C5-C6 hydrophobic face against the Trp74 indole ring was
consistently maintained during simulation (see Supplementary
Figure 7). FIGURE 4 | Site maps of a BambL subunit showing binding site residues
involved in docked pose interactions. Residues involved in 5% or fewer
interactions are colored white; residues involved in 20% or greater interactions
are colored red (for hydrogen bonding) or blue (for van der Waals). Residues
with intermediate involvement are shaded according to the color scale. with crystallographic complexes, identifying multiple residues
in the fucose binding pocket known to interact with fucose
in crystallographic structures (PDB IDs: 3ZW2, 3ZZV, 3ZWE,
and 3ZW1; Audfray et al., 2012; Topin et al., 2013). Across the
docked pose ensemble, hydrogen bonds were frequently formed
to Arg15 (27.9%), Ala38 (11.6%), and Glu26 (13.7%), all located
within the fucose-binding pocket. Surprisingly, Trp79 (4.9%),
also in the crystallographic fucose pocket, was not often involved
throughout the docked pose ensemble. van der Waals (vdW)
interactions were frequently made with Trp74 (14.6%) in the Hydrogen bonds to non-fucose portions of the carbohydrate
ligands were formed at low to moderate occupancies (20–50%)
with fucose-binding residue Trp74 (Ley: 44%, Lea: 23%, Lex: 22%)
and surface residue Asp30 (B: 37%, H type 1: 44%, Leb: 24%, Ley:
31%). Glycosidic linkage conformations explored during MD
simulations occupy global, and occasionally secondary, minima Frontiers in Pharmacology | www.frontiersin.org June 2017 | Volume 8 | Article 393 7 Molecular Simulations of BambL-Carbohydrate Recognition Dingjan et al. FIGURE 5 | Hydrogen bond interactions in BambL-saccharide complexes during MD simulations shown for A trisaccharide, B trisaccharide, H type 1 and H type 2
blood groups. Atoms are named to the conventions of the PDB exchange dictionary. Grid cells are colored and labeled by average occupancy from three replicate
simulations. Occupancy values were calculated by dividing the number of frames in which the hydrogen bond exists by the total number of simulation frames. (Figure 7). As observed in docking, the Fucα1-2Gal linkage is
again an exception, adopting a position intermediate between
the two minima for the entire duration of simulation in the
H type 1, H type 2, Leb and Ley complexes. Molecular Dynamics Simulations of
BambL-Blood Group Complexes In the H type
1 and Leb complexes, this linkage explores a narrower range
is due to the presence of the protein. Force field-based energy
contours describe the energetic behavior of each linkage as an
unbound disaccharide in vacuum (Imberty et al., 1995), while
simulation of the bound complex introduces protein, water, and
other saccharide units within the tri- or tetrasaccharide, all of FIGURE 5 | Hydrogen bond interactions in BambL-saccharide complexes during MD simulations shown for A trisaccharide, B trisaccharide, H type 1 and H type 2
blood groups. Atoms are named to the conventions of the PDB exchange dictionary. Grid cells are colored and labeled by average occupancy from three replicate
simulations. Occupancy values were calculated by dividing the number of frames in which the hydrogen bond exists by the total number of simulation frames. FIGURE 5 | Hydrogen bond interactions in BambL-saccharide complexes during MD simulations shown for A trisaccharide, B trisaccharide, H type 1 and H type 2
blood groups. Atoms are named to the conventions of the PDB exchange dictionary. Grid cells are colored and labeled by average occupancy from three replicate
simulations. Occupancy values were calculated by dividing the number of frames in which the hydrogen bond exists by the total number of simulation frames. is due to the presence of the protein. Force field-based energy
contours describe the energetic behavior of each linkage as an
unbound disaccharide in vacuum (Imberty et al., 1995), while
simulation of the bound complex introduces protein, water, and
other saccharide units within the tri- or tetrasaccharide, all of
which influence conformational behavior. A recent example of
the influence of protein binding on carbohydrate conformation
is the Lex saccharide, which occupies well-characterized “closed” (Figure 7). As observed in docking, the Fucα1-2Gal linkage is
again an exception, adopting a position intermediate between
the two minima for the entire duration of simulation in the
H type 1, H type 2, Leb and Ley complexes. In the H type
1 and Leb complexes, this linkage explores a narrower range
of higher-energy conformations compared to H type 2 and
Ley. It is possible that this difference between the calculated
energetic minima and the conformations observed in simulation June 2017 | Volume 8 | Article 393 Frontiers in Pharmacology | www.frontiersin.org 8 Molecular Simulations of BambL-Carbohydrate Recognition Dingjan et al. Molecular Dynamics Simulations of
BambL-Blood Group Complexes FIGURE 6 | Hydrogen bond interactions in BambL-saccharide complexes during MD simulations shown for Lewis group saccharides. Atoms are named to the
conventions of the PDB exchange dictionary. Grid cells are colored and labeled by average occupancy from three replicate simulations. Occupancy values were
calculated by dividing the number of frames in which the hydrogen bond exists by the total number of simulation frames. conformations in solution and “open” conformations when
bound to the RSL lectin (Topin et al., 2016; defined by the relative
positions of the fucose and galactose rings). In the present study,
the Lex saccharide maintained an open conformation during MD
simulation, corresponding to shapes “Open V” and “Open II”
in the scheme defined by Topin et al. (2016) consistent with its
continuous occupation of the binding site during simulation (see
Supplementary Figure 8)
In
the
A
and
B
trisaccharide
simulations,
the
N-
acetylgalactosamine and non-reducing end galactose move
more freely than the saccharide occupying the same position in
the other ligands. The Fucα1-2Gal glycosidic linkage in these two
saccharides occupies two conformations, defined by variation
in the ψ-angle between −60◦and +100◦. The A trisaccharide
explores both, while the B trisaccharide only occupies the former
conformation (Figure 7) FIGURE 6 | Hydrogen bond interactions in BambL-saccharide complexes during MD simulations shown for Lewis group saccharides. Atoms are named to the
conventions of the PDB exchange dictionary. Grid cells are colored and labeled by average occupancy from three replicate simulations. Occupancy values were
calculated by dividing the number of frames in which the hydrogen bond exists by the total number of simulation frames. conformations in solution and “open” conformations when
bound to the RSL lectin (Topin et al., 2016; defined by the relative
positions of the fucose and galactose rings). In the present study,
the Lex saccharide maintained an open conformation during MD
simulation, corresponding to shapes “Open V” and “Open II”
in the scheme defined by Topin et al. (2016) consistent with its
continuous occupation of the binding site during simulation (see
Supplementary Figure 8)
In
the
A
and
B
trisaccharide
simulations,
the
N-
acetylgalactosamine and non-reducing end galactose move
more freely than the saccharide occupying the same position in
the other ligands. The Fucα1-2Gal glycosidic linkage in these two
saccharides occupies two conformations, defined by variation
in the ψ-angle between −60◦and +100◦. Docking and MD Simulations of Complexes
with Blood-Group-Related Carbohydrates binding modes were observed among the stable complexes,
exhibiting different hydrogen-bonding patterns (Figure 8). In
some complexes (2, 6, 34, 30), very few hydrogen bonds were
formed and were observed for only up to 30% of MD runs. These binding modes, while stable, did not feature significant
hydrogen-bonding interactions with BambL. y
g
g
In four cases (5, 19, 18, 20), the ligand was found to interact
with the fucose-binding pocket via a non-fucose saccharide
(galactose or N-acetylgalactosamine). While these non-fucose
binding modes do include hydrogen bonds to the three fucose
pocket residues (Arg15, Glu26, and Trp79), these interactions
are not as highly occupied as those made by fucose-containing
saccharides (10, 9, 1, 17). In non-fucose binding modes,
hydrogen-bond occupancies over 70% were observed for only
one or two interactions per ligand; for fucose-mediated binding,
all three pocket residues are engaged more than 70% of the time. The remaining 20 carbohydrates bound in a fucose-
dominated manner, forming hydrogen bonds at over 70%
occupancy between a fucose and all three residues of the
fucose-binding pocket. In most cases, additional hydrogen bonds
were formed with loop residues outside the fucose-binding
pocket, with occupancies ranging from 10 to 90%. The highly
stable (>70% occupancy) non-fucose hydrogen bonds involved
residues Asp30 and Thr36, located on loop 4. The acidic
sidechain of Asp30 projects toward the fucose-binding pocket,
accepting hydrogen bonds from saccharides not directly bonded
to the buried fucose. Thr36 is located further away from the
fucose-binding pocket, and accepts hydrogen bonds via the In four cases (5, 19, 18, 20), the ligand was found to interact
with the fucose-binding pocket via a non-fucose saccharide
(galactose or N-acetylgalactosamine). While these non-fucose
binding modes do include hydrogen bonds to the three fucose
pocket residues (Arg15, Glu26, and Trp79), these interactions
are not as highly occupied as those made by fucose-containing
saccharides (10, 9, 1, 17). In non-fucose binding modes,
hydrogen-bond occupancies over 70% were observed for only
one or two interactions per ligand; for fucose-mediated binding,
all three pocket residues are engaged more than 70% of the time. The potential for non-fucose binding interactions to form
in BambL-saccharide complexes was explored by simulating
complexes of 36 blood-group-related carbohydrates to the
protein (i.e., a focused carbohydrate library). Molecular Dynamics Simulations of
BambL-Blood Group Complexes The A trisaccharide
explores both, while the B trisaccharide only occupies the former
conformation (Figure 7) FIGURE 6 | Hydrogen bond interactions in BambL-saccharide complexes during MD simulations shown for Lewis group saccharides. Atoms are named to the
conventions of the PDB exchange dictionary. Grid cells are colored and labeled by average occupancy from three replicate simulations. Occupancy values were
calculated by dividing the number of frames in which the hydrogen bond exists by the total number of simulation frames. FIGURE 6 | Hydrogen bond interactions in BambL-saccharide complexes during MD simulations shown for Lewis group saccharides. Atoms are named to the
conventions of the PDB exchange dictionary. Grid cells are colored and labeled by average occupancy from three replicate simulations. Occupancy values were
calculated by dividing the number of frames in which the hydrogen bond exists by the total number of simulation frames. In
the
A
and
B
trisaccharide
simulations,
the
N-
acetylgalactosamine and non-reducing end galactose move
more freely than the saccharide occupying the same position in
the other ligands. The Fucα1-2Gal glycosidic linkage in these two
saccharides occupies two conformations, defined by variation
in the ψ-angle between −60◦and +100◦. The A trisaccharide
explores both, while the B trisaccharide only occupies the former
conformation (Figure 7). conformations in solution and “open” conformations when
bound to the RSL lectin (Topin et al., 2016; defined by the relative
positions of the fucose and galactose rings). In the present study,
the Lex saccharide maintained an open conformation during MD
simulation, corresponding to shapes “Open V” and “Open II”
in the scheme defined by Topin et al. (2016) consistent with its
continuous occupation of the binding site during simulation (see
Supplementary Figure 8). June 2017 | Volume 8 | Article 393 Frontiers in Pharmacology | www.frontiersin.org 9 Molecular Simulations of BambL-Carbohydrate Recognition Dingjan et al. FIGURE 7 | Glycosidic dihedral angles in BambL-blood group saccharide complexes during MD simulations. Dihedrals are defined as: ϕ, O5-C1-O1-Cx;
ψ, C1-O1-Cx-Cx+1. Contour plots color coding: gray, calculated energy landscapes of constituting linkages; brown, A; light blue B; green H1; dark blue, H2; cyan
Lea; pink, Leb; orange Lex; red Ley. Contour plot lines mark intervals of 1 kcal/mol. FIGURE 7 | Glycosidic dihedral angles in BambL-blood group saccharide complexes during MD simulations. Dihedrals are defined as: ϕ, O5-C1-O1-Cx;
ψ, C1-O1-Cx-Cx+1. Frontiers in Pharmacology | www.frontiersin.org Molecular Dynamics Simulations of
BambL-Blood Group Complexes Contour plots color coding: gray, calculated energy landscapes of constituting linkages; brown, A; light blue B; green H1; dark blue, H2; cyan
Lea; pink, Leb; orange Lex; red Ley. Contour plot lines mark intervals of 1 kcal/mol. Docking and MD Simulations of Complexes
with Blood-Group-Related Carbohydrates
Interactions
between
BambL
and
blood
group/tissue
carbohydrates was mediated mainly via the single buried
fucose, with occasional hydrogen bonds formed between non-
fucose atoms and residues on loops surrounding the binding
pocket. Identifying these non-fucose binding interactions may
provide opportunities to improve inhibitor affinity for BambL
beyond the current fucose-based inhibitors. Docking and MD Simulations of Complexes
with Blood-Group-Related Carbohydrates The related
carbohydrates ranged in size from di- to heptasaccharides
and
were
composed
of
fragments
of
blood
group
and
tissue determinant carbohydrates and elongated versions of
blood group carbohydrates bearing additional saccharides (for
structures of all library members, see Supplementary Figure 1). Most of these structures contain fucose moieties and were
expected to interact with BambL via the fucose-dominated mode
observed in crystallographic structures. To explore how non-
fucose residues (such as galactose and N-acetylgalactosamine)
might occupy the fucose-binding site, a selection of di- and
trisaccharides lacking fucose were also evaluated. Complexes
with BambL were assembled by docking and simulated in explicit
solvent for 400 ns. The remaining 20 carbohydrates bound in a fucose-
dominated manner, forming hydrogen bonds at over 70%
occupancy between a fucose and all three residues of the
fucose-binding pocket. In most cases, additional hydrogen bonds
were formed with loop residues outside the fucose-binding
pocket, with occupancies ranging from 10 to 90%. The highly
stable (>70% occupancy) non-fucose hydrogen bonds involved
residues Asp30 and Thr36, located on loop 4. The acidic
sidechain of Asp30 projects toward the fucose-binding pocket,
accepting hydrogen bonds from saccharides not directly bonded
to the buried fucose. Thr36 is located further away from the
fucose-binding pocket, and accepts hydrogen bonds via the Of the 36 complexes simulated, 28 remained stably engaged
without dissociation of the ligand into bulk solvent. Multiple June 2017 | Volume 8 | Article 393 Frontiers in Pharmacology | www.frontiersin.org 10 Molecular Simulations of BambL-Carbohydrate Recognition Dingjan et al. FIGURE 8 | Hydrogen bonding occupancy of blood-group-related saccharides during MD simulations. Saccharide names indicate the ligand moieties interacting with
BambL during simulation. backbone carbonyl oxygen atom. A less-occupied hydrogen bond
(up to 67%) is formed to the indole nitrogen of Trp74, concurrent
with hydrophobic stacking against a buried fucose. Finally, Tyr35
donates a hydrogen bond via the phenolic hydroxyl to compound
28 and 33 (and additionally to the non-fucose compound 5). The
fucose-dominated binding modes featuring highest occupancy of
non-fucose hydrogen bonds involved carbohydrates 21 and 33,
illustrated in Supplementary Figure 9. from their extreme flexibility, a large number of hydroxyl
groups, leading to the formation of (often) extensive hydrogen-
bonding networks, and the formation of crucial CH/π stacking
interactions between the C-H bonds of the carbohydrates (on
their hydrophobic faces) and aromatic side chains of the protein
(Agostino et al., 2009a, 2012a). Docking and MD Simulations of Complexes
with Blood-Group-Related Carbohydrates Also, carbohydrate ligands are
modular, and different residues (e.g., galactose vs. glucose) are
able to establish highly similar interactions with the binding
site. We have previously validated Glide and tested a range of
other docking programs for structural prediction of carbohydrate
complexes with antibodies (Agostino et al., 2009a, 2012b) and
lectins (Agostino et al., 2011). We have demonstrated that, as the
result of all the above-mentioned challenges, docking programs
and scoring functions are not always able to predict the native
binding pose faithfully as the top docked pose. To overcome
this shortcoming and to harness the recognition information
embedded in the docking output, we have developed a site
mapping methodology that takes into account an ensemble
of docked poses and identifies binding site residues critically
involved in recognition of a ligand or ligand family (Yuriev et al.,
2001, 2002; Agostino et al., 2013; Dingjan et al., 2015a). Combining all the BambL residues involved in hydrogen
bonds
to
fucose
and
non-fucose
saccharides
presents
a
perspective of the target site that incorporates a wider view of
BambL-saccharide recognition, considering multiple interaction
points across the protein surface (Figure 9). This view of the
BambL binding site presents opportunities for future inhibitor
design to consider regions outside the fucose-binding pocket. Frontiers in Pharmacology | www.frontiersin.org DISCUSSION We have investigated the molecular aspects of carbohydrate
recognition of the B. ambifaria lectin by computational methods:
docking, site mapping, and MD. Molecular docking has been
shown to be extremely useful for structural predictions, if not
affinity calculations (Yuriev et al., 2015). However, docking
carbohydrate ligands presents a number of challenges stemming In this study, docking with Glide produced reasonable top
poses for a range of BambL complexes with blood group
carbohydrates (Table 2). Using the BambL structure from PDB Frontiers in Pharmacology | www.frontiersin.org June 2017 | Volume 8 | Article 393 11 Molecular Simulations of BambL-Carbohydrate Recognition Dingjan et al. FIGURE 9 | BambL binding site showing residues implicated in saccharide binding. Purple: Residues which form hydrogen bonds with the buried fucose saccharide. Orange: Residues which form hydrogen bonds with non-fucose saccharides. Green: Residues which participate in both hydrophobic and hydrogen bonding
interactions. FIGURE 9 | BambL binding site showing residues implicated in saccharide binding. Purple: Residues which form hydrogen bonds with the buried fucose saccharide. Orange: Residues which form hydrogen bonds with non-fucose saccharides. Green: Residues which participate in both hydrophobic and hydrogen bonding
interactions. FIGURE 9 | BambL binding site showing residues implicated in saccharide binding. Purple: Residues which form hydrogen bonds with the buried fucose saccharide. Orange: Residues which form hydrogen bonds with non-fucose saccharides. Green: Residues which participate in both hydrophobic and hydrogen bonding
interactions. FIGURE 9 | BambL binding site showing residues implicated in saccharide binding. Purple: Residues which form hydrogen bonds with the buried fucose saccharide. Orange: Residues which form hydrogen bonds with non-fucose saccharides. Green: Residues which participate in both hydrophobic and hydrogen bonding
interactions. ID: 3ZZV gave accurate complex prediction for the B, H type
1 and H type 2 saccharides and accurate fucose placement for
the A, Lea, Leb, and Ley determinants. All these complexes
featured a buried fucose residue (Fucα1-2Gal), providing the
majority of hydrogen-bonding interactions, and conformational
ranges reflective of predicted energetic minima (Imberty et al.,
1995) and relevant experimental structures (Yuriev et al., 2005;
Dingjan et al., 2015b). Notably, the distances between fucose
carbon atoms and the geometric centers of the imidazole phenyl
and pyrrole component ring systems of Trp74 (Supplementary
Material, Table S1) are similar to reported geometries for fucose
CH/π dispersion interactions of a closely related lectin, RSL
(Wimmerova et al., 2012). Frontiers in Pharmacology | www.frontiersin.org DISCUSSION As in the RSL-fucose complex, the
C6 atom interacts with the pyrrole part of the imidazole ring
(distance of 3.76 ± 0.3 Å), while C3 is further than 4 Å away. Unlike the RSL complex, C5 also interacts with the pyrrole ring
(distance of 3.83 ± 0.1 Å), rather than the phenyl ring, which is
further than 4 Å from the entire non-polar plane. to consider alternative binding modes while MD also explicitly
accounts for the role of water, mediating interactions of BambL
to carbohydrates. We have identified the atomic scale binding interactions
that facilitate recognition of fucosylated human blood group
saccharides by BambL. A network of hydrogen bonds combined
with a single hydrophobic stacking interaction between the
buried fucose and amino acids in the fucose-binding pocket
account for the majority of binding interactions (Figure 3). These structural features of the fucose-driven recognition closely
agree with experimental characterization of BambL-carbohydrate
binding profile by glycan array, which has demonstrated a
preference for short, fucose-bearing saccharides, with the fucose
monosaccharide among the most highly ranked binders (Audfray
et al., 2012). However, this fucose-driven recognition motif
does not explain the specificity profile of BambL compared to
other related fucose-binding lectins. Namely, the interactions
between BambL and fucosylated saccharides are highly similar
to those found in complexes featuring other six-bladed β-
propeller fucose-binding lectins: found in fungi [Aleuria aurantia
lectin, AAL (Fujihashi et al., 2003; Wimmerova et al., 2003);
Aspergillus fumigatus lectin, AFL (Houser et al., 2013); Aspergillus
oryzae lectin, AOL (Makyio et al., 2016)] and bacteria [Ralstonia
solanacearum lectin, RSL (Kostlánová et al., 2005)]. Members of Detailed elaboration of structural aspects of molecular
recognition requires expanding the single snapshot view afforded
by crystal structures or top docked poses. To that effect, we
have undertaken site mapping and MD investigations in order to
identify BambL residues critical for recognition of blood group
carbohydrates. The advantage of site mapping lies in its ability June 2017 | Volume 8 | Article 393 12 Molecular Simulations of BambL-Carbohydrate Recognition Dingjan et al. this lectin family bind fucose via the same interactions: hydrogen
bonds between O2 and a backbone amide proton, O3 and indole
nitrogen, O3 and O4 to a shared carboxylate moiety, and O4 and
O5 to a shared guanidinium moiety. In a previous docking study
of RSL-fucose recognition by Mishra et al. (2012) the same suite
of interactions was reported. SUPPLEMENTARY MATERIAL In summary, the present work details the recognition of
fucosylated human blood group determinants by BambL,
quantifies the occupancy of hydrogen bonding interactions,
and identifies opportunities for targeting residues outside
the fucose-binding pocket. Recognition mainly involves the The Supplementary Material for this article can be found
online
at:
http://journal.frontiersin.org/article/10.3389/fphar. 2017.00393/full#supplementary-material DISCUSSION fucose monosaccharide through a network of highly occupied
hydrogen-bonding interactions to Arg15, Glu26, and Trp79, and
a lower occupancy interaction with Ala38. An additional stacking
interaction between the fucose hydrophobic face and Trp74 is
also highly occupied in MD simulations. Hydrogen bonds to
non-fucose saccharides were formed in complexes with Ley, Leb,
Lea, H1, H2, and B trisaccharide and in multiple complexes
involving blood-group-related saccharides. The most occupied
interactions involved Asp30, Thr36, Trp74, and to a lesser
degree Tyr35. Carbohydrate recognition by BambL is therefore
proposed to be driven by interactions in the fucose-binding
site and further stabilized by satellite interactions between non-
fucose saccharides and surface residues outside the fucose-
binding pocket. The analysis of carbohydrate recognition by
BambL presented in this study lays the foundation for the
development of fucomimetic molecules able to bind to BambL. Such molecules have potential as anti-adhesives for the treatment
of B. ambifaria infection in cystic fibrosis patients. p
Despite the common binding mode, these lectins prefer
different blood group determinants: AAL exhibits broad
specificity, while AFL prefers Ley, and RSL prefers saccharides
featuring Fucα1-2 and Fucα1-6 moieties (blood group A,
B, and H and core of N-glycans). Varied blood group
specificity has been proposed to arise from steric hindrance
around the fucose-binding pocket, preventing strong binding
to most branched carbohydrate structures (Fujihashi et al.,
2003). Glycan array screening shows generally decreased
binding to branched carbohydrates compared to mono- and
disaccharides for these lectins, emphasizing the importance
of steric effects (Houser et al., 2013). Additionally, the non-
selective AAL lacks steric hindrance around the fucose-binding
pocket: in a bound complex featuring the disaccharide Fucα1-
6GlcNAcβ1-OMe, transferred NOE experiments confirmed
conformational flexibility around the glycosidic linkage (Weimar
and Peters, 1994). However, steric hindrance alone does not
fully explain blood group selectivity in this lectin family. AFL binds the difucosylated Ley more strongly than the
corresponding monofucosylated saccharide, H-type 2, despite
similar steric complementarity to the binding site (Houser
et al., 2013). We suggest that stabilizing interactions outside the
fucose-binding pocket (as observed in simulations of BambL
complexed with blood-group-related saccharides) play a role in
saccharide binding in the 6-bladed β-propeller lectin family more
generally. FUNDING This
work
was
supported
by
a
Victorian
Life
Sciences
Computation Initiative (VLSCI) grant number VR0250 on its
Peak Computing Facility at the University of Melbourne, an
initiative of the Victorian Government, Australia, and with
the assistance of resources from the National Computational
Infrastructure (NCI), which is supported by the Australian
Government, and the Multi-modal Australian ScienceS Imaging
and Visualization Environment (MASSIVE) via grant Y96. TD is supported by an Australian Postgraduate Award (APA)
scholarship. AI and SP are supported by CNRS, Université
Grenoble Alpes through Glyco@Alps (ANR-15-IDEX-02) and
Labex ARCANE (ANR-11-LABX-0003-01). PR is supported
by an RMIT University Vice Chancellor’s Senior Research
Fellowship. Interactions with non-fucose residues are not as highly
occupied as interactions with the fucose. However, they
contribute to a wider view of BambL-carbohydrate recognition,
considering multiple interaction points across the protein
surface. They include hydrogen bonding to Asp30, Tyr35, Thr36,
and Trp74 and hydrophobic contacts with Tyr35 (Figure 9). These contacts outside the fucose-binding pocket could be
employed in future inhibitor design for BambL to address issues
of opportunistic infections. AUTHOR CONTRIBUTIONS Each author has contributed significantly to the submitted
work. TD and EY conceived and designed the experiments. TD
performed the experiments. TD, EY, and PR analyzed the data. TD, AI, SP, EY, and PR wrote the paper. All authors read and
approved the final manuscript. p
j j
g
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conducted in the absence of any commercial or financial relationships that could
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terms. Copyright © 2017 Dingjan, Imberty, Pérez, Yuriev and Ramsland. This is an open-
access article distributed under the terms of the Creative Commons Attribution
License (CC BY). The use, distribution or reproduction in other forums is permitted,
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032.x June 2017 | Volume 8 | Article 393 Frontiers in Pharmacology | www.frontiersin.org 16
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Real World Evidence in Medical Cannabis Research
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Therapeutic innovation & regulatory science
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cc-by
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Rishi Banerjee1 · Simon Erridge1,2 · Oliver Salazar1 · Nagina Mangal1 · Daniel Couch3 · Barbara Pacchetti4 ·
Mikael Hans Sodergren1,2,4,5 Received: 25 July 2021 / Accepted: 12 October 2021 / Published online: 8 November 2021
© The Author(s) 2021 Real World Evidence in Medical Cannabis Research Rishi Banerjee1 · Simon Erridge1,2 · Oliver Salazar1 · Nagina Mangal1 · Daniel Couch3 · Barbara Pacchetti4 ·
Mikael Hans Sodergren1,2,4,5 Therapeutic Innovation & Regulatory Science (2022) 56:8–14
https://doi.org/10.1007/s43441-021-00346-0 Therapeutic Innovation & Regulatory Science (2022) 56:8–14
https://doi.org/10.1007/s43441-021-00346-0 COMMENTARY Abstract Background Whilst access to cannabis-based medicinal products (CBMPs) has increased globally subject to relaxation of
scheduling laws globally, one of the main barriers to appropriate patient access remains a paucity of high-quality evidence
surrounding their clinical effects. f
Discussion Whilst randomised controlled trials (RCTs) remain the gold-standard for clinical evaluation, there are notable
barriers to their implementation. Development of CBMPs requires novel approaches of evidence collection to address these
challenges. Real world evidence (RWE) presents a solution to not only both provide immediate impact on clinical care, but
also inform well-conducted RCTs. RWE is defined as evidence derived from health data sourced from non-interventional
studies, registries, electronic health records and insurance data. Currently it is used mostly to monitor post-approval safety
requirements allowing for long-term pharmacovigilance. However, RWE has the potential to be used in conjunction or as
an extension to RCTs to both broaden and streamline the process of evidence generation. p
g
Conclusion Novel approaches of data collection and analysis will be integral to improving clinical evidence on CBMPs. RWE can be used in conjunction or as an extension to RCTs to increase the speed of evidence generation, as well as reduce
costs. Currently, there is an abundance of potential data however, whilst a number of platforms now exist to capture real
world data it is important the right tools and analysis are utilised to unlock potential insights from these. Abbreviations
CBD
Cannabidiol
CBMPS
Cannabis-based medicinal products
FDA
Food and Drug Administration
NHS
National Health Service
NIHR
National Institute for Health Research
RCTs
Randomised Controlled Trials
RWE
Real world evidence
SATs
Supplemented single arm trials
THC (−)
Trans-Δ9-tetrahydrocannabinol
Background Cost Production methods and import costs mean that CBMPs
are typically expensive, adding further to high research
costs [18]. Research has therefore focused on compounds
under patent as opposed to generic CBMPs where research
outcomes fail to provide a similar return on investment for
licensed producers and pharmaceutical companies. Histori-
cally, clinical trials on CBMPs were funded privately, which
may be associated with potential reporting biases [19]. Background Cannabis-based medicinal products (CBMPs) are a collec-
tive term to describe a preparation or other product that
contains cannabis or its derivatives for medicinal use in
humans [1]. There are significant barriers to the integra-
tion of CBMPs within treatment pathways including ongo-
ing stigma, cost, education, complex pharmacology and a
paucity of evidence to inform international and national
guidelines [2, 3]. Limited evidence, does, however, support
the role of CBMPs in conditions such as chronic pain, neu-
rological disorders, and psychiatric disease [4]. There is also
growing evidence of side effects and how the severity and
incidence of side effects may differ between patients [4]. Vol:1 .(12345678
3 Therapeutic Innovation & Regulatory Science (2022) 56:8–14 9 The quality of evidence, however, is often insufficient in
the opinion of insurers, regulators, and guideline bodies [5]. active compounds. Heat exposure and vaporisation of dried
flower or extracted oils changes the underlying phytocan-
nabinoid composition compared to the original unprocessed
dry flowers, increasing the proportion of decarboxylated
cannabinoids [15, 16]. Assessment of efficacy using RCTs
in isolation will therefore ultimately fail to identify the most
appropriate CBMP for each clinical scenario [17]. The National Institute for Health and Care Excellence in
the UK has only recommended licensed CBMPs for a limited
range of indications [6]. Changes to scheduling as recom-
mended by the World Health Organisation, and within indi-
vidual countries, recognises the potential medicinal value of
cannabis and removes barriers for clinical and research use
[1, 7]. However, widespread stigma, complex pharmacol-
ogy, funding, and challenges in sustaining adequate supply
of consistent products continue to act as barriers for clinical
research. Barriers to Controlled Clinical Trials
for Medical Cannabis RCTs are not infallible—they are expensive and time con-
suming. Globally $100 billion USD is spent on biomedical
research [9]. In the UK, the National Institute for Health
Research (NIHR) provides £80 million GBP in funding for
clinical trials [10]. Yet, their narrow scope can lack ecologi-
cal validity to real-world circumstances and therefore lack
generalisability in more diverse populations. There are also
specific barriers to conducting RCTs using CBMPs. RCTs are possible with CBMPs; however, the above
issues present legitimate challenges. In many chronic dis-
eases there is a need for novel therapeutics and CBMPs are
therefore being utilised based on best available evidence. Due to the challenges in developing CBMPs through a
traditional drug development pipeline, the exploration of
its utility should not be limited to traditional methods. It
is important that we capture a suite of real-world evidence
(RWE) to inform prescribing guidelines, regulations, and
clinical trials. By leaning on RWE there is an opportunity
to improve the quality and design of RCTs and clinical evi-
dence in general, via a top-down approach [20]. Complex Pharmacology In addition to cannabidiol (CBD) and (−)-trans-Δ9-
tetrahydrocannabinol (THC) there are over 140 cannabi-
noids, as well as flavonoids, terpenes, and other compounds
within the flower of different cannabis plants [8]. These
can each potentially affect the clinical outcomes observed
between CBMPs due to their individual and collective
effects [11]. The concentrations of each compound are
influenced by the genetics and environment each plant is
grown in producing a distinct chemical profile. The result
of a clinical trial for one finished pharmaceutical product,
therefore, cannot be extrapolated to all CBMPs, due to their
heterogeneity. However, current evidence reviews often fail
to account for this [12, 13].f Placebo‑control An appropriately blinded assessment against placebo or
active therapy is the optimal design for RCTs. It has been
difficult to identify a placebo that cannot be distinguished
against an active CBMP according to absence of both vaso-
active and psychoactive effects, as well as the typical aroma
associated with cannabis [15]. This presents a challenge to
adequate blinding. Randomised controlled trials (RCTs) are necessary and
should continue to be the standard against which medical
evidence is upheld. However, they are expensive, time con-
suming and subject to their own limitations [8]. Whilst these
are awaited, there is a requirement to generate evidence of
potential benefits and harms to inform policy and clinical
practice. Real World Evidence RWE is defined as evidence derived from health data
sourced from non-interventional studies, registries, elec-
tronic health records and insurance data as opposed to the
highly controlled setting of RCTs [21]. There is an abun-
dance of this unstructured data, however, the necessary
frameworks and governance are needed for the application
of this data [22]. It is currently used extensively to monitor
post-approval pharmacovigilence [23]. There is clear evi-
dence of benefit in using population-based data to detect The route of administration further affects the pharma-
cokinetics of CBMPs and the associated outcome of any
trial. CBMPs can be administered sublingually, trans-
dermally, via inhalation, or orally [14]. This subsequently
affects the distribution, biotransformation and elimination of 1 3 Therapeutic Innovation & Regulatory Science (2022) 56:8–14 10 safety events associated with specific medications to imple-
ment restrictions to reduce harm [21]. depression and pain the study also reported negative aspects
of cannabis consumption, for example driving under the
influence (72% of patients) [27]. These are findings which
are unlikely to be reported by patients in controlled clinical
trials for fear of repercussions, or strict inclusion criteria. It can also be useful in collecting data in rare conditions
whereby recruitment to RCTs can be limited by the need for
defined trial sites.fi Consistent use of RWE to aid regulatory decision mak-
ing is yet to be normalised, but the promise is apparent [21]. Recently, regulator-supported initiatives have highlighted
the desire to incorporate RWE into licensing and guidelines,
developing a framework which can incorporate its insights
into decisions regarding safety and effectiveness [21, 22]. It is important that studies standardise their methodology
according to those set out by regulatory authorities to ensure
research has the greatest impact [21, 22]. Moreover, they
should seek to directly address questions set out by govern-
ing bodies as areas where there is insufficient research [24]. i
RWE can improve the efficiency of clinical trials by gen-
erating hypotheses, refining eligibility criteria, and explor-
ing drug development tools. Registries can be used to form
an infrastructure to conduct a clinical trial, lowering costs
whilst maintaining high evidence quality [28]. In supple-
mented single arm trials the controls are derived from RWE-
data sets, providing the opportunity for patient centric study
designs. RCTs can also be augmented with real-world data to
increase the size of the control group to increase the power
of the study. Limitations of Real‑World Evidence RWE is already being incorporated into the scientific
literature on cannabis (Table 1). Early examples utilised
state-level records to examine the effects of cannabis laws
on opioid misuse. Subsequently there have been examples of
online and self-administered survey tools analysing national
outcomes. More recently there has been a focus on collect-
ing evidence from clinical registries and databases with evi-
dence generated from patient-reported outcome measures
and long-term pharmacovigilance. RWE, however, does have limits to its utility. There is varia-
tion in the quality and provenance of the data stored in elec-
tronic medical records [5]. Furthermore, insurance records
typically use coding specific for reimbursement purposes
and may not provide all clinically relevant information. RWE can require complex statistical expertise to deduce
valid conclusions. Another limitation is the lack of randomisation, con-
trolled variables and internal validity. This can make it
more difficult to derive causative mechanisms behind clini-
cal outcomes. However, this is also one of the strengths of
these studies, allowing for generalisability to true clinical
practice [22]. Treatment assignment based on the physician
as opposed to randomisation, creates selection bias and more
specifically stigma biases. RCTs, therefore, are still neces-
sary to establish a strong causal relationship between medi-
cation and outcomes [30]. Real World Evidence These study designs are particularly useful for
rare diseases where participant recruitment is challenging
[29]. Types of Real‑World Evidence for Medical
Cannabis NHS England and NHS improvement published a review on
the barriers to accessing CBMPs in the UK [3]. Their recom-
mendations included the need for the collection of structured
data, and the development of methods to further support the
generation of new evidence, for patients who cannot enrol
onto relevant RCTs. Comparison of Real‑World Evidence
and Controlled Clinical Trials Between these study designs it is important to be aware
of potential divergence in reported outcomes. RWE has
broader inclusion criteria, accounting for factors like non-
standard dosing, and is not limited by scope of disease,
thereby improving ecological validity [25]. However, some
studies have concluded there is little difference between
results obtained via RCTs and observational studies [26]. RWE typically has longer patient follow-up and may conse-
quently capture rare but important adverse effects that are
not detected within RCTs. Pharmacovigilance is therefore
widely accepted as one of the most important roles of RWE. Conclusion [38]
Gulbransen et al. [39]
Rapin et al. [40]
Analysis of registries
Habib et al. [27]
Ueberall et al. [41]
Mahabir et al. [42]
Databases
Quebec Cannabis Registry (Canada) [43]
Medical Cannabis Real World Evidence [44–46
UK Medical Cannabis Registry (UK) [47, 48]
Project Twenty21 (UK) [49, 50]
Emerald Clinics (Australia) [51–53] Anonymous online survey analysing medical cannabis users and which conditions they
use it to treat focusing on patient perception of efficacy, and physical and mental health
Patients who were registered to a federally authorized licenced producer in Canada were
requested to fill out a self-administered survey i
Cannabis As Medicine Survey (CAMS-18) was an online, anonymous survey given to
patients recruited mainly via social media in Australia. The consequent analysis looked
at patterns of use and perspectives on medical cannabis Time series analysis of medical cannabis laws and state-level death certificate data in the
United States from 1999 to 2010 Time series analysis of medical cannabis laws and state-level death certificate data in the
United States from 1999 to 2010 Analysed data from drug arrest data from the Maine Diversion Alert Program regarding
diversion of prescription medications subject to scheduling, such as cannabis Analysed data from drug arrest data from the Maine Diversion Alert Program regarding
diversion of prescription medications subject to scheduling, such as cannabis Retrospective cohort study looking at the association between enrolment in the New
Mexico Medical Cannabis Program and opioid prescription use Retrospective cohort study looking at the association between enrolment in the New
Mexico Medical Cannabis Program and opioid prescription use Cross sectional study of medical cannabis use from a cannabis dispensary in California. The study highlighted perceived benefits in conditions where medical cannabis had not
yet been licensed An audit of all patients who presented to a Cannabis Care clinic in New Zealand. This
study highlighted the anxiolytic effects of cannabidiol A retrospective observational study looking at patients who had been prescribed CBD
treatment from data collated from a network of clinics Looked at the efficacy of medical cannabis for treatment of fibromyalgia. It used data
from two hospital registries. The relevant patients also filled out a questionnaire Evaluated RWD collected from the German Pain e-Registry. It looked at the efficacy
and tolerability of an oromucosal spray composed of THC: CBD. Conclusion The registry was
developed by the Institute of Neurological Sciences on behalf of the German Pain
Association Analysed data from a registry which contained data on some medical cannabis evalua-
tion clinics in the United States. Database owned by CB2 Insights Analysed data from a registry which contained data on some medical cannabis evalua-
tion clinics in the United States. Database owned by CB2 Insights Collated clinical data collected from users of dried medical cannabis with 6000 partici-
pants in Quebec Collated clinical data collected from users of dried medical cannabis with 6000 partici-
pants in Quebec A Canadian, prospective, non-interventional, observational study led by the University
Health Network in Toronto. It aims to explore the benefits of medical cannabis in an
observational setting for adults with conditions such as chronic pain, anxiety or depres-
sion. The study will evaluate the efficacy and safety of a range of CBMPs selected by
the patients themselves after assessment by a clinician. The study currently aims to
recruit 2000 participants, with completion due in 2022 A Canadian, prospective, non-interventional, observational study led by the University
Health Network in Toronto. It aims to explore the benefits of medical cannabis in an
observational setting for adults with conditions such as chronic pain, anxiety or depres-
sion. The study will evaluate the efficacy and safety of a range of CBMPs selected by
the patients themselves after assessment by a clinician. The study currently aims to
recruit 2000 participants, with completion due in 2022 Captures clinical data on prescribed CBMP formulations, adverse events and patient-
reported outcome measures across a myriad of conditions at defined timepoints. The
principal aim is to generate evidence demonstrating the effect of CBMPs on health
status or health-related quality of life and generate more accurate calculations of
adverse effects. Published early experience of improvement in health-related quality of
life, sleep and anxiety outcomes across a range of conditions Registry platform for data collection from patients provided prescriptions by licensed
producers at subsidised cost for enrolment. Limited to six specific conditions, openly
aiming to change healthcare funding decisions. Aims to recruit 20,000 patients with
initial data analysis due in 2022 Registry platform for data collection from patients provided prescriptions by licensed
producers at subsidised cost for enrolment. Limited to six specific conditions, openly
aiming to change healthcare funding decisions. Conclusion CBMPs are a complex range of pharmaceuticals which
pose challenges to traditional pathways of drug develop-
ment and translation. Development of CBMPs requires
novel approaches of evidence collection to address these
challenges. RWE can be used in conjunction or as an
extension to RCTs to both broaden and streamline the
process of evidence generation. Currently, there is an RWE can bring further clarity on questions that remain
unanswered in RCTs. A recent study utilised anonymised
surveys of patients with fibromyalgia who consumed canna-
bis flower [27]. In addition to reporting positive outcomes on RWE can bring further clarity on questions that remain
unanswered in RCTs. A recent study utilised anonymised
surveys of patients with fibromyalgia who consumed canna-
bis flower [27]. In addition to reporting positive outcomes on 1 3 Therapeutic Innovation & Regulatory Science (2022) 56:8–14 11 Table 1 Examples of Real World Evidence Generation for Cannabis-Based Medicinal Products
Regional/National Survey Studies
Ware et al. [31]
Analysis of self-administered questionnaire study conducted in the UK between 1998
and 2002
Sexton et al. [32]
Anonymous online survey analysing medical cannabis users and which conditions they
use it to treat focusing on patient perception of efficacy, and physical and mental health
Lucas et al. [33]
Patients who were registered to a federally authorized licenced producer in Canada were
requested to fill out a self-administered survey
Lintzeris et al. [34]
Cannabis As Medicine Survey (CAMS-18) was an online, anonymous survey given to
patients recruited mainly via social media in Australia. The consequent analysis looked
at patterns of use and perspectives on medical cannabis
Government records analysis
Bachhuber et al. [35]
Time series analysis of medical cannabis laws and state-level death certificate data in the
United States from 1999 to 2010
Piper et al. [36]
Analysed data from drug arrest data from the Maine Diversion Alert Program regarding
diversion of prescription medications subject to scheduling, such as cannabis
Vigil et al. [37]
Retrospective cohort study looking at the association between enrolment in the New
Mexico Medical Cannabis Program and opioid prescription use
Analysis of clinic/dispensary data
Bonn Miller et al. [38]
Cross sectional study of medical cannabis use from a cannabis dispensary in California. The study highlighted perceived benefits in conditions where medical cannabis had not
yet been licensed
Gulbransen et al. [39]
An audit of all patients who presented to a Cannabis Care clinic in New Zealand. Conclusion This
study highlighted the anxiolytic effects of cannabidiol
Rapin et al. [40]
A retrospective observational study looking at patients who had been prescribed CBD
treatment from data collated from a network of clinics
Analysis of registries
Habib et al. [27]
Looked at the efficacy of medical cannabis for treatment of fibromyalgia. It used data
from two hospital registries. The relevant patients also filled out a questionnaire
Ueberall et al. [41]
Evaluated RWD collected from the German Pain e-Registry. It looked at the efficacy
and tolerability of an oromucosal spray composed of THC: CBD. The registry was
developed by the Institute of Neurological Sciences on behalf of the German Pain
Association
Mahabir et al. [42]
Analysed data from a registry which contained data on some medical cannabis evalua-
tion clinics in the United States. Database owned by CB2 Insights
Databases
Quebec Cannabis Registry (Canada) [43]
Collated clinical data collected from users of dried medical cannabis with 6000 partici-
pants in Quebec
Medical Cannabis Real World Evidence [44–46]
A Canadian, prospective, non-interventional, observational study led by the University
Health Network in Toronto. It aims to explore the benefits of medical cannabis in an
observational setting for adults with conditions such as chronic pain, anxiety or depres-
sion. The study will evaluate the efficacy and safety of a range of CBMPs selected by
the patients themselves after assessment by a clinician. The study currently aims to
recruit 2000 participants, with completion due in 2022
UK Medical Cannabis Registry (UK) [47, 48]
Captures clinical data on prescribed CBMP formulations, adverse events and patient-
reported outcome measures across a myriad of conditions at defined timepoints. The
principal aim is to generate evidence demonstrating the effect of CBMPs on health
status or health-related quality of life and generate more accurate calculations of
adverse effects. Published early experience of improvement in health-related quality of
life, sleep and anxiety outcomes across a range of conditions
Project Twenty21 (UK) [49, 50]
Registry platform for data collection from patients provided prescriptions by licensed i Table 1 Examples of Real World Evidence Generation for Cannabis-Based Medicinal Products Analysis of self-administered questionnaire study conducted in the UK between 1998
and 2002 Ware et al. [31]
Sexton et al. [32]
Lucas et al. [33]
Lintzeris et al. [34]
Government records analysis
Bachhuber et al. [35]
Piper et al. [36]
Vigil et al. [37]
Analysis of clinic/dispensary data
Bonn Miller et al. Funding g
Not Applicable. Acknowledgements
Not Applicable. g
Not Applicable. Conclusion It
will supply medical cannabis to 3000 patients over 2 years and aims to examine real
life use of therapeutic cannabis Medical Cannabis Access Programme (Ireland) [60]
A 5-year pilot programme in which certain CBMPs can be prescribed with information
on prescribing collected by Ireland’s Health Service Executive Medical Cannabis Access Programme (Ireland) [60]
A 5-year pilot programme in which certain CBMPs can be prescribed with information
on prescribing collected by Ireland’s Health Service Executive abundance of potential data, however, it is important the
right tools and analysis are utilised to unlock potential
insights from these. Availability of Data and Materials
Not Applicable. 1. Case P. The NICE guideline on medicinal Cannabis: keeping Pan-
dora’s box shut tight? Med Law Rev. 2020;28(2):401–11. 2. Alexander SPH. Barriers to the wider adoption of medicinal Can-
nabis. Br J Pain. 2020;14(2):122–32. https://doi.org/10.1177/
2049463720922884. Author Contributions RB and SE prepared the manuscript. OS, NM, DC, BP, MS read and
approved the final manuscript. Conclusion Aims to recruit 20,000 patients with
initial data analysis due in 2022 Real world evidence platform utilising clinical registries and patient generated health
data to inform clinical care and aid bespoke drug development. They have utilised this
as part of a drug development platform for specific CBMP preparations in psychologi-
cal disease (EMD-003) and irritable bowel syndrome (EMD-004) in top-down driven
drug development Real world evidence platform utilising clinical registries and patient generated health
data to inform clinical care and aid bespoke drug development. They have utilised this
as part of a drug development platform for specific CBMP preparations in psychologi-
cal disease (EMD-003) and irritable bowel syndrome (EMD-004) in top-down driven
drug development 1 3 Therapeutic Innovation & Regulatory Science (2022) 56:8–14 12 Minnesota Medical Cannabis Programme (US) [54]
This registry collects data on the benefits, risks and outcomes regarding the therapeutic
use of medical cannabis. It is run by the Minnesota Department for Health
BfArM (Germany) [55]
A non-interventional survey on the use of these cannabis medicines, which must be
completed by any German physician prescribing medical cannabis
CB2 Insights (US) [56]
CB2 Insights is a healthcare technology company that uses real world data to monitor,
assess, and generate insights to help improve patient outcomes
Medical Cannabis Pilot Programme (Denmark) [57]
A four-year pilot programme which aimed study patients’ use of medicinal cannabis. The success of the programme has recently led to an extension of the trial period
Italian Medicines Agency (Italy) [58]
In Italy, prescriptions and patient data are registered with the Italian Medicines Agency. In 2019 26,042 medical cannabis prescriptions, attributed to 12,998 patients had been
registered
Cannabis a` usage medical programme (France) [59] Initiated by the French National Agency for Medicines and Health Products Safety, the
registry will collate information on efficacy and safety data on medical cannabis. It
will supply medical cannabis to 3000 patients over 2 years and aims to examine real
life use of therapeutic cannabis
Medical Cannabis Access Programme (Ireland) [60]
A 5-year pilot programme in which certain CBMPs can be prescribed with information
on prescribing collected by Ireland’s Health Service Executive Cannabis a` usage medical programme (France) [59] Initiated by the French National Agency for Medicines and Health Products Safety, the
registry will collate information on efficacy and safety data on medical cannabis. Open Access This article is licensed under a Creative Commons Attribution 4.0
International License, which permits use, sharing, adaptation, dis-
tribution and reproduction in any medium or format, as long as you
give appropriate credit to the original author(s) and the source, pro-
vide a link to the Creative Commons licence, and indicate if changes
were made. The images or other third party material in this article are
included in the article’s Creative Commons licence, unless indicated
otherwise in a credit line to the material. If material is not included in
the article’s Creative Commons licence and your intended use is not
permitted by statutory regulation or exceeds the permitted use, you will
need to obtain permission directly from the copyright holder. To view a
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Elementos traqueais de cinco táxons de Cactaceae da caatinga pernambucana, Brasil
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Acta Botanica Brasílica/Acta Botânica Brasílica
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1 Mestranda pela Universidade de São Paulo
2 Departamento de Botânica, Centro de Ciências Biológicas, Universidade Federal de Pernambuco, Av. Prof. Moraes Rego s/n, Cidade
Universitária, CEP 50632-970, Recife, PE, Brasil
3 Instituto de Biociências, Universidade de São Paulo, C. Postal 4005, CEP 0161-970, São Paulo, SP, Brasil
4 Autor para correspondência: emilia.arruda@bol.com.br Recebido em 29/10/2003. Aceito em 24/03/2004 RESUMO – (Elementos traqueais de cinco táxons de Cactaceae da caatinga pernambucana, Brasil). Este trabalho traz a caracterização
morfológica de elementos de vaso e WBT (“traqueíde com espessamento largo”) de raiz e cladódio de cinco táxons de Cactaceae (Harrisia
adscendens (Gurke) Britton & Rose, Melocactus × horridus Wedermann Notizbl., M. zehntneri (Britton & Rose) Luetzelb., Tacinga
inamoena (Schumann) N.P. Taylor & Stuppy e T. palmadora (Britton & Rose) N.P. Taylor & Stuppy) ocorrentes em um trecho de
caatinga do Estado de Pernambuco, Brasil. Os elementos de vaso observados tanto nas raízes quanto nos cladódios apresentam-se
praticamente idênticos entre os táxons estudados, sendo observadas pontoações alternas, opostas e escalariformes, além de placas de
perfuração simples transversal e obliqua. As traqueídes (WBT) foram registradas apenas nos cladódios de quatro dos táxons estudados,
exceto em Harrisia adscendens. As WBTs apresentam espessamento anelar ou helicoidal e o espessamento misto ocorre apenas em
Melocactus. Os elementos traqueais foram medidos, mostrando que em Melocactus tanto a raiz como o cladódio, apresentam elementos
de vaso longos e quanto as WBT essas são maiores no cladódio de Melocactus. Palavras-chave: anatomia, Cactaceae, caatinga, elementos traqueais ABSTRACT – (Traqueary elements of five taxons of Cactaceae of the caatinga from Pernambuco State, Brazil). This work brings a
morphological characterization of traqueary elements (vessels and tracheids) in root and cladode of five taxons of Cactaceae (Harrisia
adscendens (Gurke) Britton & Rose, Melocactus × horridus Wedermann Notizbl., M. zehntneri (Britton & Rose) Luetzelb., Tacinga
inamoena (Schumann) N.P. Taylor & Stuppy and T. palmadora (Britton & Rose) N.P. Taylor & Stuppy) occurent in the caatinga of
Pernambuco State, Brazil. The vessels elements observed in the roots and cladodes seem almost same among the studied taxons, were
observed alternate, opposite and scalariform pitting, and simple perforation plate transversal or oblique. The tracheids (WBT) were
exclusively noted in the cladodes of four studied taxons, except Harrisia adscendens. The WBTs with annular and helical thickening are
present in all taxons and the mix thickening is present only in Melocactus. The vessel elements and tracheids were measured and showing
that the vessel elements are longer in roots and cladodes of Melocactus and the longest WBTs were only observed in cladode of
Melocactus. Key words: anatomy, Cactaceae, caatinga, traqueary elements placas de perfuração simples (Metcalfe & Chalk 1950;
Gibson 1973; 1977; Gibson & Nobel 1986; Soffiatti &
Angyalossy 2003). Acta bot. bras. 18(4): 731-736. 2004 Acta bot. bras. 18(4): 731-736. 2004 Elementos traqueais de cinco táxons de Cactaceae da caatinga
pernambucana, Brasil Emília Cristina Pereira de Arruda1,4, Marccus Alves2 e Gladys Flávia Melo-de-Pinna3 Emília Cristina Pereira de Arruda1,4, Marccus Alves2 e Gladys Flávia Melo-de-Pinna3 Recebido em 29/10/2003. Aceito em 24/03/2004 Arruda, Alves & Melo-de-Pinna: Elementos traqueais de cinco táxons de Cactaceae da caatinga... Ainda são poucos os trabalhos realizados com
WBT, de maneira que sua origem e função ainda são
incertas (Conde 1975), embora alguns pesquisadores
relacionem sua ocorrência com a suculência (Boke
1944; Mauseth 1993). Porém, alguns trabalhos vêm
sendo desenvolvidos para demonstrar que estas células
podem ser importantes para as relações filogenéticas
das Cactaceae (Mauseth et al. 1995; Mauseth &
Landrum 1997; Landrum 2002). Quanto às dimensões
de elementos de vaso e fibras, alguns trabalhos foram
desenvolvidos (Mauseth 1993; Mauseth & Plemons-
Rodriguez 1998). Gibson & Nobel (1986) mencionaram
a possível correlação entre o comprimento e o diâmetro
destas células com hábito das plantas. Cactoideae, enquanto que Tacinga inamoena e
T. palmadora da subfamília Opuntioideae (Hunt &
Taylor 1990). A classificação do hábito dos táxons
estudados segue a proposta por Gibson & Nobel (1986)
para a família Cactaceae. Após a coleta, parte do material foi destinado à
confecção de exsicatas, segundo as normas usuais
(Mori et al. 1989) e depositadas no Herbário UFP. Parte do material vegetativo (raízes e cladódios)
coletado foi fixado em FAA 50 e, após 24h, transferido
ao etanol 70% (Johansen 1940), para sua preservação. A partir deste material, foi utilizada a técnica de
dissociação, tanto das raízes como dos cladódios, com
solução de peróxido de hidrogênio e ácido acético 1:1
(Franklin 1945). Após o material perder a coloração
natural, o mesmo foi lavado em água destilada, até a
completa remoção da solução de dissociação e
acondicionado em etanol 70% (Franklin 1945). Fragmentos deste material foram macerados sobre
lâmina histológica, sendo adicionadas gotas de safranina
1% em etanol 50% (Kraus & Arduin 1997) para a
coloração e montados em glicerina 50% (Purvis et al. 1964). O objetivo desse trabalho é caracterizar os
elementos traqueais (elementos de vaso e WBT) de
cinco táxons de Cactaceae ocorrentes em um trecho
de caatinga do Estado de Pernambuco e, por meio
destas observações, buscar detectar diferenças
diagnósticas dessas células entre os órgãos (raiz e
cladódio) bem como entre os táxons estudados. Material e métodos A análise dos elementos de vaso e das WBT foi
realizada sob microscópio de luz. A mensuração foi
efetuada em ocular milimetrada acoplada ao
microscópio de luz. Para registro das células, as lâminas
foram fotografadas em fotomicroscópio e as
representações esquemáticas foram confeccionadas
em câmara clara acoplada ao microscópio de luz. Este trabalho foi desenvolvido no Município de
Alagoinha (08º29’’S e 36º47’’W), cerca de 780m.s.m. (FIAM 1994), nas Fazendas Cajueiro Seco e Lagoa
Seca, localizadas em um trecho de caatinga do Estado
de Pernambuco, no limite entre o Agreste e o Sertão
Central, segundo a classificação proposta por Andrade-
Lima (1957; 1960). Foram realizadas excursões à área
de estudo para coleta dos órgãos vegetativos (raízes e
cladódios) e reprodutivos dos táxons selecionados bem
como observações de campo. A análise estatística dos resultados foi efetuada
com auxílio do software BioEstat 2.0 (Ayres et al. 2000). A seleção dos táxons foi efetuada por meio de
consultas às coleções disponíveis nos herbários locais
(Herbário da Universidade Federal de Pernambuco -
UFP; Herbário da Universidade Federal Rural de
Pernambuco - PEUFR e Herbário da Empresa
Pernambucana de Pesquisa Agropecuária - IPA). Devido à abundância na área de estudo, variedade de
formas e pertencentes a diferentes subfamílias foram
escolhidos os seguintes táxons: Harrisia adscendens
(Gurke) Britton & Rose, com hábito procumbente;
Melocactus × horridus Wedermann Notizbl.;
M. zehntneri (Britton & Rose) Luetzelb., ambas com
hábito globoso; Tacinga inamoena (Schumann) N.P. Taylor & Stuppy e T. palmadora (Britton & Rose)
N.P. Taylor & Stuppy ambas com hábito arbustivo. H. adscendens, Melocactus × horridus e
M. zenhtneri são representantes da subfamília Introdução O sistema vascular das Cactaceae encontra-se
constituído basicamente por células condutoras do
xilema e do floema como nos demais grupos de plantas
vasculares. Porém em Cactaceae, geralmente, o tecido
xilemático apresenta maior proporção de células
parenquimáticas se comparadas à células de paredes
lignificadas (elementos de vaso e fibras) compondo
desta forma o chamado parênquima aqüífero (Soffiatti
& Angyalossy 2003; Mazzoni-Viveiros & Costa 2003). Além de elementos de vaso, fibras e células
parenquimáticas, algumas espécies de Cactaceae
podem apresentar no xilema, um tipo especial de
traqueíde denominada “wide-band tracheid” – WBTs
(Metcalfe & Chalk 1950; Gibson 1973; Mauseth 1999). Este tipo de célula é caracterizado pela ausência de
perfurações em suas paredes e presença de rígidas
bandas de espessamento secundário, as quais podem
ser anelares, helicoidais ou em dupla hélice, voltadas
para o lume celular (Gibson 1977; Mauseth 1989;
Landrum 2001). Alguns dos principais caracteres dos elementos
de vaso das Cactaceae incluem pontoações
escalariformes, alternas a irregulares e presença de 732 Arruda, Alves & Melo-de-Pinna: Elementos traqueais de cinco táxons de Cactaceae da caatinga... Arruda, Alves & Melo-de-Pinna: Elementos traqueais de cinco táxons de Cactaceae da caatinga. Arruda, Alves & Melo-de-Pinna: Elementos traqueais de cinco táxons de Cactaceae da caatinga... presentes nestas células, apresentam-se de três tipos:
helicoidal (Fig. 1D), anelar (Fig. 1E) e misto (Fig. 1F),
cujos espessamentos anelar e helicoidal ocorrem na
mesma célula. As WBT com espessamento helicoidal
e anelar foram observadas em Melocactus ×
horridus, M. zehntneri, Tacinga inamoena e
T. palmadora, enquanto as com espessamento misto
foram registradas apenas nos cladódios de
Melocactus. É possível notar ainda variações quanto H. adscendens
M. × horridus
M. zehntneri
T. inamoena
T. palmadora
Figura 3. Variações do comprimento (µm) dos elementos de vaso
na raiz dos cinco táxons estudados, pertencentes aos gêneros
Harrisia, Melocactus e Tacinga. 0
100
200
300
400
500
H
i i
M l
t
M
h t
i
T
i
T
l
d Figura 3. Variações do comprimento (µm) dos elementos de vaso
na raiz dos cinco táxons estudados, pertencentes aos gêneros
Harrisia, Melocactus e Tacinga. 0
100
200
300
400
Harrisia adscendens
Melocactus x
M zehntneri
Tacinga inamoena
T palmadora
Figura 4. Variações do comprimento (µm) dos elementos de vaso
no cladódio dos cinco táxons estudados, pertencentes aos gêneros
Harrisia, Melocactus e Tacinga. H. adscendens
M. × horridus
M. zehntneri
T. inamoena
T. palmadora Figura 2. A-U. Representação esquemática dos principais tipos
de elementos de vaso de raízes e cladódios de Cactaceae. A-C. Cladódio. A. Tacinga palmadora (Britton & Rose). B. Melocactus × horridus Wedermann Notizbl. C. Tacinga
inamoena (Schumann) N.P. Taylor & Stuppy. D. Raiz, Harrisia
adscendens (Gurke) Britton & Rose. E-F. Cladódio. E. Tacinga
inamoena. F. Tacinga palmadora. G-H. Raiz. G. Harrisia
adscendens. H. Tacinga palmadora. I. Cladódio, Melocactus
zehntneri (Britton & Rose Luetzelb. J. Raiz, Harrisia adscendens. K-L. Cladódio. K. Melocactus × horridus. L. Melocactus zehntneri. M-N. Raiz. M. Tacinga palmadora. N. Tacinga inamoena. O-S. Cladódio, Melocactus zehntneri. P. Melocactus × horridus. Q. Harrisia adscendens. R. Melocactus zehntneri. S. Harrisia
adscendens. T. Raiz, Melocactus × horridus. U. Cladódio, Harrisia
adscendens. Barra = 150µm. Figura 4. Variações do comprimento (µm) dos elementos de vaso
no cladódio dos cinco táxons estudados, pertencentes aos gêneros
Harrisia, Melocactus e Tacinga. 0
100
200
300
400
M l
t
h
id
M
h t
i
T
i
i
T
l
d
Figura 5. Traqueides com espessamento largo - WBT (“wide-
band tracheids”) no cladódio: variações do comprimento (µm) em
quatro táxons pertencentes aos gêneros Tacinga e Melocactus. M. × horridus
M. Resultados Os elementos de vaso dos táxons analisados, tanto
nas raízes como nos cladódios, caracterizam-se por
apresentar placas de perfuração simples com posição
transversal ou oblíqua, pontoações escalariformes,
alternas e opostas e os apêndices também pontoados
(Fig. 1A-C, 2A-U). Nas Figuras 3 e 4 é possível
observar a variação no comprimento destas células,
em raízes e cladódios, respectivamente. Os testes estatísticos mostraram que elementos
de vaso mais longos observados nas raízes pertencem
a Melocactus, sendo esta diferença estatisticamente
significativa (p < 0,05) em quase todas as combinações
realizadas, exceto entre H. adscendens e T. inamoena
(Fig. 3). Nos cladódios, os maiores valores encontrados
para os elementos de vaso ocorrem em H. adscendens 733 Acta bot. bras. 18(4): 731-736. 2004 a 1. A-B. Elemento de vaso de raíz. A. Tacinga palmadora (Britton & Rose) N.P. Taylor & Stuppy evidenciando placa de perfu
es e pontoações escalariformes. B. Melocactus × horridus Wedermann Notizbl. evidenciando placa de perfuração simples e ponto
C H
i i
d
d
(G
k ) B itt
& R
l
t d
d
l dódi
id
i
d
t
õ
lt
D F M l Figura 1. A-B. Elemento de vaso de raíz. A. Tacinga palmadora (Britton & Rose) N.P. Taylor & Stuppy evidenciando placa de perfuração
simples e pontoações escalariformes. B. Melocactus × horridus Wedermann Notizbl. evidenciando placa de perfuração simples e pontoações
alternas. C. Harrisia adscendens (Gurke) Britton & Rose, elemento de vaso de cladódio evidenciando pontoações alternas. D-F. Melocactus,
WBT. D. Melocactus zehntneri (Britonn & Rose) Luetzelb., evidenciando espessamento helicoidal. E. Melocactus zehntneri, evidenciando
espessamento helicoidal. F. Melocactus × horridus Wedermann Notizbl. evidenciando espessamento misto. Al - Pontoações alternas; An -
Espessamento anelar; Ap - Apêndice pontoado; Es - Pontoações escalariformes; Hl - Espessamento helicoidal; Ps - Placa de perfuração
simples. Barra: A, B, D = 20µm; C = 15µm; E = 25µm; F = 30µm. 734 Discussão Os elementos de vaso dos cladódios e raízes dos
táxons analisados apresentam-se basicamente como
descritos na literatura das Cactaceae (Metcalfe &
Chalk 1950; Gibson 1977; Soffiatti & Angyalossy 2003),
não havendo especificidade de tipos entre os táxons
estudados. A ocorrência de WBT em Cactaceae é
mencionada por vários autores (Metcalfe & Chalk
1950; Gibson & Nobel 1986; Mauseth 1999; Landrum
2002). A função e origem destas células ainda são
incertas (Conde 1975), embora alguns autores
correlacionem sua ocorrência com o grau de suculência
da planta (Boke 1944; Gibson 1977; Fahn & Cutler
1992; Mauseth 1993), enquanto outros com a evolução
da mesma (Mauseth et al. 1995; Landrum 2002). Segundo Gibson (1977), quando estas células estão
presentes há tendência tanto à redução da proporção
de elementos de vaso, quanto à ausência de fibras no
xilema. Embora as WBT tenham permitido a distinção
dos táxons analisados, tendo-se em vista sua ausência
unicamente em H. adscendens, as afirmações feitas
pelos autores acima citados sugerem que estudos
devem ser realizados nestes e em outros táxons para
comprovar a real função destas estruturas. Quanto aos padrões dimensionais, foi observado
que o comprimento dos elementos de vaso dos táxons
analisados neste trabalho, está dentro dos valores
esperados para as Cactaceae (100-500µm) como
mencionado por Metcalfe & Chalk (1950). Gibson & Nobel (1986), por meio de análise em
119 espécies de 50 gêneros da subfamília Cactoideae
e 35 espécies de 17 grupos de Platyopuntias, constata-
ram a correlação entre os comprimentos dos elementos
de vaso e de fibras com o porte da planta, sendo estas
células mais longas quanto maior o porte da planta. Entretanto, Carlquist (1975) e Zimmerman (1983)
evidenciaram a ocorrência de elementos de vaso mais
curtos em espécies arbustivas de regiões áridas, como
é encontrado na maioria das Cactaceae. Nos táxons
estudados não parece haver correlação entre o compri-
mento dos elementos de vaso e o porte das plantas,
tendo-se em vista uma tendência destas células serem
mais longas, tanto em raízes como em cladódios, em
Melocactus, as menores plantas analisadas. Embora
H. adscendens, planta de maior porte em estudo,
apresente elementos de vaso mais longos nos cladódios,
é possível notar padrão similar em M. zehntneri. p
ç
Devido às WBT ocorrerem em tecidos
excessivamente suculentos, a possível função das
rígidas bandas de espessamento secundário é o
fortalecimento deste tecido, auxiliando na sustentação
(Bailey 1966, apud Conde 1975). Segundo Mauseth
et al. Arruda, Alves & Melo-de-Pinna: Elementos traqueais de cinco táxons de Cactaceae da caatinga. zehntneri
T. inamoena
T. palmadora Figura 5. Traqueides com espessamento largo - WBT (“wide-
band tracheids”) no cladódio: variações do comprimento (µm) em
quatro táxons pertencentes aos gêneros Tacinga e Melocactus. e em Melocactus, sobretudo em M. zehntneri, sendo
esta relação também estatisticamente significativa em
quase todas as combinações realizadas, exceto entre
M. × horridus e M. zehntneri (Fig. 4). Apenas no xilema dos cladódios de Melocactus
× horridus, M. zehntneri, Tacinga inamoena e
T. palmadora, os quais são mais suculentos dentre os
táxons estudados, foram observadas WBT; em
Harrisia adscendens, o cladódio é menos suculento. As rígidas bandas de espessamento secundário, ao comprimento das WBT entre os táxons estudados
(Fig. 5), sendo que as mais longas ocorrem em
Melocactus, principalmente em M. × horridus. Esta
relação é estatisticamente significativa para todas as
combinações realizadas. 735 Acta bot. bras. 18(4): 731-736. 2004 condução, sobretudo em plantas de ambientes secos,
como as aqui estudadas. Discussão (1995), estas bandas impedem o colapso das
paredes primárias da célula mantendo o lume apto a
conduzir mesmo em períodos de estresse hídrico. Ainda
de acordo com o autor, devido a estas propriedades, as
WBT podem representar importantes adaptações para
plantas suculentas em ambientes xéricos. Os
espessamentos ocorrentes nestas células podem ser
anelares, helicoidais ou em dupla-hélice (Metcalfe &
Chalk 1950; Gibson & Nobel 1986; Landrum 2001). Gibson (1977) mencionou a ocorrência de WBT
tipicamente estreitas e fusiformes e com espessamento
helicoidal em Cactoideae, embora o espessamento
anelar possa ocorrer em representantes de pequeno
porte da referida subfamília. Já as Opuntioideae,
embora apresentem WBT estreitas e fusiformes, o
espessamento destas células nestas espécies é anelar
ou, raramente, anelar-helicoidal. Não há especificidade
entre os tipos de espessamento das WBT dentre os
táxons estudados, pois todos apresentam espessamento
anelar e helicoidal, exceto quanto ao anelar-helicoidal,
descrito aqui como misto, o qual ocorre unicamente
em Melocactus. Conde (1975) evidenciou comprimento dos
elementos de vaso relativamente constante nas cinco
espécies de Opuntia por ele analisadas. Segundo o
autor, isto pode ser devido à relativa similaridade entre
as espécies. Este aspecto pode, em parte, ser
corroborado no presente estudo, quando comparados
os dois táxons de Tacinga (Tacinga inamoena -
132µm e T. palmadora - 161µm), bem como os dois
de Melocactus (M. × horridus - 185µm e
M. zehntneri - 198µm), embora o número de
representantes de cada gênero seja pequeno. Bailey (1957) mencionou tendência evolutiva geral
de redução do comprimento dos elementos de vaso e
das fibras em Cactaceae. Com base neste caráter as
plantas do presente estudo, que seriam mais derivadas,
as Tacinga, são na realidade consideradas menos
derivadas do que os Melocactus (Hunt & Taylor 1990),
os quais apresentam elementos de vaso mais longos. Segundo Mauseth (1993), elementos de vaso curtos
evitam o embolismo e a conseqüente interrupção na Com base nos resultados obtidos é possível indicar
que os elementos traqueais aqui observados corroboram
as referências sobre formas e tipos descritos para as
Cactaceae. Devido à similaridade destes elementos em Arruda, Alves & Melo-de-Pinna: Elementos traqueais de cinco táxons de Cactaceae da caatinga... Arruda, Alves & Melo-de-Pinna: Elementos traqueais de cinco táxons de Cactaceae da caatinga... 736 raízes e cladódios, bem como entre os táxons estudados,
não é possível a diagnose dos mesmos com base neste
caráter. Discussão Como as WBT estão ausentes nas raízes de
todos os táxons estudados e no cladódio de
H. adscendens, estas podem representar um caráter
diagnóstico ou, ainda estarem relacionadas com a
suculência. Quanto ao padrão de espessamento, o tipo
misto restrito aos cladódios de Melocactus também
permite a diagnose dos táxons estudados. Franklin, G. 1945. Preparation of thin sections of synthetic
resins and wood - resin composites and a new macerating
method for wood. Nature 155(39): 51. Gibson, A. 1973. Comparative anatomy of secundary xylem
in Cactoideae (Cactaceae). Biotropica 5(1): 29-65. Gibson, A. 1977. Wood anatomy of Opuntias with cylindrical
to globular stems. Botanical Gazette 138(3): 334-351. Gibson, A. & Nobel, P. 1986. The cactus primer. Cambridge,
Havard University Press. Hunt, D. & Taylor, N. 1990. The genera of Cactaceae:
progress towards consensus. Bradleya 8: 85-107. Contudo, deve ser ressaltado que o reduzido
número de táxons analisados, torna necessária a
ampliação de estudos sobre os elementos traqueais em
outros representantes da família, visando fixar os
caracteres observados nestes táxons, além de
estabelecer outros também úteis à diagnose. Johansen, D. 1940. Plant microtechnique. New York,
McGraw-Hill Book Co. Inc. Kraus, J. & Arduin, M. 1997. Manual básico de métodos em
morfologia vegetal. Rio de Janeiro, EDUR. Landrum, J.V. 2001. Wide-band tracheids in leaves of genera
in Aizoaceae: the systematic occurence of a novel cell
type and its implications for the monophyly of the
subfamily Ruschioideae. Plant Systematics and
Evolution 277: 46-61. Referências bibliográficas Mauseth, J. 1999. Comparative anatomy of Espostoa,
Pseudoespostoa, Thrixanthocereus and Vatricania. Bradleya 17: 27-37. Andrade-Lima, D. 1957. Estudos fitogeográficos de
Pernambuco. Recife, IPA. Andrade-Lima, D. 1957. Estudos fitogeográficos de
Pernambuco. Recife, IPA. Andrade-Lima, D. 1960. Estudos fitogeográficos de
Pernambuco. Revista Arquivo do Instituto de Pesquisa
Agronômica 5: 305-341. Mauseth, J.D. & Landrum, J.V. 1997. Relictual vegetative
anatomical characters in Cactaceae: the genus Pereskia. Journal of Plant Research 110: 55-64. Andrade-Lima, D. 1960. Estudos fitogeográficos de Andrade Lima, D. 1960. Estudos fitogeográficos de
Pernambuco. Revista Arquivo do Instituto de Pesquisa
Agronômica 5: 305-341. Mauseth, J.D. & Plemons-Rodriguez, B.J. 1998. Evolution
of extreme xeromorphic characters in wood: a study of
nine evolutionary lines in Cactaceae. American Journal
of Botany 85(2): 209-218. Ayres, M.; Ayres Jr., M.; Ayres, D. & Santos, A. 2000. BioEstat 2.0: Aplicações estatísticas nas áreas de
ciências biológicas e médicas. Brasília, Sociedade Civil
Mamirauá e CNPq. Mauseth, J.D.; Uozumi, Y.; Plemons, B.J. & Landrum, J.V. 1995. Structural and systematic study of an ununsual
tracheid type in cacti. Journal of Plant Research 108:
517-526. Bailey, I.W. 1957. The potentialities and limitation of wood
anatomy in the study of the phylogeny and classification
of Angiosperms. Journal of the Arnold Arboretum 38:
243-254. Mazzoni-Viveiros, S. & Costa, C. 2003. Periderme. In:
Anatomia vegetal. B. Appezzato-da-Glória & S. Carmello-
Guerreiro. Viçosa, Ed. UFV. Boke, N.H. 1944. Histogenesis of the leaf and areole in
Opuntia cylindrica. American Journal of Botany 31(6):
299-316. ç
Metcalfe, C. & Chalk, L. 1950. Anatomy of the Dicotyledons. v.II. Oxford, Claredon Press. Mori, S.A.; Silva, L.A.M.; Lisboa, G. & Coradin, L. 1989. Manual de manejo de herbário fanerogâmico. Ilhéus,
CEPLAC Metcalfe, C. & Chalk, L. 1950. Anatomy of the Dicotyledons. v.II. Oxford, Claredon Press. Carlquist, S. 1975. Ecological strategies of xylem evolution. Berkeley, University California Press. Mori, S.A.; Silva, L.A.M.; Lisboa, G. & Coradin, L. 1989. Manual de manejo de herbário fanerogâmico. Ilhéus,
CEPLAC. Conde, L. 1975. Anatomical comparisons of five species of
Opuntia (Cactaceae). Annals of the Missouri Botanical
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techniques in botany. London, Butterworths. Fahn, A. & Cutler, D. 1992. Xerophytes. BerlinGebrüder
Borntraeger. Soffiatti, P. & Angyalossy, V. 2003. Stem anatomy of
Cipocereus (Cactaceae). Bradleya 21: 39-48. FIAM. Fundação de Desenvolvimento Municipal do Interior
de Pernambuco. 1994. Perfil Municipal do Interior de
Pernambuco. Recife, PE. Zimmerman, M. 1983. Xylem structure and the ascent of
sap. Agradecimentos Landrum, J.V. 2002. Four succulent families and 40 million
years of evolution and adaptation to xeric environments:
What can stem and leaf anatomical characters tell us
about their phylogeny? Taxon 51: 463-473. Os autores agradecem ao Dr. Nigel Taylor, pela
gentileza na identificação do material botânico; ao
PIBIC/CNPq/UFPE (Proc. 20310), pela concessão e
renovação da bolsa de iniciação científica concedidas
à primeira Autora. Mauseth, J. 1989. Comparative structure-function studies
within a strongly dimorphic plant, Melocactus inortus
(Cactaceae). Bradleya 7: 1-12. Mauseth, J. 1993. Medulary bundles and the evolution of
cacti. American Journal of Botany 80: 928-932. Referências bibliográficas New York, Springer, Berlin Heidelberg. Versão eletrônica do artigo em www.scielo.br/abb
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Solanum lycopersicum, a Model Plant for the Studies in Developmental Biology, Stress Biology and Food Science
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foods
Review
Solanum lycopersicum, a Model Plant for the Studies in
Developmental Biology, Stress Biology and Food Science
Wei Liu 1,2 , Kui Liu 1,2 , Daoguo Chen 1,2 , Zhanquan Zhang 1,2 , Boqiang Li 1,2 , Mohamed M. El-Mogy 3 ,
Shiping Tian 1,2 and Tong Chen 1,2, *
1
2
3
*
Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China
University of Chinese Academy of Sciences, Beijing 100049, China
Vegetable Crops Department, Faculty of Agriculture, Cairo University, Giza 12613, Egypt
Correspondence: chentong@ibcas.ac.cn
Abstract: Fruits, vegetables and other plant-derived foods contribute important ingredients for
human diets, and are thus favored by consumers worldwide. Among these horticultural crops,
tomato belongs to the Solanaceae family, ranks only secondary to potato (S. tuberosum L.) in yields
and is widely cultivated for fresh fruit and processed foods owing to its abundant nutritional
constituents (including vitamins, dietary fibers, antioxidants and pigments). Aside from its important
economic and nutritional values, tomato is also well received as a model species for the studies
on many fundamental biological events, including regulations on flowering, shoot apical meristem
maintenance, fruit ripening, as well as responses to abiotic and biotic stresses (such as light, salinity,
temperature and various pathogens). Moreover, tomato also provides abundant health-promoting
secondary metabolites (flavonoids, phenolics, alkaloids, etc.), making it an excellent source and
experimental system for investigating nutrient biosynthesis and availability in food science. Here,
we summarize some latest results on these aspects, which may provide some references for further
investigations on developmental biology, stress signaling and food science.
Citation: Liu, W.; Liu, K.; Chen, D.;
Zhang, Z.; Li, B.; El-Mogy, M.M.;
Tian, S.; Chen, T. Solanum
Keywords: development; food; fruit quality; fungal pathogen; metabolite
lycopersicum, a Model Plant for the
Studies in Developmental Biology,
Stress Biology and Food Science.
Foods 2022, 11, 2402. https://
doi.org/10.3390/foods11162402
Academic Editor: Stefania Masci
Received: 19 July 2022
Accepted: 9 August 2022
Published: 10 August 2022
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affiliations.
Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1. Introduction
Tomato (Solanum lycopersicum) is one of the best studied cultivated dicotyledonous
plants [1]. Its domestication has mainly experienced two stages, represented by S. pimpinellifolium and S. lycopersicum var. cerasiforme as the ancestor, while persistent breeding of
modern cultivars mainly concerns the combinations of various traits to meet with market
requirements [1,2]. As one of the common vegetables originated from Central and South
America, tomato is favored by numerous consumers for its delicious taste and high nutritional value, and have thus become one of the most economically important crops since
the 16th century [1]. According to the report of FAO, the total yield of tomato equals up to
182.05 million tons worldwide and the cultivation area is 5.06 million hectares in 2020; developing countries in Asia contribute more than 1/3 to the total yield [3]. Because of
their economic importance, agronomic traits directly affecting ultimate fruit yield, such
as branching pattern, inflorescence architecture and fruit development, have been paid
additional attention by scientists [4]. Many genes involved in yield-related traits have been
identified in tomato, including SELF PRUNING (SP), LOCULE NUMBER (LC), FASCIATED
(FAS), TERMINATING FLOWER (TMF), etc. [4,5].
In addition, tomato has plenty of nutritional traits, making it popular among people
all over the world. According to USDA statistics, it is one of the low-calorie vegetables (extremely low fat and zero cholesterol), with only 18 calories per 100 g [6]. Tomato is also an
excellent source of antioxidants, dietary fiber, minerals, and vitamins. Taking antioxidants,
for example, tomato has a total oxygen radical absorbance capacity at 367 µmol TE/100 g,
Foods 2022, 11, 2402. https://doi.org/10.3390/foods11162402
https://www.mdpi.com/journal/foods
Foods 2022, 11, 2402
2 of 15
attributed to the high levels of vitamin A and flavonoid antioxidants, such as α and ßcarotenes, xanthins and lutein. These antioxidants have potent efficacies in scavenging
harmful oxygen-free radicals, thus improving people’s night vision and protecting them
from aging, tumors and other health-related problems [6]. It should be noted that domestication and genetic breeding of tomato during the past several decades have resulted in
decreased genetic diversity and unexpected loss of some important traits, including flavor,
aroma and resistance [7,8]. With the continuous change in lifestyle and living standard,
people have made efforts to reintroduce the lost traits from wild relatives into commercial
cultivars [5,9,10]. Because of tomato’s virtues and the aforementioned situations, tomato
has been received as a model species for studies in life science and food science.
2. S. lycopersicum Is Used for Investigating Inflorescence Branching and Fate
Determination of Shoot Apical Meristem
Tomato is a species which undergoes sympodial growth, whose stem cell activity
directly determines plant architecture, flowering time, inflorescence structure, fruit size,
harvest index and final fruit yield [11,12], composing the developmental basis for molecular
design and genetic breeding of tomato cultivars. Therefore, tomato has been widely used
as an excellent material for investigating the mechanisms underlying inflorescence branch,
flowering and fate determination of shoot apical meristem (SAM) [13,14]. Among the
developmental regulators, reactive oxygen species (ROS) produced simultaneously with
respiration have important roles in regulating growth, development and responses to
unfavorable conditions of plants [15,16]. As the most stable form of ROS, H2 O2 is also
accumulated in the SAM of normally grown tomato plants, while the accumulation site
coincides with the expression site of TMF, a transcription factor controlling the maturation
of SAM, suggesting that ROS may be related to the maturation of SAM [17]. It was found
that locally accumulated H2 O2 in SAM drove reversible liquid–liquid phase separation
of TMF in tomato [13]. In response to cellular redox status variation, cysteine residues
in TMF may form disulfide bonds, which are necessary for connecting multiple TMF
molecules and further increasing intrinsically disordered regions to trigger phase separation.
Such phase separation enables TMF to suppress the expression of ANANTHA, a floral
identity gene [13]. This characteristic reversible biomolecular condensate may provide
a suitable microenvironment for rapid transcriptional programming via redox-regulated
phase separation. These results well correlate liquid–liquid phase separation of proteins,
redox signaling and cell fate determination of SAM, thereby assuring tomato as an excellent
example for dissecting complex agronomic traits encompassing developmental robustness
and stress responses.
Functional redundancy of gene families is always caused by gene duplications and
gene family expansion, resulting in so-termed phenotypic robustness [18]. However, how
such phenotypic robustness is maintained in the specific cellular contexts is still seldom
studied in plants. The fate transition of SAM is a well-regulated programmed developmental process which is required to be initiated and completed at the right time and right
place [17]. Untimely transition may lead to failure to adapt to environmental changes or
crop yield loss [11]. The precise control of tomato stem cell fate transition is determined
by ALOG transcription factors. TMF, the first ALOG transcription factor identified from
tomato, functions to maintain meristem at a vegetative state [19]. During gene duplication and gene family expansion, some mutations were introduced in the cis-regulatory
sequences and coding sequences of ALOG genes, maintaining the activity of the four TFAM
genes (TMF family member) (TFAM1/2/3/11) in SAM [19]. TFAM1/2/3/11 proteins all
contain typical intrinsically disordered regions (IDRs) and undergo liquid–liquid phase
separation [14]. Interestingly, although the ALOG domain is conserved, many mutations
occur to the N-terminal and C-terminal IDRs, leading to differences in protein phase separation and transcriptional regulation [14]. Following site-directed mutation, IDR replacement
and transcriptional activity analysis, it was confirmed that the realization of the robustness
of tomato cell fate transition depends on the ALOG transcription factors to form hetero-
Foods 2022, 11, 2402
occur to the N-terminal and C-terminal IDRs, leading to differences in protein phase separation and transcriptional regulation [14]. Following site-directed mutation, IDR replacement and transcriptional activity analysis, it was confirmed that the realization of the robustness of tomato cell fate transition depends on the ALOG transcription factors to form
3 of 15
heterogeneous biomolecular condensates through protein phase separation, thereby precisely regulating the spatio-temporal expression of ANATHA (AN) [14]. Consequently, tomatoes
canbiomolecular
blossom and bear
fruit in the
right place
at the
rightseparation,
time. This work
represents
geneous
condensates
through
protein
phase
thereby
precisely
another
excellent
example for investigating
fate
determination
of SAM
using tomato
as a
regulating
the spatio-temporal
expression of
ANATHA
(AN) [14].
Consequently,
tomatoes
model
system.
can blossom and bear fruit in the right place at the right time. This work represents an-
other excellent example for investigating fate determination of SAM using tomato as a
3.model
S. lycopersicum
system. Is a Model Material for Studies on Fruit Ripening Regulation
Some fruits, particularly climacteric fruit, are harvested at lower maturity and serve
S. lycopersicum
Is aatModel
Material
for Studies
on Fruit Ripening
Regulation
as3.foods
for consumers
appropriate
maturity
by postharvest
processing
at harvest, storage, delivery
and
retailing
[20].
Therefore,
it
is
necessary
to
manipulate
the
ripening
pro-as
Some fruits, particularly climacteric fruit, are harvested at lower maturity
and serve
cessing
andconsumers
maintain the
fruit qualitymaturity
to provide
foods
at the right
time forstorage,
confoods for
at appropriate
by delicious
postharvest
processing
at harvest,
sumers
[21].
to [20].
a relatively
small
(~900
Mb), a relatively
short life
cycle,
delivery
andOwing
retailing
Therefore,
it isgenome
necessary
to manipulate
the ripening
processing
well
genetic
extensive
germplasm
collection
andfor
mature
experianddissected
maintain the
fruit background,
quality to provide
delicious
foods at the
right time
consumers
[21].
mental
for genetic
transformation,
tomato
has become
a model
plantdissected
for the
Owingprocedures
to a relatively
small genome
(~900 Mb),
a relatively
short life
cycle, well
geneticon
background,
extensive
germplasm
collection
mature
experimental
procedures
studies
developmental
biology
of climacteric
fruit and
[20,22].
Tomato
fruit development
forbe
genetic
transformation,
has become a model
plant early
for thefruit
studies
on developmencan
categorized
into two tomato
major developmental
processes:
development
and
tal biology
of whereas
climacteric
[20,22]. Tomato
fruit development
can are
be categorized
into
fruit
ripening,
thefruit
morphological
structure
and size of fruit
mainly detertwo major
processes: stage
early fruit
and fruit ripening,
whereas
mined
at thedevelopmental
early fruit development
[23]. development
The early development
of tomato
fruit
thebemorphological
structure
and
size of
aredevelopment
mainly determined
atsetting;
the early
decan
further divided
into three
stages:
(1)fruit
ovary
and fruit
(2)fruit
rapid
velopment
stage
[23].
The
early
development
of
tomato
fruit
can
be
further
divided
into
division of the cells and significant increase in the number of cells; (3) trigger of growth
three
stages: (1) of
ovary
development
andfruit
fruitripening
setting; has
(2) rapid
division
of the cells and
and
development
tomato
fruit. Tomato
obvious
stage characteristics,
significant
increase
in
the
number
of
cells;
(3)
trigger
of
growth
and
development
tomato
by which the ripening process is always categorized as immature green (IMG), of
mature
fruit.(MG),
Tomato
fruit ripening
has obvious
stage
by which
thechanges
ripeninginprocess
green
breaker
(Br), orange
(Or) and
red characteristics,
ripe (RR) according
to the
fruit
is always
categorized
immature
(IMG), mature
green (MG),
breaker (Br),by
orange
color
and firmness
[21]as(Figure
1A).green
Importantly,
fruit ripening
is accompanied
the
(Or)
and
red
ripe
(RR)
according
to
the
changes
in
fruit
color
and
firmness
[21]
(Figure
1A).
formation of many fruit quality-related traits, such as firmness, color, aroma, vitamins and
Importantly,
fruit
ripening
is
accompanied
by
the
formation
of
many
fruit
quality-related
others [22].
traits, such as firmness, color, aroma, vitamins and others [22].
Figure 1. Tomato fruit development and virus-induced gene silencing. (A) Tomato fruit development.
Figure 1. Tomato fruit development and virus-induced gene silencing. (A) Tomato fruit develop(B) Phenotypes for VIGS-PDS fruit (S. lycopersicum cv MicroTom and S. lycopersicum Del/Ros1 cv
ment. (B) Phenotypes for VIGS-PDS fruit (S. lycopersicum cv MicroTom and S. lycopersicum Del/Ros1
cvMicroTom).
MicroTom).
Among the methods for analyzing ripening-related traits, virus-induced gene silencing
(VIGS) has been proven as an efficient technique for rapid examination of gene functions in
plants, particularly for fruit color and other obviously visible phenotypes [24]. In terms of
tomato fruit, VIGS offers a rapid alternative for knocking down the expression of a specific
gene without tedious genetic transformation through tissue culture [25]. The TRV-based
VIGS system can be efficiently introduced by infiltration into detached tomato fruit by
either vacuum infiltration or by injection into the fruit attached to the host plant via car-
Foods 2022, 11, 2402
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popodium or stem [26]. As definite examples, VIGS-SlPDS, SlEIN2, SlCTR1 and SlEILs
(the genes related to lycopene biosynthesis and ethylene signaling) all resulted in similar
ununiform color phenotypes as observed later by stable transformation with RNAi or
CRISPR-Cas9 constructs [26], thereby significantly shortening the time required for preliminary identification of gene functions. Similarly, VIGS technique has also been applied
to develop a visual reporter system in some specific transgenic tomato lines based on
anthocyanin accumulation, such as Del/Ros1 [27]. The Del/Ros1 transgenic line expresses
Delila and Rosea1 from the ornamental flower snapdragon (Antirrhinum majus L.) under the
control of the fruit-specific E8 promoter, showing abundant anthocyanin and characteristic
purple fruit. VIGS-Del/Ros1 impairs the original purple fruit phenotype of Del/Ros1 line
(Figure 1B), whereas simultaneous VIGS of Del/Ros1 and a certain gene of interest may provide a directly visible control for gene expression analysis and precise sampling of different
sectors. This may facilitate the functional dissection for the genes whose knock-down or
knockout manipulation may not lead to obviously direct-visible variations.
In terms of the mutants, omics approaches have revealed more insights into regulatory
machinery upstream of ethylene and ripening-related signaling systems. The transcription factor MADS-RIN (RIPENING INHIBITOR) is a ripening-related key regulator that
has been extensively investigated during the past several decades [28,29]. Rin mutant
has defects in fruit softening and does not exhibit the characteristic respiratory rise and
ethylene peak [30]. However, it has been suggested that rin mutation resulted in the fusion
of truncated RIN to Macrocalyx (RIN-MC), which produced a gain-of-function chimeric
protein transcriptionally repressing fruit ripening [28,31]. Coincidently, knockout of SBPCNR and NAC-NOR transcription factors by CRISPR/Cas9 genome editing only led to
postponed or partially non-ripening phenotypes, which were significantly different from
the phenotypes observed in the spontaneous mutants [32]. No alteration was detected
in the SPL-CNR DNA sequence; however, its promoter accumulated hypermethylated
modification, giving rise to inhibition on the transcription of the SPL-CNR gene and the
Cnr mutant phenotype [33]. Similarly, the non-ripening (nor) mutant of tomato also harbors
a truncated protein (NOR186) composed of 186 amino acids due to two adenine deletion
in NAC-NOR, leading to a gain-of-function mutant in which the transcriptional activation
domain was interrupted but the DNA-binding domain was retained (US Patent, No. US
6762347B1). NAC-NOR overexpression in the nor mutant did not fully complement the
defects in ripening-related phenotypes, while the truncated NOR186 competed with the
wild-type NOR for binding to the promoter regions of 1-aminocyclopropane-1-carboxylic
acid synthase2 (SlACS2), pectate lyase (SlPL) and geranylgeranyl diphosphate synthase2
(SlGgpps2) [34]. Collectively, previous models depicting RIN, NOR and other regulators as
indispensable for fruit ripening should be reconsidered [20,29,31,34], while the integration
of state-of-art molecular biological techniques and data mining may provide solutions to
these unsolved questions in tomato fruit.
4. S. lycopersicum Is a Classic Model Species for Studies on Responses to
Abiotic Stress
Under significant global climate changes, plants are persistently confronted with
various unfavorable environmental conditions (such as high light, low/high temperature,
drought, salinity, etc.) during their life cycles, which oblige them to successfully evolve
sophisticated signaling network to cope with such harsh conditions [35,36]. Tomato has also
been well utilized to investigate the responsive machinery to environmental stimuli [36].
Responses to low light and high light. Light is above all the most important signal
for plants, and is indispensable for photosynthetic biomass production and resistance to
pathogens [37]. A recent study demonstrated that tomato was more susceptible to Pst
DC3000 under low light, which was attributed to the decreased apoplastic glucose. Further studies revealed that the regulator of G protein signaling 1 (RGS1)/heterotrimeric G
protein was activated to negatively regulate the defense responses of tomato; however,
this regulation was independent of SA or JA signaling [38]. These results correlate the
Foods 2022, 11, 2402
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signaling of plants in response to abiotic and biotic stress, which may provide certain clues
for developing future strategies for efficient production of protected vegetables. In contrast,
high light intensity may lead to photooxidative stress, bursts of reactive oxygen species
(ROS) and subsequent responses [39]. In a lipidomics study to identify lipophilic molecules
that enhance tolerance after exposure to combined high-temperature and high-light stress,
alpha-tocopherol and plastoquinone/plastoquinol were the most significantly upregulated,
which may contribute to the protection of photosystem II (PSII) over photodamage under
environmental stress [40]. Interestingly, certain light intensity and quality may induce
the accumulation of specific metabolites [41,42], among which steroidal glycoalkaloids
(SGAs) and anthocyanins are two groups. SGAs are specialized secondary metabolites
mainly produced in Solanaceous species [8], which are toxic for humans and contribute to
defense responses. It was found that SlHY5 (ELONGATED HYPOCOTYL 5) and SlPIF3
(PHYTOCHROME INTERACTING FACTOR3) directly bound to respective elements in the
promoter regions and transcriptionally activated the key genes involved in SGA biosynthesis, namely GAME1, GAME4 and GAME17, thus modulating the transcript abundance of
these enzymes [42]. These results provide a potential genetic basis for genetic manipulation
of SGA level in Solanaceous crops. As major water-soluble pigments, anthocyanins have
important roles in protecting plant tissues from being damaged by high light, UV radiation, and other unfavorable environmental factors [27]. High light induced a nonuniform
anthocyanin pigmentation pattern in the Pro35S:BrTT8 tomato fruit. This was attributed to
the induction of SlAN2 expression at high level in the upper part of fruit after exposure
to high light, resulting in efficient local assembly of MBW complex (a protein complex
composed of MYB, bHLH and WDR protein) with BrTT8 and SlAN11 [41]. In contrast, the
low light treatment failed to activate the genes involved in anthocyanin biosynthesis, while
the genes involved in chlorophyll biosynthesis were also markedly suppressed [41], further
assuring the feasibility for genetical manipulation of secondary metabolites under artificial
illumination environment.
Responses to salinity. Most tomato cultivars are sensitive to salinity during their
life, particularly at the seed germination and early seedling stages, while natural variation in salinity tolerance has been detected in wild relatives, including S. pimpinellifolium,
S. pennellii, S. habrochaites and S. lycopersicoides [43]. A couple of famous genes are successively identified in tomato to alleviate the toxicity of excessive Na+ [44–49]. The plasma
membrane Na+ /H+ antiporter SlSOS1 is essential for salinity tolerance by promoting the
transport of Na+ from underground parts to aboveground parts [44], while the overexpression of SlNHX2, a K+ /H+ antiporter, led to an enhanced K+ sequestration capacity
and higher salinity tolerance [45]. In contrast, the SlSOS2 (a gene encoding a protein
kinase) overexpressing plants displayed higher salinity tolerance by promoting Na+ export
and compartmentation [46]. Attempts have been made to enhance salinity tolerance in
tomatoes by genetic engineering on these genes that encode Na+ /H+ antiporters SlSOS1
and SlNHX2, as well as regulatory proteins (including SlSOS2). As a major challenge
for plant growth, development and productivity, salt stress often triggers significant transcriptional reprogramming, underpinning the importance of transcription factors in salt
stress responses [50]. Transcription factors of diverse families are closely correlated with
the responses of tomato to salt stress. SlAREB1 and SlAREB2 (the genes encoding ABAresponsive element-binding proteins) were induced by salinity in both roots and leaves.
The genes encoding oxidative stress-related proteins, lipid transfer proteins and late embryogenesis abundant proteins were up-regulated in the SlAREB1-overexpressing plants,
which may be responsible for the higher salt tolerance [47]. SlARS1 (altered response to salt
stress 1) was a R1-type MYB transcription factor [48]. The ars1 mutant displayed defects in
stomatal closure in an abscisic acid (ABA)-dependent manner, while the SlARS1 overexpression line did not show chlorosis but improved water use efficiency, further implying a
potential role of ABA signaling in the salt tolerance conferred by SlARS1. SlDREB2 encoded
a dehydration-responsive element-binding protein, which improved the salt tolerance by
elevating K+ /Na+ ratio as well as proline and polyamine contents, while ABA signaling
Foods 2022, 11, 2402
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was also involved in the tolerance conferred by SlDREB2 [49]. It should be noted that some
key components as mentioned above are involved in the responses to abiotic and biotic
stress, further suggesting the necessity of dissecting the crosstalk of these signaling events
and exploring key determinants involved [51].
Responses to cold stress. Cold stress is among the major abiotic stress restricting geographical distribution, normal growth and crop yield [52]. Many cold-activated signaling
pathways are induced to protect plants from injuries during cold stress. Similar to its
counterpart in Arabidopsis, ICE1-CBF-COR module is conserved and also works in cold
responses of tomato, as the expression of SlCBF1 and SlCBF2 are significantly upregulated
after 3 h at 10 ◦ C during cold acclimation in tomato. Moreover, overexpression of SlICE1
enhanced chilling tolerance by elevating the expression of SlCBF1, SlDRCi7 and SlP5CS and
the antioxidant capacity of tomato [53]. The transgenic SlICE1 overexpressing lines also
accumulated higher levels of pigments and antioxidants, including β-carotene, lycopene
and ascorbic acid, as well as certain amino acids and amines, suggesting that the enhanced
chilling tolerance may be partly attributed to the improved antioxidant capacity [54]. In
addition, cold stress may also affect fruit flavor and other quality traits, as refrigeration
at low temperature can delay fruit ripening and prolong the storability of tomato [55,56].
Consumers have begun to complain about the loss of flavor after cold storage during
production. An integrative analysis on the transcriptome and flavor metabolome of tomato
fruit showed that flavor-related volatiles lost after low temperature storage, while fruit
sugar and acid contents did not change. The reduction in the expression of volatile synthesis genes and transcription factor genes related to ripening was associated with the
methylation status in the promoter regions [56].
Responses to drought. Water shortage is one of the major threats for sustainable yield
output, while increasing aridity has further aggravated the situation [57]. Solanum lycopersicum cv. M82 has relatively poor tolerance to abiotic stress, while some of its wild relatives,
such as S. pennellii LA716, has higher tolerance [58]. Two excellent studies generated a
collection of 50 introgression lines (ILs) of S. pennellii, which greatly facilitated the dissection
of the mechanisms underlying drought tolerance in tomato [9,10]. Along with the recurrent
parent S. lycopersicum cv. M82, the transcriptional profiling for two drought-tolerant lines
identified from the introgression lines showed that the genes involved in transcriptional
regulation, signalling, cell wall structure, wax biosynthesis, gluconeogenesis, purine and
pyrimidine nucleotide biosynthesis, tryptophan degradation, starch degradation, methionine biosynthesis and superoxide radical scavenging were specifically affected by drought
stress [59]. Using the near-isogenic lines (NILs) carrying the resistance genes Ol-1, Ol-2 and
Ol-4 to Oidium neolycopersici, Sunarti et al. revealed that the growth reduction caused by
drought stress was not further aggravated upon powdery mildew (PM) infection, while
more severe drought stress resulted in less biomass of PM. Upon drought stress, the NILs
harboring Ol-2, and Ol-4 displayed normal resistance and cell death phenotype, while the
NIL carrying Ol-1 showed mildly attenuated resistance [60]. Interestingly, many genes
related to hormonal signaling and wound responses, such as SlNCED, SlPR1, SlACS2 and
SlLIN6, were significantly induced, further implying a complex cross-talk of abscisic acid
(ABA), salicylic acid (SA), jasmonic acid (JA), ethylene (ET) and wound signaling during
combined stress conditions.
5. S. lycopersicum Is a Classic Model Species for Studies on Responses to Biotic Stress
Tomato has always been a model plant for studying the defense responses of plants to
pathogenic microorganisms and insects [61,62], which may provide important reference for
the practices in maintaining fruit resistance and reducing food loss. The first Pto gene (a
resistance gene encoding a serine/threonine kinase) for verifying the “gene to gene” theory
was cloned from tomato [63]. Until now, many important resistance-related genes have
been cloned from tomato, which are involved in the resistance to fungi, nematodes, aphids,
bacteria and viruses [64–67] (as summarized in Table 1). From injury stimulation to hormonal signaling and resistant gene expression, tomato has been extensively used to explore
Foods 2022, 11, 2402
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the jasmonate (JA)-regulated immunity of plants in response to insect attack and other
mechanical damages. JA is a phytol-oxylipin hormone modulating growth and immunity
of plants by triggering transcriptional programming, in which the basic helix-loop-helix
transcription factor MYC2 and MED25 (a subunit in the Mediator complex) play a central
role. In the presence of the JA ligand jasmonoyl-isoleucine (JA-Ile), JASMONATE-ZIM
DOMAIN (JAZ) proteins form a co-receptor complex with CORONATINE-INSENSITIVE
1 (SCFCOI1 ) in a JA-Ile dependent manner, while the SCFCOI1 -dependent degradation of
JAZs leads to the release of MYC2; MYC2 thereby activates JA-mediated transcriptional
reprogramming of downstream genes [68]. Most of the MYC-targeted genes were transcription factors (MYC2-traget transcription factors, MTFs) and proteins related to early
JA responses. MYC2 and the MTF JA2-like preferentially regulate wound-responsive
genes, while MYC2 and ETHYLENE RESPONSIVE FACTOR.C3 (also an MTF) preferentially regulate pathogen responsive genes [64], suggesting a differential activation of
wound-responsive and pathogen-responsive transcriptional modules encompassing MYC2.
The transcriptional activation of JA-responsive genes also depends on MED25, a subunit
in the Mediator transcriptional activation complex [68]. Moreover, MYC2 also regulate
the termination of JA signaling by regulating several MYC2-targeted bHLH (MTB) TFs,
which exert antagonistic effects on the MYC2-MED25 transcriptional activation complex by
competing with MYC2 for its targets [66]. In addition to MYC2 and RNA polymerase II,
MED25 also interacts with other regulators involved in MYC2-dependent transcription,
including hormone receptor activation, transcriptional termination and epigenetic regulation (Figure 2) [68], thereby affecting downstream responses of tomato to pathogen and
insect invasion.
Table 1. Representative resistance/tolerance genes from different tomato cultivars.
Gene Name
Pathogen
Disease
Tomato Cultivar
Reference
Fungal disease
Asc
Alternaria alternata f. sp. lycopersici
Alternaria stem canker
S. esculentum
[69]
Py
Pyrenochaeta lycopersici
Corky root
S. hirsutum
[70]
Frl
Fusarium oxysporum f. sp.
radicis-lycopersici
Crown and root rot
S. peruvianum
[71]
Ph1, Ph2, Ph3, Ph4, Ph5, Ph6
Phytophthora infestans
Late blight
S. pimpinellifolium,
S. habrochaites
[72]
Cf (Cf-2, Cf-5, Cf-9, etc.)
Cladosporium fulvum
Leaf mold
S. peruvianum
[73]
Sm
Stemphylium lycopersici, S. solani, S.
floridanum
Gray leaf spot
S. pimpinellifolium
[74]
Lv
Leveillula taurica
S. chilense
[75]
Ol-1, Ol-3, Ol-4, Ol-5, Ol-6
Oidium neolycopersici
S. hirsutum
[76]
I, I2
Fusarium oxysporum f. sp. lycopersici
race 1
S. pimpinellifolium
[77,78]
S. pennellii
[77,78]
S. esculentum
[79]
Bacterial speck
S. pimpinellifolium
[80]
Bacterial spot of tomato
S. pimpinellifolium (Rx4 and
RxLA1589), Hawaii7981
(Xv3), S. pennellii (RXopJ4)
[81,82]
Powdery mildew
I3, I7
Fusarium oxysporum f. sp. lycopersici
race 2
Ve1, Ve2
Verticilliium dahliae
Wilt
Verticillium wilt
Bacterial disease
Pto
Rx4, RxLA1589, Xv3, RXopJ4
Pseudomonas syringae pv. tomato
Xanthomonas
perforans race T3
ulation to hormonal signaling and resistant gene expression, tomato has been extensively
used to explore the jasmonate (JA)-regulated immunity of plants in response to insect attack and other mechanical damages. JA is a phytol-oxylipin hormone modulating growth
and immunity of plants by triggering transcriptional programming, in which the basic
Foods 2022, 11, 2402
8 of 15
helix-loop-helix transcription factor MYC2 and MED25 (a subunit in the Mediator complex) play a central role. In the presence of the JA ligand jasmonoyl-isoleucine (JA-Ile),
JASMONATE-ZIM DOMAIN (JAZ) proteins form a co-receptor complex with CORONATINE-INSENSITIVE
1 (SCFCOI1) in a JA-Ile dependent manner, while the SCFCOI1-dependTable 1. Cont.
ent degradation of JAZs leads to the release of MYC2; MYC2 thereby activates JA-mediGene Name
Pathogen reprogramming of downstream
Disease
Tomato
Reference
ated transcriptional
genes [68].
MostCultivar
of the MYC-targeted
genes were transcription factors
(MYC2-traget transcription factors, MTFs) and proteins
Viral disease
related
early
JA(ToMV),
responses.
Tomato to
mosaic
virus
tomatoMYC2 and the MTF JA2-like preferentially regulate woundchloroticgenes,
spot virus
(TCSV),
responsive
while
MYC2 and ETHYLENE RESPONSIVE FACTOR.C3 (also an MTF)
groundnut ringspot virus (GRSV),
Mosaic
S. hirsutum, S. peruvianum
[83]
Tm-1, Tm-2, Tm22
preferentially
regulate
pathogen
responsive genes [64], suggesting a differential activation
and chrysanthemum stem necrosis
of wound-responsive
virus (CSNV) and pathogen-responsive transcriptional modules encompassing
MYC2.
transcriptional
activation of
JA-responsive
genes S.
also
depends on MED25,
Sw (Sw-5a and Sw-5b)
TomatoThe
spotted
wilt virus (TSWV)
Spotted
wilt
peruvianum
[84] a
subunit in the Mediator transcriptional activation complex
[68].
Moreover,
MYC2
also
S. chilense (Ty-1, Ty-3, Ty-4,
Tomato yellow leaf curl
several
MYC2-targeted
bHLH
Yellow by
leaf regulating
curl
Ty-6),
S. habrochaites
(Ty-2),
[85]
Ty-1, Ty-2, Ty-3, Ty-4, Ty-5, Ty-6 regulate the termination of JA signaling
virus (TYLCV)
S. peruvianum
(Ty-5)
(MTB) TFs, which exert antagonistic effects on the MYC2-MED25
transcriptional
activaNematode
disease for its targets [66]. In addition to MYC2 and RNA
tion complex by competing
with MYC2
Mi1, Mi3, Mi5, Mi9
Meloidogyne
incognita
S. peruvianum
[86]
polymerase
II, MED25
also interacts with other regulators involved
in MYC2-dependent
transcription, including hormone
receptor activation, transcriptional termination and epAphids
igenetic
regulation
(Figure 2) [68], thereby affecting downstream
responses of tomato
Mi1
Macrosiphum
euphorbiae
S. peruvianum
[86] to
pathogen
and
insect
invasion.
Modified and updated from [35].
Figure
2. Tomato
as aasmodel
experimental
system
for for
investigating
host–pathogen
interaction.
Upon
Figure
2. Tomato
a model
experimental
system
investigating
host–pathogen
interaction.
Upon
insect
attack
or wounding,
systemin
is processed
from
prosystemin
following
proteolytic
cleavages
insect
attack
or wounding,
systemin
is processed
from
prosystemin
following
proteolytic
cleavages
andand
further
binds
to its
putative
receptor
STR1/2,
thus
triggering
thethe
octadecanoid
pathway
forfor
JA JA
further
binds
to its
putative
receptor
STR1/2,
thus
triggering
octadecanoid
pathway
COI1 receptor perceives JA, further triggers JAZ repressor degradation and thus relieve
synthesis.
SCF
COI1
synthesis. SCF
receptor perceives JA, further triggers JAZ repressor degradation and thus relieve
the transcription factors to induce the expression of JA-responsive genes for defense.
the transcription factors to induce the expression of JA-responsive genes for defense.
Tomato is significantly affected by an array of pathogens (including bacteria, fungi,
oomycetes, viruses, and nematodes) during cultivation and at postharvest stage [35]. Rapid
advances in integrative omics techniques and other novel techniques have facilitated a
comprehensive elucidation of the molecular machinery underlying the interaction between
tomato and certain pathogens [8,62]. Importantly, tomato fruit–pathogen interaction is
an excellent system for investigating the transition from resistance to susceptibility in
plants [35,87,88]. Transcriptomic, proteomic and metabolomic techniques have been employed to document the global responses of tomato to infections [8,87,88]. Generally,
fruit at low maturity are more resistant to pathogens, but gradually become susceptible
when the ripening program is triggered [88]. Accompanied with substantial biochemical
and physiological variations, fruit ripening is coordinated by complex signaling events
at transcriptional, post-transcriptional and epigenetic levels [20,89]. As revealed by integrative transcriptomic and metabolomic analysis on 580 introgression lines, S. pennellii
introgressions resulted in the variations for a bulk of transcripts and metabolites in ripeningrelated processes [8]. α-tomatine, a major glycoalkaloid in tomato, was transformed into
Foods 2022, 11, 2402
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esculeosides and lycoperosides during fruit development, which may be important for
reducing the bitter taste and the defense responses of fruit. Meanwhile, specific subsets
of susceptibility- and resistance-related genes also showed significant variations, while
transient knock-down of ACO5, ACD2 and 4CL-like genes resulted in bigger necrotic
lesions following Botrytis cinerea inoculation, suggesting that these genes may be crucial
for the resistance to fungal pathogens in tomato fruit at higher maturity [8]. Blanco-Ulate
et al. performed an RNAseq analysis on tomato fruit following Botrytis cinerea infection,
aiming at examining the gene expression profiles for the signaling of ethylene (ET), abscisic
acid (ABA), salicylic acid (SA) and JA [87]. Notably, in a study using different pathogens to
infect tomato fruit at varied maturity, besides the common factors (chitinases, PR proteins,
WRKY transcription factors, and reactive oxygen species) responding to pathogen infection, susceptible ripe fruit also displayed strong immune responses [88]. As revealed by
transcriptional profiling and hormone quantitation, susceptible tomato genotypes failed to
maintain the cellular redox homeostasis, whereas endogenous JA production and signaling
were normally activated as observed in resistant fruit. These results collectively suggest
that the interaction between tomato plants (fruit) and pathogens may provide important
reference for modulating quality traits and alleviating fungal diseases.
6. S. lycopersicum Is an Excellent Material for Studies on Nutritional Metabolite
Synthesis and Other Topics in Food Science
As an important component in human diets, tomato has also been an excellent experimental material for food science owing to abundant beneficial nutrients and antioxidants [1,5]. Besides sugars, organic acids, amino acids and other primary metabolites,
tomato is also abundant in pigments, flavonoids, volatiles and other secondary metabolites [90,91]. Moreover, tomato also has some specific secondary metabolites that accumulate at millimolar levels, which display antimicrobial and antinutritional activity, such as
α-tomatine [92].
Tomato fruit and its processed products always contain high levels of lycopene and
β-carotene, thus having been regarded as important sources of lycopene and provitamin
A in human diet [7]. Owing to the presence of health-promoting phytochemicals, such as
tocopherols and flavonoids in tomato, tomato and its processed products are catalogued
as functional foods [6]. Tomato cultivars and transgenic lines have been generated with
higher levels of lycopene, β-carotene zeaxanthin and lutein after domestication and genetic breeding [93]. Anthocyanins are also natural pigments ubiquitously distributed in
plants [94–96]. Butelli et al. generated a characteristic purple tomato line accumulating anthocyanin at high level by ectopic expression of two transcription factors (Del and Rosea1)
from A. majus. Importantly, these transgenic fruits also significantly prolonged the life span
of cancer-susceptible mice, implying additional health-promoting effects and potential
benefits of application [97]. Similar anthocyanin-enriched fruits have also been reported
for S. lycopersicum lines carrying Aft gene, atv gene and both genes (Aft × atv plants) as
well as S. lycopersicum cv. MicroTom overexpressing AtMYB75 and SlMYB75 [94–96,98,99].
As reported, the Pro35S:BrTT8 tomato fruit accumulated abundant anthocyanins in an
uneven pattern under natural high light, but remained anthocyaninless under low light
conditions [41]. Notably, the endogenous SlTT8 was significantly upregulated in the
Pro35S:BrTT8 transgenic line, implying that SlTT8 may be regulated by BrTT8 and other
potential regulators, rather than a director target of high light signaling [41], which deserves further clarification. Nevertheless, all these cultivars may serve as excellent materials
for the study on anthocyanin pigmentation patterns in fruits exposed to light, cold and
other environmental factors, thereby substantially facilitating metabolic engineering on
tomato cultivars.
As narrated in a GWAS mapping of quality traits for tomato fruit, robust phenotyping
and metabolite profiling technologies have enabled high-throughput quantitation of many
growth parameters and variants [4,5]. Meanwhile, the results also demonstrated that
genetic breeding globally affected fruit metabolite content as selection of alleles for fruit
Foods 2022, 11, 2402
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size and color significantly altered metabolite profiles [7]. In a recent study, Szymanski et al.
performed transcriptional, metabolomic and phenotypic profiling in combination with QTL
analysis on tomato fruit. The integrated transcriptomic and metabolomic analysis identified
a bulk of genomic loci associated with specific transcripts and metabolites. These variations
were associated with key constituents in Solanum glycoalkaloid biosynthesis, as well as transcripts and metabolites involved in responses to pathogens, thereby providing important
data for correlating transcriptional and metabolic reprogramming with pathogen resistance
in tomato fruit [8]. Li et al. constructed a global map of for major metabolic variations
throughout the growth cycle by integrating spatio-temporal metabolomic and transcriptomic data, thus providing resources for investigating metabolic processes in tomato [90].
These results are valuable for dissecting regulatory mechanisms and further metabolic engineering for flavonoids, polyphenols, steroidal glycoalkaloids, phenylpropanoids and other
compounds. These compounds have diversified functions in many important biological
processes in plants, including pigmentation of fruits and vegetables, cell wall biofortification, defense responses to pathogen invasion, shields over high-light, drought, chilling
and salinity, as well as precursors for aromatic volatiles [100]. Moreover, these compounds
comprise an indispensable component of our daily diets, as revealed by numerous benefits
of fruits and vegetables in terms of preventing cardiovascular diseases, vitamin deficiency,
obesity and other chronic diseases [101,102]. Metabolic engineering has brought about
many surprises and may fundamentally change our lifestyle in the near future. Given
the newly reported existence of a ‘duplicate’ pathway for steroidal glycoalkaloid (SGA)
biosynthesis in tomato, an excellent study reported exciting results for promoting the accumulation of provitamin D3 in tomato by gene editing for 7-dehydrocholesterol reductase
gene (Sl7-DR2) [103]. As compared with the wild type of fruit, the Sl7-DR2-knockout
homozygous lines accumulated 7-dehydrocholesterol at higher levels in both leaves and
mature green fruit, which is known as provitamin D3 , thereby modifying phytosterol
biosynthesis pathway in tomato fruit [103]. According to estimates, the provitamin D3 in
tomato may be comparable to that in two eggs of average size, a dose recommended for
vitamin D by FDA [104]. These results may provide biofortified foods conveniently and
efficiently, thus making it possible to alleviate potential risks of neurocognitive diseases,
tumors and even mortality.
7. Conclusions
In summary, as an excellent material for developmental biology, stress responses and
food science, tomato has substantially expanded our understandings of the unknown
mysteries in life science and food science. However, we are still confronted with some
refractory situations nowadays, such as insufficient food supply and malnutrition in some
poverty areas, as well as obesity, diabetes, cardiovascular diseases and other health-related
problems due to major changes of lifestyles. Given the rapid progresses in scientific and
technological breakthroughs, we can imagine that further in-depth studies using wild
tomato relatives and cultivars may undoubtedly update our knowledge and provide more
health-promoting benefits.
We believe that several aspects should be paid additional attention. Firstly, as the
global climate changes, extreme weather occurs more frequently and severely than ever,
while abiotic and biotic stresses may simultaneously take place during practical tomato
production. It is urgent to find out how plants can stimulate their intrinsic defense machineries in response to multiple stresses, so as to provide references for the development
of efficient, environmental-friendly and safe strategies to improve broad-spectrum resistance/tolerance in agricultural production. Secondly, CRISPR-Cas9 (clustered regularly
interspaced short palindromic repeats/CRISPR-associated proteins 9) technology and other
genome editing/modification tools have exhibited great potential to increase food yields
and improve food quality, while many selection marker-free transgenic lines have been
generated [62,105]. All these genetically edited varieties still require more flexibility and
attempts in policy regulations from the authorities. Topically, online sales of genome-
Foods 2022, 11, 2402
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editing tomato fruits containing a high level of GABA (G-aminobutyric acid) have started
early in Japan this year. This is the first agricultural product in the world to be launched
using CRISPR/Cas9 technology [105]. Thirdly, classical breeding techniques should be
organically combined with new techniques, thus enriching the modern toolbox used to
facilitate the improvements in agronomic traits and nutritional traits [106]. With continuous
improvements in people’s requirements for better flavor and longer shelf-life, the way of
consuming tomato gradually becomes diversified, and the demand for tomato flavor is
increasingly higher no matter whether it is consumed as cooking vegetables or fresh fruit.
It is well received by the science community that the research retrieving previously lost
quality traits by genetic manipulation and metabolic engineering is beneficial for enriching
tomato germplasm resources.
Author Contributions: Conceptualization and original figure preparation: W.L., S.T. and T.C.
Writing—original draft preparation: W.L., K.L. and D.C. Writing—review and discussion on the
manuscript: B.L. and Z.Z. Writing—review and editing: S.T., M.M.E.-M. and T.C. All authors have
read and agreed to the published version of the manuscript.
Funding: This work is supported by grants from Beijing Natural Science Foundation (6212025) and
National Natural Science Foundation of China (32072637, 31930086).
Data Availability Statement: Not applicable.
Acknowledgments: We apologize for the inadvertent omission of any pertinent original research due
to space limitation. The authors would like to thank the three anonymous reviewers for their critical
comments and constructive suggestions, and sincerely thank Cathie Martin for sharing valuable
experimental materials.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or
in the decision to publish the results.
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http://repository.uin-suska.ac.id/63887/1/Artikel-Islam%20on%20the%20air%20the%20struggle%20for%20salafism%20through%20radio%20in%20Indonesia.pdf
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Islam on the air: the struggle for salafism through radio in Indonesia
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Indonesian Journal of Islam and Muslim Societies/Indonesian Journal of Islam and Muslim Sociaties
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Imron Rosidi Universitas Islam Negeri Sultan Syarif Kasim Riau
Email: imronrosidi@uin-suska.ac.id Toni Hartono Universitas Islam Negeri Sultan Syarif Kasim Riau
E-mail: toni.hartono@uin-suska.ac.id
DOI:10.18326/ijims.v12i1.59-84 Indonesian Journal of Islam and Muslim Societies
Vol. 12, no. 1 (2022), pp. 59-84 doi : 10.18326/ijims.v12i1. 59-84 Indonesian Journal of Islam and Muslim Societies
Vol. 12, no. 1 (2022), pp. 59-84 doi : 10.18326/ijims.v12i1. 59-84 Akh. Muzakki Universitas Islam Negeri Sunan Ampel Surabaya
E-mail: akh.muzakki@uinsby.ac.id Masduki Universitas Islam Negeri Sultan Syarif Kasim Riau
E-mail: masduki@uin-suska.ac.id Akh. Muzakki
Universitas Islam Negeri Sunan Ampel Surabaya
E-mail: akh.muzakki@uinsby.ac.id Abstract Indonesian Islam has become the point of contestation of ideologies, particularly
between the so-called globally-inspired and locally- rooted views of Islam. This
article deals with the Salafism struggle in da’wah on the airwaves through the
radio as locally rooted in Indonesia with a special reference to the Salafi radio
highly popular in Batam of Riau, Hang Radio. It analyzes two main issues, 59 IJIMS: Indonesian Journal of Islam and Muslim Societies, Volume 12, Number 1, June 2022: 59-84 first on the growth of religious thinking in Indonesian Islam and its relationship
with media propaganda, and second on the Salafi struggle for Islamic identity by
means of broadcasting through radio. It argues that through the radio the Salafists
implement their ideology as part of their socio-religious identity in a public sphere. Through a hermeneutical-phenomology analaysis, this article finds that the
Salafism struggle of Islamic identity by means of radio is fragmented rather than
cohesive and solid. Moreover, this struggle is not immune to capitalism. Above
all, this struggle is also influenced by transnational and local elements. Islam Indonesia telah menjadi topik kontestasi ideologi, khususnya antara
pandangan tentang Islam yang terinspirasi global dan pandangan yang berakar
dari lokal. Artikel ini fokus pada perjuangan dakwah kelompok Salafi melalui
Radio di Batam Riau yakni Hang Radio. Artikel ini menganalisis dua isu
utama, pertama tentang perkembangan pemikiran keagamaam di Indonesia dan
hubungannya dengan propaganda media, dan kedua tentang perjuangan identitas
Islam Salafi melalui radio. Artikel ini menjelaskan bahwa melalui radio kelompok
Salafi mengimplementasikan ideologi mereka sebagai bagian dari identitas
sosial-keagamaannya di ruang publik. Melalui analisis hermeneutis-fenomologi,
artikel ini menemukan bahwa perjuangan identitas Islam Salafi melalui radio
lebih terfragmentasi ketimbang kohesif dan solid. Apalagi perjuangan ini tidak
kebal terhadap kapitalisme. Lebih dari itu, perjuangan ini juga dipengaruhi oleh
unsur transnasional dan lokal. Keywords: Da’wah Radio, Islamic Identity, Salafism, Indonesian Islam 1Mukhlis Rahmanto, “Rowing In The Flow of Khalaf; Indonesian Salafism Response 6Sunarwoto, “ Salafi Dakwah Radio : A contest for religious authority,” Archipel 91
(2016), 203-230. 5Mariam Amashukeli, En/Countering Stigma of Terrorism: The Case of Pankisi, Tbilisi: Eastern
Partnership Civil Society Facility, 2018, 8-9. 4Henri Lauzière, “Islamic Nationalism through the Airwaves: Taqi> al-Di>n al-Hila>li>’s
Encounter with Shortwave Radio, 1937–39”, Die Welt des Islams, Volume 56, Number 1
(2016), 6-33. 2Alex Thurston, “Abubakar Gumi’s al-’Aqıda al-Sah}iha bi-Muwafaqat al-Sharı’a: Global
alafism and Locally Oriented Polemics in a Northern Nigerian Text”, Islamic Africa, Volume
, Number 2 (2011), 12. https://doi.org/10.5192/21540993020209 Towards Contemporary Islamic Economics”, Humanities & Social Sciences Reviews, Volume 7,
Number 4 (2019), 969. https://doi.org/10.18510/hssr.2019.74132. ,
(
),
p //
g/
/
3Justin Qi, “Rap & Revolution: From The Arab Spring to ISIL and Beyond,” Harvard
nternational Review, Volume 40, Number 2 (2019), 22-23. Towards Contemporary Islamic Economics , Humanities & Social Sciences Reviews, Volume 7,
Number 4 (2019), 969. https://doi.org/10.18510/hssr.2019.74132.
2Alex Thurston, “Abubakar Gumi’s al-’Aqıda al-Sah}iha bi-Muwafaqat al-Sharı’a: Global
Salafism and Locally Oriented Polemics in a Northern Nigerian Text”, Islamic Africa, Volume
2, Number 2 (2011), 12. https://doi.org/10.5192/21540993020209
3Justin Qi, “Rap & Revolution: From The Arab Spring to ISIL and Beyond,” Harvard
International Review, Volume 40, Number 2 (2019), 22-23.
4Henri Lauzière, “Islamic Nationalism through the Airwaves: Taqi> al-Di>n al-Hila>li>’s
Encounter with Shortwave Radio, 1937–39”, Die Welt des Islams, Volume 56, Number 1
(2016), 6-33.
5Mariam Amashukeli, En/Countering Stigma of Terrorism: The Case of Pankisi, Tbilisi: Eastern
Partnership Civil Society Facility, 2018, 8-9.
6Sunarwoto, “ Salafi Dakwah Radio : A contest for religious authority,” Archipel 91
(2016), 203-230. 2Alex Thurston, “Abubakar Gumi’s al-’Aqıda al-Sah}iha bi-Muwafaqat al-Sharı’a: Global
Salafism and Locally Oriented Polemics in a Northern Nigerian Text”, Islamic Africa, Volume
2, Number 2 (2011), 12. https://doi.org/10.5192/21540993020209
3Justin Qi, “Rap & Revolution: From The Arab Spring to ISIL and Beyond,” Harvard
International Review, Volume 40, Number 2 (2019), 22-23.
4Henri Lauzière, “Islamic Nationalism through the Airwaves: Taqi> al-Di>n al-Hila>li>’s
Encounter with Shortwave Radio, 1937–39”, Die Welt des Islams, Volume 56, Number 1 8Media Zainul Bahri, “Islamisme Terselubung” Versus Moderat Islam: Mencermati Geliat
Radio-Radio Dakwah di Batam”, in Din Wahid and Jamhari Makruf (eds.) Suara Salafisme:
Radio Dakwah di Indonesia, Jakarta: Prenadamedia Grup, 2017, 11-60. 7Chris Chaplin, “ Salafi Islamic Piety as Civic Activism : Wahdah Islamiyah and
Differentiated Citizenship in Indonesia”, Citizenship Studies, Volume 22, Number 2 (2018),
208-223. Introduction Many methods and media have been made use of in conducting the
da’wah by both the globally-inspired and locally-rooted ideologies of
Islam. Salafism, as an example of a globally-inspired ideology of Islam,
has developed several media for the da’wah, not only through in-person
religious learning but also technologically facilitated instruments, including
exclusively radio.1 This can be seen in different parts of the Islamic world. 60 Islam on the air: the struggle for Salafism through radio in Indonesia (Masduki, et.al) In Nigeria, for example, Salafism under the leadership of Abubakar
Gumi has been employing the preaching by means of radio since 1967.2
In Tunisia, Salafism developed a local radio station for Islamic preaching
and the so-called revolutionary identity struggle.3 Furthermore, radio is
also made use of to signify the struggle for Egyptian nationalism-based
Islamic ideology, as exemplified by a Moroccan exiled in Nazi Germany,
Taqi> al-Di>n al-Hila>li>, by maximizing preaching activities through shortwave
radio.4 Moreover, radio has been used to provide local people with “an
alternative source of information” occasionally produced by the majority
of media enterprises which puts a negative stigma on Muslims, particularly
those having a close ideological connection to Salafism such as those
Muslims of ethnic Chechens in Georgia, as being terrorists.5 In fact, Salafi
radios are not monolithic because, as Sunarwoto shows in his research in
Surakarta, Central Java, there is the internal dynamism on these radios. There are three Salafi radios in Surakarta which have competed in gaining
legitimacy for their position within the Salafi da’wah movement.6 Chris
Chaplin, however, does not focus on Salafi radios. Instead, he discusses the
way in which Salafists shape the nature and interpretation of citizenship
by focusing on Wahdah Islamiyah, a Salafi organisation with over 120 61 IJIMS: Indonesian Journal of Islam and Muslim Societies, Volume 12, Number 1, June 2022: 59-84 9Muhammad Harfin Zuhdi, “ Tipologi Pemikiran Hukum Islam : Pergulatan Pemikiran
dari Tradisionalis hingga Liberalis”, Ulumuna: Jurnal Studi Keislaman, Volume 16, Number
1 (2012), 43-45. branches in Indonesia.7 branches in Indonesia.7 branches in Indonesia.7 To add an analysis of Islamic ideology of Salafism as globally inspired
and developed as such, this article deals with Salafism struggle in da’wah
on the airwaves through the local radio in Indonesia. It focuses on Salafi
radio highly popular in Batam of Riau, Hang Radio. Characteristically,
Hang Radio controlled certain segments by bringing Salafi religious
understandings.8 This is different from other Salafi radios in Central
Java, East Java and South Sulawesi. The differences can be seen from the
local context in which Hang Radio is managed. Batam is an industrial
city having a heterogonous society. Its population is plural due to the
coming of many people from Java, Sumatra and other locations. This
article aims to elaborate further on the struggle for Islamic identity
carried out by Hang Radio. This Islamic identity struggle is related to the
question of how to be a Muslim amidst modernization, secularization
and globalization that could weaken the spirituality of the Batam Muslim
community. By using hermeneutic phenomenology analysis, the data from
observations and interviews are interpreted to understand the struggles
of Islamic identities carried out by Hang Radio in Batam City so
that the da’wah struggle can be understood. This article explains two main parts. First, on religious understanding
growth in Indonesia and its relationship with media propaganda. It is
necessary to have a closer look historically at important matters on what
has happened in Indonesia linked to the emergence of religious thought
and how the media respond to this propaganda. Second, about the struggle 62 Islam on the air: the struggle for Salafism through radio in Indonesia (Masduki, et.al) for Islamic identity carried out by Hang Radio, this part deals with the
issue of how the radio can play the role of propaganda in the public sphere
and how this radio uses religious symbols to strengthen a certain Islamic
identity, as well as how the ideology of the so-called da’wah radio attracts
public attention. The ideological contestation and the rise of da’wah radio Currently, Indonesia is home to multiple Islamic ideologies. Each
Islamic ideology is disseminated through media and organization. This
brings an ideological contestation. The ideological contestation within
Muslim communities can be traced back to Prophet Muhammad’s era
when Muslims at that time were involved in a heated debate as to who
was worthy of being the substitute for Prophet Muhammad’s leadership. Interpretation and understanding of different religious teachings are not
only related to politics, but also to legal issues which give birth to many
schools of Islamic law (fiqh),9 theology (kalam), and Sufism along with its
tariqah (Sufi Order). Diverse religious ideas later became the foundation and the world view
of various Muslim communities in Indonesia. However, the choice of
the Muslim community towards religious ideas that are characterized
by Shafi’iyah in the field of law turns out to be a majority compared to
other understandings. Likewise the choice tendency towards the practice
of the tariqah mu’tabarah (recognized Sufi Order) is far more desirable
than the ghairu mu’tabarah (non-recognized Sufi Order), especially the
Qadiriyah and Naqsabandiyah orders. In the archipelago, religious beliefs
(aqeedah) relating to human relations with Allah are much influenced
by the teachings of theology (kalam) developed by al-Imam al-Ash’ari 63 63 IJIMS: Indonesian Journal of Islam and Muslim Societies, Volume 12, Number 1, June 2022: 59-84 and al-Imam al-Maturidi compared to Mu’tazilah or Jabariyah. The
religious view based on the Shafi’i schools of thought practiced by the
Qadiriyah and Naqsabandiyah orders and this Ash’ariyah theology was
later called the followers of Ahl al-Sunnah wa al-Jama’ah.10 In Indonesia,
this term is more popularly referred to as “Indonesian Islam.” The use
of the term “Indonesian Islam” tends to be differentiated from the term
“Islam in Indonesia.” Azyumardi Azra suggests that the choice of the
term Indonesian Islam shows that “Muslims in Indonesia believe in the
same pillars of Islam, but in the embodiment of the social life of Islamic
culture and has its own distinctions which are not found anywhere else
in the Muslim World.”11 Along with its self-representation as the site of Islamic ideological
contestation, Indonesian Islam has witnessed the rise of non-in-person
da’wah activities, particularly those through airwaves. The rise of non-in-
person da’wah activities means that there has been the growth of da’wah
activities done on media, namely social media (YouTube, Facebook,
Whatsapp), television, and radio. 12Martin van Bruinessen, “Introduction: Contemporary Developments in Indonesian
Islam and the “Conservative Turn” of the Early Twenty-first Century”, in Martin van
Bruinessen (ed.), Contemporary Developments in Indonesian Islam: Explaining the “Conservative
Turn, Singapore: ISEAS–Yusof Ishak Institute, 2013, 1-20. f
y
,
,
,
11Azyumardi Azra, “Islam Indonesia: Kontribusi Pada Peradaban Global,” Prisma, vol.
9, no. 04 (2010), 83. 10See Richard C. Martin and Mark R. Woodward, and Dwi S. Atmaja, Defenders of Reason
in Islam: Mu’tazilism from Medieval School to Modern Symbol, Oxford,UK: Oneworld, 1997. 13See also Didik Novi Rahmanto, Adrianus Eliasta Meliala, Ferdinand Andi Lolo, “Ideology
Deconstruction of Islamic State of Iraq and Syria (ISIS) Returnees in Indonesia”, Indonesian
Journal of Islam and Muslim Societies, Volume 10, Number 2 (2020), 381-408. https://doi.
org/10.18326/ijims.v10i2.381-408 14Nurhaya Muchtar and Jeffrey A Ritchey, “Preaching, Community, and Convergence:
Use of Old and New Media by Progressive Indonesian Islamic Leaders”, The International
Communication Gazette, Volume 76, Number 4-5 (2014), 360-376. https://doi.
org/10.1177%2F1748048514524099 15Sunarwoto, “Dakwah Radio in Surakarta: A Contest for Islamic Identity”, in Jajat
Burhanuddin & Kees van Djik (eds.), Islam in Indonesia: Contrasting Images and Interpretations,
Amsterdam: Amsterdam University Press, 2013, 195-214. 16Irzum Farihah, “Radio sebagai Solusi Problem Keagamaan Muslimah,” At-Tabsyir, Volume
2, Number 2 (2014), 137-156. 2, Number 2 (2014), 137-156. 13See also Didik Novi Rahmanto, Adrianus Eliasta Meliala, Ferdinand Andi Lolo, “Ideology
Deconstruction of Islamic State of Iraq and Syria (ISIS) Returnees in Indonesia”, Indonesian
Journal of Islam and Muslim Societies, Volume 10, Number 2 (2020), 381-408. https://doi.
org/10.18326/ijims.v10i2.381-408
14Nurhaya Muchtar and Jeffrey A Ritchey, “Preaching, Community, and Convergence:
Use of Old and New Media by Progressive Indonesian Islamic Leaders”, The International
Communication Gazette, Volume 76, Number 4-5 (2014), 360-376. https://doi.
org/10.1177%2F1748048514524099
15Sunarwoto, “Dakwah Radio in Surakarta: A Contest for Islamic Identity”, in Jajat
Burhanuddin & Kees van Djik (eds.), Islam in Indonesia: Contrasting Images and Interpretations,
Amsterdam: Amsterdam University Press, 2013, 195-214.
16Irzum Farihah, “Radio sebagai Solusi Problem Keagamaan Muslimah,” At-Tabsyir, Volume
2, Number 2 (2014), 137-156. org/10.18326/ijims.v10i2.381 408
14Nurhaya Muchtar and Jeffrey A Ritchey, “Preaching, Community, and Convergence:
Use of Old and New Media by Progressive Indonesian Islamic Leaders”, The International
Communication Gazette, Volume 76, Number 4-5 (2014), 360-376. https://doi.
org/10.1177%2F1748048514524099
15Sunarwoto, “Dakwah Radio in Surakarta: A Contest for Islamic Identity”, in Jajat
Burhanuddin & Kees van Djik (eds.), Islam in Indonesia: Contrasting Images and Interpretations,
Amsterdam: Amsterdam University Press, 2013, 195-214.
16Irzum Farihah, “Radio sebagai Solusi Problem Keagamaan Muslimah,” At-Tabsyir, Volume
2, Number 2 (2014), 137-156. The ideological contestation and the rise of da’wah radio To get a better sense of a sociological
context of this phenomenon, the dynamics of state-Islam relations deserves
thorough attention. Since the era of information and political openness
after the collapse of the New Order in 1998, the religious understanding
of Muslim communities in Indonesia, which Martin van Bruinessen
calls “conservative turn”, grows and develops.12 The conservative turn
refers to the rise of conservative Muslims in Indonesia identified by the 64 Islam on the air: the struggle for Salafism through radio in Indonesia (Masduki, et.al) presence of radical Muslim groups such as the FPI (Islamic Defenders
Front) especially after the New Order period. The emergence of various religious understandings as mentioned
above seems to be driven by the interaction of local Muslim
community with the global Muslim community competing in winning
trust of the Muslim community. 13 Contestation of religious ideology can
be seen from the radio. In the Muslim community there is an
attempt to show Islamic identity in the public sphere as well as to
communicate with its followers14, including by radio in Batam. In the
industrial city of Batam with its so-called hybrid cultural character,
Salafi religious understanding has been strengthened along with the
emergence of Hang radio as one of the da’wah radios. More than that, the presence of da’wah radio has sociologically a close
connection to the local context of the representation of Islamic identity. Sunarwoto explains that the proliferation of da’wah radio in Surakarta is
not free from Islamic identity diversity in Surakarta.15 The growing presence
of da’wah radio has impacted women in that they have a growing access
to religious learning more widely than the conventional religious learning
activities so far. Irzum farihah assumes that radio has become important
to mediate da’wah to reach a community of women.16 65 IJIMS: Indonesian Journal of Islam and Muslim Societies, Volume 12, Number 1, June 2022: 59-84 18https://seindahsunnah.com/kabar-islam/daftar-radio-dan-tv-lokal-atau-internet-islami-
e-indonesia/ accessed on October 1st, 2021 at 01.57 PM. 17Muhammad Risantoso, Robeet Thadi, and Moch Iqbal, “Klasifikasi Pesan Dakwah pada
Radio Siaran L-Baas 97,6 FM,” Jurnal Dawuh, Volume 1, Mumber 1 (2020), 34-39. 19Noorhaidi Hasan, “ The Making of Public Islam: Piety, Agency and Commodification
n the Landscape of the Indonesian Public Sphere”, Contemporary Islam, Volume 3, Number
(2009), 229-250. g
23https://www.goodnewsfromindonesia.id/2021/02/25/kalahkan-medan-batam-jadi-kota
paling-makmur-di-region-sumatra accessed on October 4th, 2021 at 07.45 AM. 20https://worldpopulationreview.com/world-cities/batam-population accessed on October
1st, 2021 at 04.12 PM. 21https://regional.kompas.com/read/2020/11/24/21210891/umk-2021-kepri-ditetapkan-
tertinggi-di-batam-terendah-di-tanjungpinang accessed on October 1st , 2021 at 04.20 PM.
22https://batampos.co.id/2020/07/02/terbaru-segini-jumlah-perusahaan-asing-di-kota-
batam-sekarang/ accessed on October 4th, 2021 at 07.39 AM. p //
p p
/
/
p p
1st, 2021 at 04.12 PM.
21https://regional.kompas.com/read/2020/11/24/21210891/umk-2021-kepri-ditetapkan-
tertinggi-di-batam-terendah-di-tanjungpinang accessed on October 1st , 2021 at 04.20 PM.
22https://batampos.co.id/2020/07/02/terbaru-segini-jumlah-perusahaan-asing-di-kota-
batam-sekarang/ accessed on October 4th, 2021 at 07.39 AM.
23https://www goodnewsfromindonesia id/2021/02/25/kalahkan-medan-batam-jadi-kota- 17Muhammad Risantoso, Robeet Thadi, and Moch Iqbal, “Klasifikasi Pesan Dakwah pada
Radio Siaran L-Baas 97,6 FM,” Jurnal Dawuh, Volume 1, Mumber 1 (2020), 34-39.
18https://seindahsunnah.com/kabar-islam/daftar-radio-dan-tv-lokal-atau-internet-islami-
se-indonesia/ accessed on October 1st, 2021 at 01.57 PM.
19Noorhaidi Hasan, “ The Making of Public Islam: Piety, Agency and Commodification
on the Landscape of the Indonesian Public Sphere”, Contemporary Islam, Volume 3, Number
3 (2009), 229-250.
20https://worldpopulationreview.com/world-cities/batam-population accessed on October
1st, 2021 at 04.12 PM.
21https://regional.kompas.com/read/2020/11/24/21210891/umk-2021-kepri-ditetapkan-
tertinggi-di-batam-terendah-di-tanjungpinang accessed on October 1st , 2021 at 04.20 PM.
22https://batampos.co.id/2020/07/02/terbaru-segini-jumlah-perusahaan-asing-di-kota-
batam-sekarang/ accessed on October 4th, 2021 at 07.39 AM.
23https://www.goodnewsfromindonesia.id/2021/02/25/kalahkan-medan-batam-jadi-kota-
paling-makmur-di-region-sumatra accessed on October 4th, 2021 at 07.45 AM. ,
,
p
26Interview with Zein Alatas, Riau, 14 September 2017. ,
25Interview with Zein Alatas, Riau, 14 September 2017. 24https://www.batamnews.co.id/berita-35136-3-lokasi-mengintip-megahnya-singapura
dari-batam.html accessed on October 4th, 2021 at 07.50 AM. 24https://www.batamnews.co.id/berita-35136-3-lokasi-mengintip-megahnya-singapura-
dari-batam.html accessed on October 4th, 2021 at 07.50 AM.
25Interview with Zein Alatas, Riau, 14 September 2017.
26Interview with Zein Alatas, Riau, 14 September 2017. Hang Radio amongst the growing phenomenon of da’wah Many kinds of da’wah radio in Indonesia have been growing since the fall
of the New Order regime.17 In July 2014, the Association of Indonesian
Islamic Radio and Television published that there were 58 da’wah radios
in Indonesia.18 Hang Radio for example was founded in 2002 in Batam
at Batam Center Mall in Batam City and had 4 employees. This illustrates
that freedom of information during the reform period contributed to
the development of da’wah radio. The purpose of developing da’wah is
accommodated by government regulations that encourage freedom of
information thus it is easier to establish da’wah radio.19 In this context,
internal factors such zeal to spread the Islamic da’wah and external
factors in the form of freedom of information play an important role in
the development of da’wah radio in Batam. In 2021, the population of
Batam is 1.617.168. In fact, in 1950, its population was only 46.962.20The
standard of basic income in Batam in 2021 is Rp. 4.150.930.21 The number
of foreign companies in Batam is 537.22 Referring to the 2019 Central
Statistics Agency (BPS) data released in 2020, Batam was the richest city in
the Sumatra region. Its per capita revenue was Rp. 119.33 million/year.23 66 Islam on the air: the struggle for Salafism through radio in Indonesia (Masduki, et.al) 27Interview with Zein Alatas, Riau, 14 September 2017.
28Inaya Rakhmani, Mainstreaming Islam in Indonesia: Television, Identity & the Middle Class,
New York: Palgrave Macmillan, 2016, 4.
29Interview with Ghazali Yahya, Riau, 14 September 2017.
30Din Wahid, “ Nurturing Salafi Manhaj: A Study of Salafi Pesantren in Contemporary
Indonesia,” Wacana, vol.15, no. 2 (2014), 367-376. Islam on the air: the struggle for Salafism through radio in Indonesia (Masduki, et.al) Batam is very close to Singapore. Some places in Singapore can even be
seen from the hills in Batam.24 The trajectory of Hang Radio as a da’wah radio originated from the
path of “repentance”. This radio station used to be like other kinds
of urban radio attached to the identity of the juvenile. The broadcast
programs were filled with pop music entertainment. However, financial
problems became a turning point for this radio station to replace the so-
called “wrong” mission to the so-called “missionary” mission. The radio
did not get profit so it was bankrupt. The owner then thought that it
was due to the content of the radio programs which were not suitable
with Islamic values. Furthermore, the owner received financial assistance
from a donor. The donor convinced the owner of this radio station to
change the direction of the radio to the so-called “right” road. For the
owner, this was God’s “rebuke” for the good of himself and the people. Then, the owners approved the change request and the mission of the
broadcast program. Hang Radio is still used as a symbol of Malay which
is synonymous with Islam. Hang Radio, according to the owner of the
radio, Zein Alatas,25 is a name that precisely describes the spirit of the
great Malays of the past such as Hang Tuah and Hang Jebat. Now with 9
employees, Hang Radio is increasingly stable in finance. For Zein Alatas,
“When used for profit, this radio was closed for a long time. We live and
develop due to the donations from Muslims who care about our missionary
mission.”26 Alatas argues that the names of the people who donated are
not important. What needs to be underlined is that the struggle of da’wah
will not be a loss or ‘bankrupt’ because God will not be silent to provide 67 67 IJIMS: Indonesian Journal of Islam and Muslim Societies, Volume 12, Number 1, June 2022: 59-84 financial conveniences for those who are consistent in preaching.27 The location of Hang Radio is right in the center of the crowds of
Batam. It is located inside a trade or shopping complex. This reflects a
“marriage” between Islam and capitalism. Shops and trade are symbols
of capitalism while Hang Radio is a symbol of Islamic media that has a
mission to preach. Islam on the air: the struggle for Salafism through radio in Indonesia (Masduki, et.al) This is due to the fact that, according to Inaya Rakhmani
when discussing Islam in Indonesian television series, “rather than militant
groups determined to establish Sharia-based regimes, the intelligentsia is
using Western technology and education to push for democracy all the
while rejecting its superiority over Islamic moral values.”28 The strategy
of selecting locations in the crowd of Batam cities has become a kind of
‘stepping stone’ for the development of wider Islamic da’wah. Hang Radio
uses capitalism as an element of supporting the prophetic mission on the
one hand and rejects it as an element that contradicts the values of Islamic
law on the other one. 31Bianca J. Smith and Saipul Hamdi, “Between Sufi and Salafi Subjects: Female
Leadership, Spiritual Power and Gender Matters in Lombok,” in Bianca J. Smith and Mark
Woodward (eds), Gender and power in Indonesian Islam: Leaders, Feminists, Sufis and Pesantren Selves,
London and New York: Routledge, 2014, 25. The Salafism of Hang Radio Hang Radio does not accommodate other Islamic identities in its broadcast
programs. There will be no music in the broadcast program, even religious or
Islamic music, whether it is pop music or dangdut. This information is
strongly suggested by Ghazali Yahya, the director of Hang Radio program,
saying “the right entertainment for a Muslim is the Qur’an”.29 That speech
marks an identity that is unique in the Salafi Islamic tradition. The doctrine
of Salafism can be traced back to an earlier period, to the teachings of Ibn
Taymiyya and Ahmad ibn Hanbal.30 As a group that is closely associated 68 Islam on the air: the struggle for Salafism through radio in Indonesia (Masduki, et.al) with a literal approach to studying the Qur’an and Hadith. Salafists intend
to revive the traditions of the Prophet Muhammad and his companions
under the onslaught of modernization and globalization. According to Roel
Meijer as quoted by Bianca J. Smith and Saipul Hamdi,31 the Salafists are,
basically, “a fragmented movement that evades concrete definition.” Thus,
the term Salafi refers to movement that is fluid so that it does not have
a fixed definition. The Salafi movement is not a stable homogeneous
movement. In the global Islamic landscape, Salafi experiences an exchange
of ideas with local culture which influences its heterogeneous character. The following sub-subheadings illustrate how the Salafism of Hang
Radio comes to the forefront along with its airwave broadcast, allowing
three prominent issues, broadcast direction, the presentation of the role of
women, and the insistence of Islamic symbols, to become the subjects of
exemplary analysis. Each of the three issues is to be analytically discussed
in a very brief way one after the other just to show how Salafism came to
broadcasting. 33Noorhaidi Hasan, “Ideologi, Identitas dan Ekonomi-Politik Kekerasan: Mencari Model
Solusi Mengatasi Ancaman Radikalisme dan Terorisme di Indonesia”, Prisma, Volume 29,
No. 4 (2010), 10. 32https://www.bbc.com/indonesia/berita_indonesia/2016/08/160823_indonesia_radio_
batam_singapura accessed on October 1st, 2021 at 03.26 PM. Hang Radio’s D}a’wah broadcast direction Hang Radio does not broadcast music entertainment programs. This
reflects the characteristics of Salafi religious beliefs. Hang Radio considers
the definition of entertainment not merely in the form of music but
broad meaning. The Qur’an is the best entertainment that can be
presented. Entertainment in everyday life that develops today is a symbol
of capitalism. Hang Radio implicitly rejects capitalism that has caused a
spiritual drought. For Hang Radio, the spiritual drought is a result of the
absence of “entertainment,” namely murattal. Technically, murattal is played 69 IJIMS: Indonesian Journal of Islam and Muslim Societies, Volume 12, Number 1, June 2022: 59-84 as an “interlude” program for listeners. However, in a broader context, the
spiritual drought resulting from modernization and capitalism has basically
been utilized by Hang Radio for the survival of its broadcast programs. This favorite radio broadcast program is a matter of life. Listeners give
questions related to the problems they face, then the speaker gives answers
along with religious reasons. Basically, the listeners need answers based on
religion, especially the Qur’an and Hadith. Some broadcast programs on
Hang Radio, which are considered related to ‹sensitive› issues, are changed
and modified in accordance with public interest. The Hang Radio broadcast
program is always evaluated by Hang Radio management to anticipate
broadcast programs that are ‘hard.’ Management felt responsible for
preventing it because Singaporean media openly mentioned the influence
of the Hang Radio broadcast program in forming the radical character of
their listeners. According to the media reports, there were two Singaporeans
who wanted to join ISIS (Islamic State in Iraq and Syria; an organization
allegedly accused of as being a world’ terrorist group) because they were
affected by the Hang Radio broadcast program.32 They are loyal listeners to
Hang Radio who are likely to be influenced by the takfir doctrine. In the
Salafi group, takfir doctrine is a monotheism-based religious creed that is
widely known. This doctrine, according to Noorhaidi Hasan,33 comes from
the idea of Sayyid Qutb who wants to revive political Islam. As a Salafi radio, Hang Radio at the beginning always gives other
groups no room to carry out different religious practices. Hang Radio’s D}a’wah broadcast direction Not only is
the Singapore government worried about the impact of Hang radio
broadcast program, but also many local Muslims in Batam and they do not 70 70 Islam on the air: the struggle for Salafism through radio in Indonesia (Masduki, et.al) agree with the Islamic identity that was developed by Hang Radio. Several
other Muslim groups such as FPI (Islamic Defenders Front) felt ‘heated’ and
several times held demonstrations at the Hang Radio office. Responding
to the rejection and negative agitation of other Islamic groups, Hang Radio
made changes to its broadcasts. In the perspective of structural functional
theory, this phenomenon is common because a social system has the
ability to defend itself and adapt itself to something new, or to analyze,
borrowing the words of Giddens, “processes of evolution via mechanisms
of adaptation”.34 Moreover, the mechanism possessed by a social system
tends to show its ability to keep itself balanced. 36For comparison, see the way young Salafists actualize their identity in their daily life in
Yuyun Sunesti, Noorhaidi Hasan, and Muhammad Najib Azca, “Young Salafi-Niqabi and
Hijrah: Agency and Identity Negotiation”, Indonesian Journal of Islam and Muslim Societies,
Volume 8, Number 2 (2018), 173-197. https://doi.org/10.18326/ijims.v8i2.173-198. 34Anthony Giddens, The Constitution of Society: Outline of the Theory of Structuration, Berkeley
nd Los Angeles: University of California Press, 1984, 1. 35For comparison, Radio Fajri FM also rejects women as its presenters. See Neisya
Ghassani Sabilah and Armawati Arbi, “ Strategi Komunikasi Radio Fajri 993 FM Bogor
alam Mempertahankan Citra Radio Dakwah”, Al Munzir, Volume 13, Number 2 (2020), 141. and Los Angeles: University of California Press, 1984, 1.
35For comparison, Radio Fajri FM also rejects women as its presenters. See Neisya
Ghassani Sabilah and Armawati Arbi, “ Strategi Komunikasi Radio Fajri 993 FM Bogor
dalam Mempertahankan Citra Radio Dakwah”, Al Munzir, Volume 13, Number 2 (2020), 141.
36For comparison, see the way young Salafists actualize their identity in their daily life in
Yuyun Sunesti, Noorhaidi Hasan, and Muhammad Najib Azca, “Young Salafi-Niqabi and
Hijrah: Agency and Identity Negotiation”, Indonesian Journal of Islam and Muslim Societies,
Volume 8, Number 2 (2018), 173-197. https://doi.org/10.18326/ijims.v8i2.173-198. Hang Radio’s presentation of the role of women Hang Radio has quite clear rules in terms of gender segregation. Women are
not allowed to become employees at Hang Radio.35 If women were allowed
to work at Hang Radio, the atmosphere of religiosity in Hang Radio would
be reduced. Although the element of comfort was explicitly used as the
main reason; basically the reason was modified for the sake of strengthening
Islamic identity. In the Salafi tradition, segregation of men and women is a
necessity. Women are not allowed to move in the public domain.36 Besides
rejection of the presence of women in work activities, in Hang Radio,
there is also a strong paternalistic shade—the separation of studio entrances
and exits between males and females as an example. This limit is a symbolic
rule and at the level of reality there has been a deviation. This is in line with 71 IJIMS: Indonesian Journal of Islam and Muslim Societies, Volume 12, Number 1, June 2022: 59-84 the views of Delmus Puneri Salim who says that regulations in their basic
form are non- immune social products with social construction.37 In this context, the regulation of the separation of entrances for men
and women in Hang Radio has been reduced to the realm of reality. However, the integration in this type seems to be less in accordance with
the traditions and habits of the general public, especially in Batam. In a
cosmopolitan culture such as Batam, social interaction without segregation
of the sexes in public space become commonplace. Thus, the Hang Radio
regulation related to the separation between men and women does not
seem to be fully be implemented in social reality. 38Andrew N. Weintraub, “Introduction: The Study of Islam and Popular Culture in
Indonesia and Malaysia”, in Andrew N. Weintraub (ed.), Islam and Popular Culture in Indonesia
and Malaysia, London and New York: Routledge, 2011, 4. 37Delmus Puneri Salim, The Transnational and the Local in the Politics of Islam: The Case of
West Sumatra Indonesia, New York and London: Springer, 2015, 7-8. 39For comparison, see Zainal Abidin Achmad, “Integrasi Pesan Dakwah dan Budaya: Studi
Etnografi Virtual Mediamorfosis Radio Nada FM Sumenep Madura”, Jurnal Komunikasi Islam,
Volume 9, Number 2 (2019), 238-263. 40Larry Witham, Marketplace of the Gods: How Economics Explains Religion, Oxford: Oxford
University Press, 2010. Hang Radio’s insistence on Islamic symbols A symbol for an urban society has an important function for the struggle for
identity. Symbols also have meaning to the affiliated group. Basically,
symbols are very close to popular culture, serving as a selling point for
luring consumers to consume certain products. Since the 1990s, according
to Andrew N. Weintraub,38 “the public presence of Islamic symbols, ideas,
and texts in film, television, popular print media, music, and the Internet
in Indonesia and Malaysia has been growing.” The presence of Islamic symbols in public spaces in Indonesia signifies
two things; public piety is increasingly widespread on the one hand and
silencing the meaning of religion on the other. The first is suspected by
the symptoms of revivalism in Indonesia, while the second is motivated
and infiltrated by the spirit of capitalism. In the context of Hang Radio,
the two indications above are experiencing a process of attraction and 72 72 Islam on the air: the struggle for Salafism through radio in Indonesia (Masduki, et.al) mutual influence. The appearance of Hang Radio’s audio is interpreted
as a way to strengthen Hang Radio’s identity as an independent radio
missionary and Salafi character. Environmental governance of Hang Radio studio is also loaded with
the symbol of the purification of Islam typical of Salafism, but it is not
immune to capitalism.39 Near the parking area, there are stalls selling halal
food and drinks. In addition to various Indonesian food and beverages,
this shop sells typical Salafi products namely honey and herbal products. The nearby shops sell typical salafi attributes, such as books, headscarves,
honey, clothes, and so forth. Male visitors who enjoy food are generally
attached to salafi symbols. Female visitors wear headscarves and veils
while men wear white pants to the heels of their feet and are generally
bearded. Visitors at Hang Radio are generally filled with symbols of Islamic
identity that represent the salafi movement. Figure 1. HangFM Hang TVStation
(Source : The Straitstimes)
39For comparison, see Zainal Abidin Achmad, “Integrasi Pesan Dakwah dan Budaya: Studi
Etnografi Virtual Mediamorfosis Radio Nada FM Sumenep Madura”, Jurnal Komunikasi Islam,
Volume 9, Number 2 (2019), 238-263. Figure 1. HangFM Hang TVStation
(Source : The Straitstimes) Figure 1. HangFM Hang TVStation (Source : The Straitstimes) (Source : The Straitstimes) 73 Islam on the air: the struggle for Salafism through radio in Indonesia (Masduki, et.al) Islam on the air: the struggle for Salafism through radio in Indonesia (Masduki, et.al) program operations. Every year, Hang radio must pay rent for the studio. This significant cost certainly requires strategies based on economic value
for the continuation of the da’wah radio. Although what Hang Radio is
doing is the self-sustained economy; it is actually a part of capitalism. This
is due to the fact that Hang Radio does not only get money to support its
survival, it also receives economic profit to pay salary for its staffs. Strengthening salafi Islamic symbols can also be found in the Hang
Radio studio mushalla. Inside the mushalla there are bookshelves filled with
books. The books were obtained from visitor donations and purchased by
Hang radio employees. The ideas of the books represent the ideas of the
Salafi movement. The books do not allow the appearance or symbol of
human images. An Iqra’ book (a guide to reading Arabic letters to master
the Qur’an) whose back cover displays the picture of the author, Kiyai As’ad
Humam, is covered with white paper. For the Salafi movement, images of
human beings may not be displayed to avoid heresy. Inside the Mushalla there is a billboard that displays a number of donations
from visitors or mushalla worshipers. What is interesting is not only in
the form of rupiah (Indonesian currency) but also Singapore dollars. This
indicates that the influence of Hang radio has spread across national
borders. Visitors or worshipers from Singapore are interested in the
Hang Radio broadcast program and in turn change from listeners to
loyal followers of the Salafi identity. Loyal followers of Hang Radio are
characterized by behavior that changes totally in worship matters (religious
practice) to attributes or symbols. IJIMS: Indonesian Journal of Islam and Muslim Societies, Volume 12, Number 1, June 2022: 59-84 IJIMS: Indonesian Journal of Islam and Muslim Societies, Volume 12, Number 1, June 2022: 59-84 Food and beverage shops before entering the Hang Radio studio
represent the character of capitalism. Delicious dishes and fresh drinks
are offered. Visitors of Hang Radio studios are given a business show that
does not conflict with Islamic law. Every day, dozens of Hang Radio visitors
come from Batam, Singapore and Malaysia to see the atmosphere of the
radio studio and to improve the social solidity relations of fellow Hang
radio listeners. Even some visitors from Singapore occasionally provide
food assistance in the form of rice, instant noodles, or cakes. The donations
are distributed to those who are entitled to the ‘hand carts’ program. The
program was also broadcast by Hang Radio as an effort to attract listeners
to make donations on the one hand and efforts to improve the broadcast
programs by making them more interesting to be heard on the other side. Capitalism is an ideology that manifests and seeps to various sectors of
life along with the openness of a social system. It can manifest in various
faces and can join hands with elements of religion, so that it can easily
give rise to the so-called “economics of religion.”40 In the context of
Hang radio, both social interaction with the surrounding environment
and the influence of social structures encourage the spirit of capitalism to
infiltrate Hang Radio policies, regulations and strategies. The openness
of its relationships with generous listeners has encouraged Hang Radio
to flex its policies in terms of religion to be in line with the ideology of
capitalism. Generous listeners can contribute in the form of material to
support the symbolization of their salafi identity. The support can be in
cash or through the purchase of salafi attributes sold near the Hang Radio
studio. The value of economic benefits from the practice of buying and
selling commodities is clearly useful for the benefit of Hang radio.This is
because Hang Radio requires funds that are not small for the broadcast 74 41Imron Rosidi and Rizal Zain, “ Strategi Radio Republik Indonesia (RRI) Pekanbaru
dalam Menyampaikan Program Siaran Dakwah”, Idarotuna, Volume 1, Number 2 (2019), 1-15.
42See Sarah Genner and Daniel Süss, “Socialization as Media Effect”, in Patrick
Rössler, Cynthia A Hoffner, and Liesbet van Zoonen (eds.), The International Encyclopedia of
Media Effects, Chichester, West Sussex; Malden, MA: John Wiley & Sons, Inc., 2017, 1-15.
43Masduki and Shabri Shaleh Anwar, Filosofi Dakwah Kontemporer, Tembilahan, Riau:
PT. Indragiri Dot Com, 2018, 77-88 ff
J
y
43Masduki and Shabri Shaleh Anwar, Filosofi Dakwah Kontemporer, Tembilahan, Riau:
T. Indragiri Dot Com, 2018, 77-88 44For comparison, see Syarif Fauzi and Afif Abd. Latif, “Model Radio Dakwah dalam
Meningkatkan Pemahaman Keagamaan Masyarakat”, Tabligh: Jurnal Komunikasi dan Penyiaran
Islam, Volume 1, number 2 (2016), 89-110. 45Zaki Islam, Aef Kusnawan, and Dadan Anugrah, “ Fenomena Dakwah Salaf di Radio
Tarbiyah Sunnah 1476 AM”, Prophetica: Scientific and Research Journal of Islamic Communication
and Broadcasting, Volume 5, Number 1 (2019), 28. g,
,
,
46Interview with Zein Alatas, Riau, 14 September 2017. The trajectory of Salafism on the air Hang Radio listeners usually come from a profession that is mobile. In
Batam, the majority of listeners began an emotional connection with the
radio station through radio installed in their personal cars. They listen to 75 IJIMS: Indonesian Journal of Islam and Muslim Societies, Volume 12, Number 1, June 2022: 59-84 Hang Radio broadcasts while driving.41 In the midst of the business and
traffic jams, they need something that can be heard to avoid psychological
confusion on the one hand, and to learn religious teachings through the
airwaves on the other hand. Motivation of ‘fadness’ combined with a
passion for learning religion has complemented the process of forming
the identity of a listener and turned him into a loyal follower of the Salafi
movement. During this time, these listeners did not really understand the
teachings of Islam because most of them had a “secular” educational
background. These listeners positioned Hang Radio as a new ‘center
of religious learning’ which negated the center of traditional and non-
religious learning environments. In current development of life, the value
socialization value has shifted from conventional institutions (families and
educational institutions) towards new media (television, radio, and the
internet),42 or digital-based.43 The role of these conventional institutions
has been reduced along with the dominance of radio, the internet and
television media in the process of value dissemination. In that context, Hang Radio has functioned as a media that adds to
the diversity of religious authorities in Batam. This is influenced by the
effectiveness and efficiency of the religious learning process which does not
require much energy, time and money. Hang Radio is also a ‘challenger’
that is quite calculated towards the authority of established traditional
scholars. Crucial religious issues need not be questioned by ulama (Muslim
scholars) because Hang Radio has often provided ‘enlightenment’ of 76 76 Islam on the air: the struggle for Salafism through radio in Indonesia (Masduki, et.al) Islam on the air: the struggle for Salafism through radio in Indonesia (Masduki, et.al) the religion needed. The authority of traditional scholars is possible to
experience reduction along with the development of da’wah media. Central to Hang Radio’s struggles to come to the forefront of Islam in
Batam are its strategies to present itself as a da’wah radio, allowing itself
to be identical to no other than the promulgation of Islamic religious
learning.44 For this reason, the radio rejects to be accused of being a radio
that brings radicalism in the Muslim community. To dismiss this kind of
accusation, the radio is committed to the Islamic tradition of carrying
out the so-called “peace.” 45The owner of Hang Radio, Zein Alatas, said
“we work in the public domain; many people are watching over our work. How can we spread ideas or convey ideas that are clearly prohibited by the
State. If we want to spread radical ideas or ideologies, of course we will
work quietly in a hidden place.”46 Hang Radio uses the term public space to answer allegations of radicalism
addressed to it. The public sphere has ‘transparency’ that can be accessed by
all parties. Public space is like a transparent “aquarium” that can be seen by
everyone. In public spaces there are still private domains or domains that are
“hidden.” In this case it is clear that there is still something guarded not to
be displayed in the public space. However, Hang Radio is still in the stage
of fighting for its salafi identity in the public sphere. Basically, the role of
other Muslim communities is very dominant in suppressing the movement
of deviations that might occur in Hang Radio. The Muslim community in
Batam still strongly recognizes that Indonesian Islam is tolerant and open to
a diversity of thoughts. The accommodative nature is sometimes mistaken 77 IJIMS: Indonesian Journal of Islam and Muslim Societies, Volume 12, Number 1, June 2022: 59-84 by some scholars as a form of syncretism between religion and culture, even
though this kind of assumption has been denied by a number of circles, for
example by Azra,47 saying that syncretic-Abangan Islam in Geertz’s terms48 was
no longer relevant in the present context. The renewal and revival of Islam in
Indonesia over the last two decades have actually eliminated that assumption. Hang Radio is trying to fight for an Islamic identity in the public space. 47Azyumardi Azra, “Islam Indonesia”…, 83.
48Clifford Geertz, The Religion of Java, Chicago and London: The University of Chicago
Press, 1976.
49Sunarwoto, “Dakwah Radio in Surakarta”…, 196. 48Clifford Geertz, The Religion of Java, Chicago and London: The University of Chicago
ress, 1976. 49Sunarwoto, “Dakwah Radio in Surakarta”…, 196. 47Azyumardi Azra, “Islam Indonesia”…, 83. 47Azyumardi Azra, “Islam Indonesia”…, 83. Islam on the air: the struggle for Salafism through radio in Indonesia (Masduki, et.al) should have its own uniqueness or distinctiveness. This is related to the
increasingly tight business competition today. The uniqueness is important
to be a selling power that attracts listeners to continue to consume the
Hang Radio broadcast program. The second purpose is that the distinctive
identity is highlighted to influence listeners so that they have the same
identity they share with each other. The motive is that Hang Radio is
making efforts to influence listeners to follow the religious understanding
or religious identity they have developed. Islam on the air: the struggle for Salafism through radio in Indonesia (Masduki, et.al) Identity in this context refers to the view of Sunarwoto49 that “it is what
makes people different from one another and significant membership
of a certain society.” In that context, identity is something inherent in a
person and group. In Islam, along with the diversity of interpretations of
Islamic texts, the presence of a variety of different identities in the Muslim
community is a necessity. The Salafi ideas echoed by Hang Radio will not give birth to
homogenization in the Muslim community. This is because the influence
of Hang Radio will vary at the level of individual Muslims depending on
their socio-cultural background and education. Hang Radio listeners have
different perceptions of the Hang radio broadcast program. Especially in
Batam, besides Hang radio, there are many other da’wah radio stations
struggling with each other over the “how Muslims should be” discourse
in the onslaught of modernization and globalization. Each da’wah radio
has its own distinctive identity. The identity is highlighted in the broadcast program. The protrusion has
two purposes. The first is that the distinctive identity is set up to be a selling
power to the public. This means that the identity highlighted by Hang
Radio has its own advantages as a tool or a means of differentiation from
other da’wah radios in Batam. Every product sold in the public domain 78 78 Islam on the air: the struggle for Salafism through radio in Indonesia (Masduki, et.al) Conclusion The struggle of Hang Radio in Batam is a form of exertion to enforce
Islamic identity through the media. At first, the Hang Radio station
had a rigid mission of Islamic purification. Along with the increasing
contestation with other Muslim communities, Hang Radio softened its
self-perceived prophetic mission. Hang Radio produces ideas, regulations,
symbols, broadcast programs, and studio forms based on their Salafi Islamic
identity. Radio managers (owners and employees) continue to practice
negotiations, but substantially do not eliminate the identity of ‘Salafi
Islam,’ which is attached to Hang Radio. Hang Radio, which has Salafi
religious ideas and an audience segment of this group, has a way to fight for
Islamic identity. This is framed in the vision, mission, affiliation, programs,
and policies and so on. Hang Radio tries to deliver how Muslims should
be in the midst of modernization, secularization and globalization. As an implication, the way Hang Radio came into the broadcasting field
along with its struggles for the Salafist da’wah of Islamic ideology it adheres
to suggests the dynamics of Islam on the airwaves in Indonesia, including in
a growing industrial city of Batam. This further implies that the struggle for
a certain Islamic identity is more fragmented than intact and cohesive. This
struggle for the Islamic identity in question is not immune from the spirit of 79 79 IJIMS: Indonesian Journal of Islam and Muslim Societies, Volume 12, Number 1, June 2022: 59-84 capitalism. However, it seems to be remarkably influenced by transnational
and local elements, both ideologically and economically. Apart from that,
the presence of Hang Radio in Batam indicates that the struggle for the
Salafist Islamic identity through the da’wah media appears to be exclusively
strong, and it is justifiable to say that Salafism in Batam has presented its
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Feeding on resistant rice leads to enhanced expression of defender against apoptotic cell death (OoDAD1) in the Asian rice gall midge
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Abstract Background: The Asian rice gall midge (Orseolia oryzae) is a destructive insect pest of rice. Gall midge infestation in
rice triggers either compatible or incompatible interactions leading to survival or mortality of the feeding maggots,
respectively. In incompatible interactions, generation of plant allelochemicals/defense molecules and/or inability of
the maggots to continue feeding on the host initiate(s) apoptosis within the maggots. Unraveling these molecular
events, triggered within the maggots as a response to feeding on resistant hosts, will enable us to obtain a better
understanding of host resistance. The present study points towards the likely involvement of a defender against
apoptotic cell death gene (DAD1) in the insect in response to the host defense. Results: The cDNA coding for the DAD1 orthologue in the rice gall midge (OoDAD1) consisted of 339 nucleotides
with one intron of 85 bp and two exons of 208 and 131 nucleotides. The deduced amino acid sequence of OoDAD1
showed a high degree of homology (94.6 %) with DAD1 orthologue from the Hessian fly (Mayetiola destructor) —a
major dipteran pest of wheat. Southern hybridization analysis indicated that OoDAD1 was present as a single copy in
the genomes of the Asian rice gall midge biotypes (GMB) 1, 4 and 4 M. In the interactions involving GMB4 with Jaya
(susceptible rice host) the expression level of OoDAD1 in feeding maggots gradually increased to 3-fold at 96hai (hours
after infestation) and peaked to 3.5-fold at 96hai when compared to that at 24 hai. In contrast, expression in maggots
feeding on RP2068 (resistant host) showed a steep increase of more than 8-fold at 24hai and this level was sustained at
48, 72 and 96hai when compared with the level in maggots feeding on Jaya at 24hai. Recombinant OoDAD1,
expressed in E. coli cells, when injected into rice seedlings induced a hypersensitive response (HR) in the resistant
rice host, RP2068, but not in the susceptible rice variety, Jaya. Conclusions: The results indicate that the expression of OoDAD1 is triggered in the feeding maggots probably
due to the host resistance response and therefore, is likely an important molecule in the initial stages of the
interaction between the midge and its rice host. Keywords: Plant-insect interactions, Incompatible interactions, Hypersensitive response, Apoptosis, Orseolia oryzae,
Oryza sativa, Gall formation executed and regulated by various molecules within the
animal cell [2]. © 2015 Sinha et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. RESEARCH ARTICLE
Open Access
Feeding on resistant rice leads to enhanced
expression of defender against apoptotic cell
death (OoDAD1) in the Asian rice gall midge
Deepak K Sinha1 Isha Atray1 JS Bentur2,3 and Suresh Nair1*
Sinha et al. BMC Plant Biology (2015) 15:235
DOI 10.1186/s12870-015-0618-y RESEARCH ARTICLE
Open Access
Feeding on resistant rice leads to enhanced
expression of defender against apoptotic cell
death (OoDAD1) in the Asian rice gall midge
Deepak K Sinha1 Isha Atray1 JS Bentur2,3 and Suresh Nair1*
Sinha et al. BMC Plant Biology (2015) 15:235
DOI 10.1186/s12870-015-0618-y Sinha et al. BMC Plant Biology (2015) 15:235
DOI 10.1186/s12870-015-0618-y Open Access Abstract One such regulator, DAD1 (defender
against apoptotic cell death) was identified and proved
to be interacting with MCL1 (a member of the BCL2
protein family) providing a new perspective on its putative
role in apoptosis. The DAD1 gene was originally isolated
during complementation studies of a mutant hamster
cell line undergoing apoptosis upon incubation at non-
permissive temperatures. Further, these tsBN7 cells could
be rescued at the non-permissive temperature upon trans-
fection with the DAD1 wild-type gene [3]. Feeding on resistant rice leads to enhanced
expression of defender against apoptotic cell
death (OoDAD1) in the Asian rice gall midge Deepak K. Sinha1, Isha Atray1, JS Bentur2,3 and Suresh Nair1* * Correspondence: suresh@icgeb.res.in
1Plant Molecular Biology Group, International Centre for Genetic Engineering
and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110 067, India
Full list of author information is available at the end of the article Background Apoptosis or programmed cell death features stereotyp-
ical morphological changes such as shrinking of cell, cell
deformation, condensation of chromatin and finally cell
fragmentation into apoptotic bodies. These changes are
the consequences of several biochemical and molecular
events occurring within the cell [1]. Such events are * Correspondence: suresh@icgeb.res.in
1Plant Molecular Biology Group, International Centre for Genetic Engineering
and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110 067, India
Full list of author information is available at the end of the article Page 2 of 11 Sinha et al. BMC Plant Biology (2015) 15:235 Involvement of DAD1 in regulation of apoptosis or
apoptosis related pathways have since been widely re-
ported. DAD1 was reported to be involved during devel-
opment of C. elegans embryos [4] and Bombyx mori [5]. Involvement of DAD1 in temperature induced apoptotic
cell death was reported in Araneus ventricosus and Argo-
pecten irradians [6, 7]. In the plant kingdom, the role of
DAD1 homologues has been reported in Arabidopsis
thaliana [8], pea [9] and rice [10]. Differential regulation
of DAD1 gene was observed in flower petals during the
senescence phase [9]. Results of these investigations indi-
cated the important role played by DAD1 in apoptosis
and development in both animal and plant systems. Up-regulation of DAD1-like anti-apoptotic genes was
speculated in insect pests upon stress encountered due
to plant defense molecules [11]. Interestingly, DAD1
homologue was up-regulated in one such insect pest,
Hessian fly (Mayetiola destructor), feeding on resistant
wheat (wheat host that initiates hypersensitive mediated
defense response upon Hessian fly infestation), suggesting
its role in inhibition of unwanted apoptosis triggered due
to the defense response of the host [11]. As observed in
the Hessian fly-wheat interaction, the Asian rice gall
midge (Orseolia oryzae) infestation in rice is known to
induce a similar type of defense response. However, no
reports on the role of any anti-apoptotic genes in the
rice-gall midge interaction are available. insect during hypersensitive incompatible or compatible
interaction. This work was initiated with the hypothesis
that the maggots feeding on resistant rice plants en-
counter greater stress-induced challenge when compared
to maggots feeding on susceptible rice plants. This stress
encountered by the insect in the resistant host, generated
by the plant defense molecules [20] and the inability to
feed thereafter, leads to initiation of apoptosis within the
insect. Background In order to survive, the likely survival mechanism
in the insect would be the up-regulation of anti-apoptotic
genes [11]. DAD1 is an important apoptotic suppressor
gene and till date there has been no report on DAD1-like
genes from the Asian rice gall midge. Also, it is still un-
clear, if secretions from the gall midge feeding on the rice
pant trigger apoptosis in the host plant or not. Therefore,
we deemed it pertinent to clone, characterize and express
DAD1 from the gall midge and evaluate its role in the in-
sect and corresponding host responses, if any. The current study describes characterization of DAD1
from the Asian rice gall midge and its transcriptional
expression patterns in the insect during compatible and
incompatible interactions with its host. Orseolia oryzae
DAD1 (OoDAD1) was isolated from a cDNA library
generated from maggots feeding on susceptible and resist-
ant host varieties [21]. Transcriptional over-expression of
OoDAD1 observed in maggots during compatible and in-
compatible interactions suggested its role in regulation of
unwanted apoptosis. In addition, this study for the first
time demonstrated that the host plant recognizes DAD1
from the insect, leading to the induction of a hypersen-
sitive mediated response. Results of the present investi-
gation revealed the important role played by DAD1 in
insect-plant interaction. The Asian rice gall midge is the third major insect pest
of rice. Estimated economic loss incurred upon gall midge
attack amounts to $80 million in India alone [12]. More
recently, as a result of extensive research towards under-
standing the galling interaction [13–16] and with the
availability of vast amounts of sequence data [16], rice-gall
midge is emerging as a useful model system for under-
standing the molecular and physiological events that en-
able insects to overcome the host defense machinery. The
gall midge-rice interaction is either compatible or incom-
patible [17]. During compatible interaction, the gall midge
manipulates the host to survive and induces gall forma-
tion; whereas during incompatible interaction the host
defense overcomes the strategies adopted by the insect
leading to the mortality of the gall midge. Incompatible
interaction can be classified into two types: HR+ and
HR-. HR+ type interaction is manifested by a hypersensi-
tive response and cell death in the plant at the region
of entry of the gall midge maggots whereas HR-type
interaction is non-hypersensitive mediated defense response. Background Inability of the insect maggots to feed due to production of
plant allelochemicals/defense molecules results in the
death of the maggots in an incompatible interaction and
the maggots usually die within 96 h post egg hatching [18]. V i
di
h
d
ib d h h
i i
h Characterization of OoDAD1 The full-length cDNA of OoDAD1 consisted of 339 nu-
cleotide bases [GenBank:KP890835] coding for 113 amino
acids with a predicted molecular mass of 12.7 kDa. The
cDNA clone was designated OoDAD1 and the genomic
sequence was designated gOoDAD1 [GenBank:KP890834]. The latter consisted of one intron of 85 bp and two exons
of 208 and 131 nucleotides. The estimated pI of the predicted protein OoDAD1
was found to be 9.18. There were eight non-polar and
seven polar amino acid residues. The instability index, as
computed by ExPASy-ProtParam tool, was 36.33 that
classified the protein as a stable protein. TOPCONS pre-
dicted OoDAD1 to possess three trans-membrane heli-
ces (Additional file 1: Figure S1). Bioinformatics analysis
using SMART predicted the absence of a secretory signal se-
quence in the predicted protein while TOPCONS confirmed
that the protein was likely to be localized to membranes. Various studies have described the hypersensitive mech-
anism in the host plants [19]. However, there are few stud-
ies that investigated and compared the response of the Page 3 of 11 Sinha et al. BMC Plant Biology (2015) 15:235 (Acrythosiphon pisum) both of which are also plant
feeders. Maximum homology of the deduced amino acid sequence
was observed with DAD1 from Mayetiola destructor
(MdesDAD1; 89 %, 3e–52; Acc. No. ABY21317) DAD1
followed by DAD1 from Anopheles gambiae (78 %, 2e–47;
Acc. No. AAQ94040). Secondary structure prediction tool,
ROBETTA, predicted 5 probable structures for OoDAD1
(Additional file 2: Figure S2). Of all the predicted models
the fifth model was found to have lowest score (lowest
energy) and the maximum stability. OoDAD1 was predicted
to possess four large alpha-helices, as reported in
MdesDAD1, with no beta strands across the entire de-
duced protein sequence. Differential expression analysis of OoDAD1 in maggots
feeding on susceptible and resistant rice varieties g
p
The transcript level of OoDAD1 was assessed in a set of
compatible (Jaya-GMB4) and incompatible interactions
(RP2068-GMB4) (Fig. 4). The expression level of OoDAD1
transcripts at 24 h in maggots feeding on the susceptible
(Jaya) host was used as a baseline and the expression at all
other time points were scored relative to this. In the inter-
actions involving GMB4 with Jaya (susceptible host) and
RP2068 (resistant host), over-expression of OoDAD1 was
observed in both cases. However, the expression level in
maggots feeding on Jaya gradually increased by more than
3-fold at 72hai and peaked to 3.5-fold at 96hai [0.35
(log10) fold] (hours after infestation) in comparison to
maggots feeding on Jaya 24hai. However, in case of
maggots feeding on RP2068 there was sudden increase
of expression level to 8-fold from 24hai [0.8 (log10) fold]
and this enhanced expression was sustained at 48hai [0.65
(log10) fold], 72hai [0.70 (log10) fold] and 96hai [0.85
(log10) fold]. Southern analysis Southern hybridization, using a 339 bp OoDAD1 fragment
as probe, revealed that OoDAD1 existed as a single copy
in all the three biotypes (GMB1, GMB4 and GMB4M)
analysed (Fig. 3). A single hybridization signal was ob-
served in all the three biotypes digested with restriction
enzymes EcoRI, EcoRV and DraI. Further, no restriction
fragment length polymorphism (RFLP) was detected be-
tween these biotypes. A multiple sequence alignment of the predicted amino
acid sequence of DAD1 (Fig. 1) from different insects
with OoDAD1 revealed several homologous domains and
conserved regions. Results indicated that the protein
was highly conserved at the C-terminal region as com-
pared to the N-terminus. The highest degree of homology
(similarity score) was 94.6 % in case of Mayetiola destruc-
tor (Acc. No. ABY21317). Phylogenetic analysis
h l
l Phylogenetic analysis of OoDAD1 revealed the degree of
relationship of OoDAD1 with respect to those from other
organisms (Fig. 2). However, this study clearly classified
DAD1 of plant and animal taxa into two large clades. The
clade containing DAD1 from animals were further sub-
divided into vertebrates and invertebrates. The dipterans
were clustered in a sub-clade under the invertebrates
group. OoDAD1 was grouped within the clade containing
other insects belonging to the order Diptera. The tree also
revealed close relationship of OoDAD1 with orthologues
from the Hessian fly (Mayetiola destructor) and aphid Acyrthosiphon pisum (BAH72166)
Culex quinquefasciatus (EDS39316)
Maconellicoccus hirsutus (ABM55607)
Anopheles gambiae (AAQ94040)
Aedes aegypti (ABF18074)
Orseolia oryzae (KP890835)
Drosophila melanogaster (NP_609222)
Mayetiola destructor (ABY21317)
Acyrthosiphon pisum (BAH72166)
Culex quinquefasciatus (EDS39316)
Maconellicoccus hirsutus (ABM55607)
Anopheles gambiae (AAQ94040)
Aedes aegypti(ABF18074)
Orseolia oryzae (KP890835)
Drosophila melanogaster (NP_609222)
Mayetiola destructor (ABY21317)
Acyrthosiphon pisum (BAH72166)
Culex quinquefasciatus (EDS39316)
Maconellicoccus hirsutus (ABM55607)
Anopheles gambiae (AAQ94040)
Aedes aegypti (ABF18074)
Orseolia oryzae (KP890835)
Drosophila melanogaster (NP_609222)
Mayetiola destructor (ABY21317)
Fig. 1 Multiple sequence alignment of OoDAD1 with its orthologues from blood-feeding, sap-sucking and phytophagous insects. Black line indicates
the variable N-terminal region of the proteins compared. Shaded boxes highlight conserved domains. Accession numbers are in parenthesis. Conserved amino acid residues are shown below the shaded boxes Acyrthosiphon pisum (BAH72166)
Culex quinquefasciatus (EDS39316)
Maconellicoccus hirsutus (ABM55607)
Anopheles gambiae (AAQ94040)
Aedes aegypti (ABF18074)
Orseolia oryzae (KP890835)
Drosophila melanogaster (NP_609222)
Mayetiola destructor (ABY21317)
Acyrthosiphon pisum (BAH72166)
Culex quinquefasciatus (EDS39316)
Maconellicoccus hirsutus (ABM55607)
Anopheles gambiae (AAQ94040)
Aedes aegypti(ABF18074)
Orseolia oryzae (KP890835)
Drosophila melanogaster (NP_609222)
Mayetiola destructor (ABY21317)
Acyrthosiphon pisum (BAH72166)
Culex quinquefasciatus (EDS39316)
Maconellicoccus hirsutus (ABM55607)
Anopheles gambiae (AAQ94040)
Aedes aegypti (ABF18074)
Orseolia oryzae (KP890835)
Drosophila melanogaster (NP_609222)
Mayetiola destructor (ABY21317)
Fig. 1 Multiple sequence alignment of OoDAD1 with its orthologues from blood-feeding, sap-sucking and phytophagous insects. Black line indicates
the variable N-terminal region of the proteins compared. Shaded boxes highlight conserved domains. Accession numbers are in parenthesis. Conserved amino acid residues are shown below the shaded boxes Fig. 1 Multiple sequence alignment of OoDAD1 with its orthologues from blood-feeding, sap-sucking and phytophagous insects. Black line indicates
the variable N-terminal region of the proteins compared. Shaded boxes highlight conserved domains. Accession numbers are in parenthesis. Conserved amino acid residues are shown below the shaded boxes Sinha et al. Phylogenetic analysis
h l
l BMC Plant Biology (2015) 15:235 Page 4 of 11 Plasmodium falciparum (CAX64065)
Entamoeba histolytica (XP653360)
Chlamydomonas reinhardtii (EDP07649)
Toxoplasma gondii (EEB03219)
Cryptococcus neoformans (XP566824)
Paracoccidioides brasiliensis (EEH34703)
Penicillium marneffei (XP002150325)
Ajellomyces dermatitidis (EEQ78565)
Neosartorya fischeri(XP001265275)
Aspergillus fumigatus (EDP55725)
Caenorhabditis elegans (AAB96727)
Brugia malayi (EDP36930)
Maconellicoccus hirsutus (ABM55607)
Caligus clemensi (ACO15368)
Ixodes scapularis (AAY66894)
Schistosoma mansoni (CAZ29760)
Araneus ventricosus (AAN86571)
Aedes aegypti (ABF18074)
Drosophila melanogaster (NP609222)
Penaeus monodon (ABU54835)
Acyrthosiphon pisum (BAH7216)
Mayetiola destructor (ABY21317)
Orseolia oryzae (KP890835)
Argopecten irradians (AAX56947)
Nematostella vectensis (XP001627013)
Esox lucius (ACO14483)
Oncorhynchus mykiss (ACO07853)
Salmo salar (ACI67912)
Anoplopoma fimbria (ACQ58842)
Epinephelus awoara (ABD79023)
Tetraodon nigroviridis (CAF97730)
Osmerus mordax (ACO09768)
Homo sapiens (AAP36473)
Pongo abelii (CAH90946)
Bos taurus (AAX09015)
Sus scrofa (BAA13115)
Taeniopygia guttata (ACH43768)
Gallus gallus (NP001007474)
Xenopus laevis (BAA03652)
Danio rerio (AAI50400)
Pisum sativum (AAC77357)
Petunia x hybrida (AAO73434)
Zea mays (ACG28121)
Arabidopsis thaliana (NP565807)
Ricinus communis (XP002518988)
Citrus unshiu (BAA36555)
Oryza sativa Japonica (BAA24072)
Hordeum vulgare (CAB56224)
Solanum lycopersicum (CAB61887)
Betula pendula (CAB66329)
Picea mariana (AAC32147)
Elaeis guineensis (ACF06465)
Perkinsus marinus (XP002766749)
Paramecium caudatum (BAD13417)
Cryptosporidium muris (EEA04882)
Culex quinquefasciatus (EDS39316)
Sap
Sucking
Insects
N
O
N
C
H
O
R
D
A
T
E
S
C
H
O
R
D
A
T
E
S
A
N
I
M
A
L
I
A
P
L
A
N
T
A
E
Gladiolus hybrid cultivar 'Traveler' (BAD11075)
Nicotiana suaveolens x tabacum (BAB40809)
98
99
97
77
100
100
99
91
65
94
90
74
Anopheles gambiae ((AAQ94040)
Fig. 2 Phylogenetic tree showing relationship between orthologues of DAD1 reported from different organisms. The tree was constructed using the
Neighbour-joining method and the pair-wise distances were calculated using the Poisson-corrected distance method included in the MacVector suite
of programs. Branch lengths are arbitrary. This tree was arrived at using 1000 replications and figures at nodes represent detected bootstrap
values above 50 %. Accession numbers are in parenthesis Fig. 2 Phylogenetic tree showing relationship between orthologues of DAD1 reported from different organisms. The tree was constructed using the
Neighbour-joining method and the pair-wise distances were calculated using the Poisson-corrected distance method included in the MacVector suite
of programs. Branch lengths are arbitrary. This tree was arrived at using 1000 replications and figures at nodes represent detected bootstrap
values above 50 %. Accession numbers are in parenthesis Generation of recombinant OoDAD1 was observed in Jaya plants after injection (Additional file 3:
Figure S3). The injection region of the plants when stained
with DAB showed brown coloration in Suraksha after 48 h
post injection and after 72 h post injection in RP2068 (Fig. 7). DAB staining produces brown coloration in tissues having
increased peroxidase activity (increased production of
reactive oxygen species) and is used as a marker for
hypersensitivity in plants. The use of pET 28a vector for protein expression resulted
in the production of His-tagged OoDAD1 protein with a
molecular weight of 13.2 kDa, (including the seven His-
residues in the N-Terminal region) as expected. Anti-His
antibodies were used to confirm the expression and the
size of the protein (Fig. 5). Discussion The purified OoDAD1 protein (purified using Ni-NTA
column), the protein elution buffer and water injection
into the host variety RP2068, initiated HR. However, the
observed spread of HR in RP2068 (Fig.6a) was more in
plants injected with OoDAD1 when compared with the
plants injected with buffer and BSA. However, no HR For a better understanding of the molecular basis of insect-
plant interaction, studying the defense response initiated
by the avirulent maggots, feeding on a resistant host, is
equally important as gaining insight into the mechanism of
infestation of virulent maggots. Initiation of hypersensitive Page 5 of 11 Sinha et al. BMC Plant Biology (2015) 15:235 Fig. 3 Southern analysis of genomic DNAs of Asian rice gall midge biotypes (GMB1, GMB4 and GMB4M). The DNAs were digested with EcoRI, EcoRV
and DraI and probed with a 339 bp fragment of OoDAD1. Molecular mass (in kb) of the hybridization signals is indicated by figures on the left Fig. 3 Southern analysis of genomic DNAs of Asian rice gall midge biotypes (GMB1, GMB4 and GMB4M). The DNAs were digested with EcoRI, EcoRV
and DraI and probed with a 339 bp fragment of OoDAD1. Molecular mass (in kb) of the hybridization signals is indicated by figures on the left DAD1 from an insect which when injected into resistant
rice hosts induced a hypersensitive response. response as a result of apoptosis in the plants has been well
documented in case of plant-microbe interaction [22]. However, other than the study on Hessian fly-wheat inter-
action, there are no additional reports that delve into the
modulation of genes related to apoptosis in the insect dur-
ing its interaction with the host plant [11]. BLAST and phylogenetic analyses not only showed
high homology between DAD1 orthologues of insects
and OoDAD1 but also a high degree of conservation of
the predicted amino acid residues of OoDAD1 with
those reported for DAD1 from other species including
plants and vertebrates. Such a high degree of conserva-
tion is likely indicative of an important functional role
played by DAD1 in vertebrates, invertebrates and plants. The current study indicated that OoDAD1 is transcrip-
tionally modulated in the pest depending on whether it
is feeding on a susceptible or resistant host. In addition,
this is the first report of the heterologous expression of a Sinha et al. BMC Plant Biology (2015) 15:235 Page 6 of 11 Fig. Discussion 4 Expression of OoDAD1 in the Asian rice gall midge. Relative expression evaluated in midges feeding on susceptible [Jaya (white bars)] and
resistant [RP2068 (shaded bars)] rice varieties determined using Quantitative Real-Time PCR. The time points mentioned are 24, 48, 72 and 96hai. RQ values describe the relative expression values of transcripts with reference to expression level of OoDAD1 in maggots feeding on Jaya variety
(24hai). Error bars represent mean ± SD Fig. 4 Expression of OoDAD1 in the Asian rice gall midge. Relative expression evaluated in midges feeding on susceptible [Jaya (white bars)] and
resistant [RP2068 (shaded bars)] rice varieties determined using Quantitative Real-Time PCR. The time points mentioned are 24, 48, 72 and 96hai. RQ values describe the relative expression values of transcripts with reference to expression level of OoDAD1 in maggots feeding on Jaya variety
(24hai). Error bars represent mean ± SD Earlier reports confirm DAD1 to be a member of the oli-
gosaccharyl transferase complex responsible for N-linked
glycosylation [23]. It has also been observed that the
C-terminal amino acid residues, which are known to be
crucial for N-terminal glycosylation function [24], are con-
served in DAD1 from a wide range of organisms. However, on the resistant plants, the transcript levels
of OoDAD1 registered an instant increase and these
levels were sustained till 96hai. In previous studies in-
volving Hessian fly-wheat interactions, it was observed
that Hessian fly maggots failed to establish a feeding site
on resistant wheat varieties, and as a result stress respon-
sive genes and those involved in disruption of homeostasis
were up-regulated [20, 29]. Further, it is likely that on re-
sistant hosts the maggots face nutritional stress owing to
its inability to maintain feeding, probably due to toxic
plant compounds and/or feeding deterrents, and as a
result face starvation and eventual death. This could re-
sult in a cascade of events connected with the defense
pathways of which one could be over-expression of DAD1
to prevent it from succumbing to stress-induced apoptosis. However, despite a high degree of homology between
DAD1 from different organisms, a few dissimilar amino
acid residues amongst the proteins provide each with
specific signatures that can be used to classify DAD1 of
different organisms corresponding with their evolutionary
relationship. Moreover, these specific signatures are cap-
able of differentiating DAD1 of plants from those in the
animal kingdom. Discussion In addition, these signatures also differ-
entiate DAD1 from invertebrates and vertebrates and also
those from phytophagous and haematophagous dipterans. Interestingly, results from the plant injection assays,
using recombinant OoDAD1, showed increased HR in gall
midge resistant rice hosts which contradicts the reported
role of DAD1. A plausible explanation for the observed
results could be the presence of certain motifs in the
insect-derived DAD1 that the gall midge resistant host
recognizes and as a result initiates the hypersensitive
reaction against the foreign protein. This is also supported
by data from the phylogenetic analysis that clearly dif-
ferentiates DAD1 of plant and animal origin. Moreover,
it has also been suggested that DAD1 (a homologue of
Ost2) of one organism may not proceed with the conserved
mechanism of PCD signaling pathway in another organism
[30]. Furthermore, staining of rice tissues with DAB, after
injection of recombinant OoDAD1, showed increased HR
in Suraksha at 48hai while in RP2068 it was observed at
72hai. This may be due to inherent genotypic differences in
the two resistant rice varieties. Our earlier studies have also DAD1 has been shown to play an important role as a
suppressor of the apoptotic pathway in many organisms
[4]. Hence, the differential expression pattern of OoDAD1
in maggots feeding on susceptible and resistant hosts is
likely indicative of its important role in the apoptotic path-
way in the rice gall midge too. While controlled apoptosis
is induced in the cell during developmental stages, apop-
tosis can also occur when cell is under stress [2]. In the case
of gall midge-rice compatible interaction, the midge under-
goes its normal life cycle. On susceptible plants, maggots
establish a feeding site between 12 and 24 hai and genes in-
volved in growth and development are up-regulated, as also
observed in the Hessian fly-wheat interaction [25–27]. And
therefore, the minor increase in the OoDAD1 transcript ob-
served in maggots feeding on susceptible host during 48 to
96hai as compared to 24hai could be a part of normal
homeostasis and development [28] of maggots. Page 7 of 11 Sinha et al. BMC Plant Biology (2015) 15:235 shown that these two resistant rice varieties carry gall
midge resistance genes Gm11 and gm3, respectively. Though both rice varieties behave differently upon gall
midge attack the final outcome in both cases is mortality
of the maggots [18, 21]. Discussion Conclusions
I
l
i
th
i
t d
ib d h
h
th
M
1
2
3
kDa
10
15
25
35
40
55
70
10
15
(A)
(B)
Fig. 5 Heterologous expression of OoDAD1 and Western analysis. a Coomassie blue stained sodium dodecyl sulphate polyacrylamide
gel showing over-expression of recombinant OoDAD1 in BL21 (DE3)
pLysE Escherichia coli-based expression system using pET 28a expression
vector. Lanes: 1, lysate of induced, transformed cells with OoDAD1; 2,
supernatent of induced, transformed cells with OoDAD1; 3, pellet
of induced, transformed cells with OoDAD1. M, protein ladder. b Western
analysis of polyacrylamide gel shown in (a) and electrotransferred to a
nitrocellulose membrane and probed with anti-His tag antibodies
(see Experimental Procedures). Lane designation is same as in (a). Arrows indicate the location of the His-tagged OoDAD1. Numbers
on the left represent molecular weights in kilodaltons (kDa) In addition, plant assays involving recombinant OoDAD1
suggested that the insect protein is detected by the re-
sistant host resulting in HR. However, more detailed
studies would be required for further unraveling the role
played by OoDAD1 in the gall midge-rice interaction. In
addition, RNAi-based studies in conjunction with studies
on OoDAD1 mutants would help in understanding the
role of DAD1 in insect-plant interaction in general and
gall midge-rice interaction in particular. Besides, immuno-
localization studies with OoDAD1 would help in further-
ing our understanding of the molecular events during
insect-plant interactions. M
1
2
3
kDa
10
15
25
35
40
55
70
(A) Insect stages, dissections and collection of maggots Insect stages, dissections and collection of maggots
In order to identify differentially expressed genes maggots
were dissected out from the host at different time intervals
of 24, 48, 72 and 96hai. To determine the time intervals,
rice seedlings (15-day-old) of both Jaya and RP2068
variety were infested with GMB4 and regularly monitored. The maggots take 4–6 h to reach the apical meristem after
formation of eyespot. Individual rice seedlings were
dissected under the microscope and the maggots were
collected in RNAlater (Ambion, Austin, TX, USA) and
stored at–80C till further use. Approximately, 600 maggots
per rice variety were dissected out. shown that these two resistant rice varieties carry gall
midge resistance genes Gm11 and gm3, respectively. Though both rice varieties behave differently upon gall
midge attack the final outcome in both cases is mortality
of the maggots [18, 21]. Insect material and DNA extraction Numbers
on the left represent molecular weights in kilodaltons (kDa) Insect material and DNA extraction The experimental material consisted of Asian rice gall
midge biotype 4 (GMB4) insects maintained in greenhouse
[31] at the Directorate of Rice Research, Hyderabad, India. Adult gall midges were collected initially from different gall
midge infested areas in India. These insects were reared on
Jaya and checked on RP2068 varieties of rice under stand-
ard conditions [31]. GMB4 is virulent (forms galls on the
host plant) on Jaya (lacks gall midge resistance genes) and
avirulent (unable to form galls on the host plant leading
to subsequent mortality of the maggots) on RP2068
(possesses gall midge resistance genes) rice variety. DNA
was extracted from adult gall midges using micro pestle,
which had been chilled in liquid nitrogen. The ground
tissue was suspended in extraction buffer (1 % SDS,
0.05 M NaCl; 0.05 M Tris–HCl, pH 8.0; 0.025 M EDTA),
followed by Proteinase K and RNase treatment, and puri-
fied with phenol: chloroform: isoamyl alcohol (25:24:1),
and then with chloroform: isoamyl alcohol (24:1) [32]. The purified genomic DNA was then ethanol precipi-
tated and re-suspended in distilled water. 10
15
(B) (B) Fig. 5 Heterologous expression of OoDAD1 and Western analysis. a Coomassie blue stained sodium dodecyl sulphate polyacrylamide
gel showing over-expression of recombinant OoDAD1 in BL21 (DE3)
pLysE Escherichia coli-based expression system using pET 28a expression
vector. Lanes: 1, lysate of induced, transformed cells with OoDAD1; 2,
supernatent of induced, transformed cells with OoDAD1; 3, pellet
of induced, transformed cells with OoDAD1. M, protein ladder. b Western
analysis of polyacrylamide gel shown in (a) and electrotransferred to a
nitrocellulose membrane and probed with anti-His tag antibodies
(see Experimental Procedures). Lane designation is same as in (a). Arrows indicate the location of the His-tagged OoDAD1. Numbers
on the left represent molecular weights in kilodaltons (kDa) Fig. 5 Heterologous expression of OoDAD1 and Western analysis. a Coomassie blue stained sodium dodecyl sulphate polyacrylamide
gel showing over-expression of recombinant OoDAD1 in BL21 (DE3)
pLysE Escherichia coli-based expression system using pET 28a expression
vector. Lanes: 1, lysate of induced, transformed cells with OoDAD1; 2,
supernatent of induced, transformed cells with OoDAD1; 3, pellet
of induced, transformed cells with OoDAD1. M, protein ladder. b Western
analysis of polyacrylamide gel shown in (a) and electrotransferred to a
nitrocellulose membrane and probed with anti-His tag antibodies
(see Experimental Procedures). Lane designation is same as in (a). Arrows indicate the location of the His-tagged OoDAD1. Southern blot analysis y
Restriction digestion was performed using genomic DNA
(3 μg) isolated from three biotypes using EcoR1, EcoRV
and Dra1 restriction enzymes (New England Biolabs,
Beverly, MA, USA). The digested DNA fragments were
electrophoresed on 0.8 % agarose gel (30 V for 12 h)
and blotted onto a nylon membrane (GeneScreen Plus,
Perkin Elmer, Boston, MA, USA). Transfer of DNA was
done using the alkali transfer procedure and the blot
was probed with the 339 bp OoDAD1 fragment cloned
from GMB4. The fragment was labeled with α–32P deoxy-
cytidine triphosphate using a Nick translation kit (Invitro-
gen, Carlsbad, CA, USA). Hybridization with the probe and
washing of the blot were followed as described by
Mohan et al., 1994 [34]. RNA isolation and genomic clone recovery RNA isolation and genomic clone recovery
RNA was isolated using RNeasy Plus Micro Kit (Qiagen,
GmbH, Hilden, Germany) following the manufacturer’s
protocol. Two biological replicates, that were temporally
separated, were included in this study. First strand cDNA In conclusion, the experiments described here show the
crucial role played by OoDAD1 in gall midge-rice inter-
action. OoDAD1 is transcriptionally up-regulated in mag-
gots, feeding on resistant host, in an attempt to overcome
the challenge faced by the maggots in the resistant host. Page 8 of 11 Sinha et al. BMC Plant Biology (2015) 15:235 Fig. 6 HR response of gall midge resistant rice injected with recombinant OoDAD1. Plant injection assay showing induction of HR response in the gall
midge resistant rice variety, RP2068, upon injection with recombinant OoDAD1. RP2068 injected with a purified protein; b protein elution buffer;
c BSA dissolved in protein elution buffer. Black dots indicate the markings made prior to injection for easy localization of injected regions Fig. 6 HR response of gall midge resistant rice injected with recombinant OoDAD1. Plant injection assay showing induction of HR response in the gall
midge resistant rice variety, RP2068, upon injection with recombinant OoDAD1. RP2068 injected with a purified protein; b protein elution buffer;
c BSA dissolved in protein elution buffer. Black dots indicate the markings made prior to injection for easy localization of injected regions synthesis was performed using Superscript III RT enzyme
(Invitrogen, Carlsbad, CA, USA) according to the manu-
facturer’s protocol. A cDNA library was prepared as
mentioned in Sinha et al., 2011 [21]. Genome walking
and RACE were performed using the published protocol
[33]. Details of primers used in this study are provided
in Table 1. PCR products were cloned in Topo TA
cloning Vector (Invitrogen, Carlsbad, CA, USA) and se-
quenced by M/s Macrogen Inc., Seoul, South Korea. distance/neighbor-joining method provided in the Mac-
Vector suite of programs. 1000 replications were used to
obtain bootstrap values for the branches. The evolutionary
distances were computed using Poisson-corrected distances
and the gaps were distributed proportionally. Sequence and phylogenetic analysis Sequence assembly was carried out using Phred and Phrap
included in the MacVector suite of programs (MacVector
Inc., Cary NC, USA; V: 12.0.5). Sequence similarity and
annotations were performed using web-based BLAST
programs on the National Centre for Biotechnology
Information (NCBI; http://www.ncbi.nlm.nih.gov/) servers. Secretion signal peptide analysis of the predicted amino
acid sequence was performed using SMART software
(http://smart.embl-heidelberg.de). Molecular weight and pI
was calculated using ExPASy-ProtParam tool (http://
web.expasy.org/protparam/). PSORT II analysis (Prediction
of Protein Sorting Signals and Localization Sites in Amino
Acid Sequences, http://psort.hgc.jp/form2.html) was used
for identifying the localization sites. In the absence of any
matching models, structures were predicted using Rosetta
fragment insertion method. The de novo protein threading
program ROBETTA (http://robetta.bakerlab.org) was used
to predict the secondary structure of OoDAD1. Real-Time PCR and statistical analyses Real Time expression profile was performed for OoDAD1
gene during different stages of insects feeding on suscep-
tible (Jaya) and resistant (RP2068) hosts. Primer Express
(version 3.0; Applied Biosystems, Foster City, CA, USA)
was used to design the Real-Time PCR primers for
OoDAD1 and control genes (Table 1). Equal quantity of
total RNA (20 ng; as estimated by NanoVue spectro-
photometer [GE Healthcare, Little Chalfont, UK]), from
different stages were reverse transcribed using Superscript
III RT enzyme (Invitrogen, Carlsbad, CA, USA) and oligo
(dT) primers according to the manufacturer’s protocol. A phylogenetic tree was constructed using the predicted
amino acid sequence of OoDAD1 and reported homo-
logues in other organisms. The homologues of DAD1
from vertebrates, invertebrates and plants were used for
this analysis. The tree was constructed using the inbuilt Page 9 of 11 Sinha et al. BMC Plant Biology (2015) 15:235 Page 9 of 11 Fig. 7 DAB staining confirms HR response in resistant rice varieties injected with OoDAD1. DAB (3,3′-diaminobenzidine)-staining of recombinant
OoDAD1-injected rice plants to show HR reaction in gall midge resistant rice varieties (Suraksha and RP2068) compared with the gall midge sus-
ceptible variety, Jaya, at 24, 48 and 72hai post-injection with water (w), buffer (b) and recombinant OoDAD1 (p) (see Experimental Procedures). Two of the resistant rice varieties (Suraksha and RP2068) showed HR (browning of DAB-stained tissue) Fig. 7 DAB staining confirms HR response in resistant rice varieties injected with OoDAD1. DAB (3,3′-diaminobenzidine)-staining of recombinant
OoDAD1-injected rice plants to show HR reaction in gall midge resistant rice varieties (Suraksha and RP2068) compared with the gall midge sus-
ceptible variety, Jaya, at 24, 48 and 72hai post-injection with water (w), buffer (b) and recombinant OoDAD1 (p) (see Experimental Procedures). Two of the resistant rice varieties (Suraksha and RP2068) showed HR (browning of DAB-stained tissue) Amplification efficiency of the designed primers was
checked using serially diluted cDNA samples. Actin gene
was selected as the internal control after evaluating several
candidates using GENORM [35]. identify primer dimers and contamination. The ampli-
fied fragments were cloned in pCR4-TOPO-TA vector
(Invitrogen, Carlsbad, CA, USA) and sequenced to
confirm their identity. The quantification of mRNA of
OoDAD1 in the maggots was estimated using Relative
Standard Curve method. Output data were analyzed
using the 2-ΔΔCtmethod, inbuilt into the StepOne real-
time PCR analysis software (Applied Biosystems) and
results displayed as Relative Expression Values (REVs). p
p
bAmplicon size when genomic DNA was used as template aAmplicon size when cDNA was used as template
b Heterologous expression of OoDAD1 protein and Western
blot analysis fine needle (26G; 0.45 × 13 mm) into the stem of the rice
plant carefully. The region to be injected was marked be-
fore the injection of the protein in order to localize the
injected area. Ten plants per rice variety per time point
(24, 48, 72 and 96 h) in three biological replicates were
used for this experiment. The injected portion was sliced
off after 24, 48, 72 and 96 h post-injection and stained
with DAB (3,3′-diaminobenzidine) staining solution as
described earlier [37] with minor modifications. fine needle (26G; 0.45 × 13 mm) into the stem of the rice
plant carefully. The region to be injected was marked be-
fore the injection of the protein in order to localize the
injected area. Ten plants per rice variety per time point
(24, 48, 72 and 96 h) in three biological replicates were
used for this experiment. The injected portion was sliced
off after 24, 48, 72 and 96 h post-injection and stained
with DAB (3,3′-diaminobenzidine) staining solution as
described earlier [37] with minor modifications. y
Bacterial expression vector pET 28a (Novagen, Darmstadt,
Germany) and BL21 (DE3) pLysE E.coli competent cells
were used for the production of recombinant OoDAD1. The full-length cDNA, coding for OoDAD1, was PCR
amplified using forward and reverse primers (Table 1). The primers were designed with BamH1 and XhoI restric-
tion sites to enable the cloning of the PCR fragment into
their corresponding sites in the pET 28a vector. Chem-
ically competent BL21 (DE3) pLysE E. coli were trans-
formed with the designed expression plasmid. LB medium
(10 ml) containing 50 μg/ml kanamycin was inoculated
with overnight culture (1 %) of transformed cells. LB
medium was incubated at 37 °C till the optical density
(OD) of the medium reached to 0.6 (OD600). 0.5 mM
isopropyl-b-D-thiogalactoside (IPTG; Merck, Darmstadt,
Germany) was used to induce expression of the His-tagged
OoDAD1 protein. The cells were pelleted by centrifugation
at 10,000 g for 10 mins after 4 h of induction. The resultant
pellet was re-suspended in lysis buffer [25 mM Tris, 10 Mm
NaCl, 10 mM benzamidine, 15 % glycerol, 1 mM phenyl-
methylsulfonyl fluoride (PMSF), 2 mg/ml lysozyme] and
centrifuged at 13 000 g for 15 min. Availability of supporting data The nucleotide sequences of full-length OoDAD1 have
been submitted to GenBank under accession numbers
KP890835 (cDNA clone) and KP890834 (genomic clone). Other data related to this article are included within the
article and its additional files. Additional files Additional file 1: Figure S1. Consensus prediction of membrane protein
topology using TOPCONS server indicated the presence of three
trans-membrane helices (grey and white boxes in the graph) in the
predicted amino acid sequence of OoDAD1. TOPCONS predicted the
topology of OoDAD1 from five different topology prediction algorithms:
SCAMPI (single sequence mode), SCAMPI (multiple sequence mode),
PRODIV-TMHMM, PRO-TMHMM and OCTOPUS. The output of these five
algorithms were used as input for the TOPCONS Hidden Markov Model
(HMM) (shown in maroon), which provided a consensus prediction for
the protein together with a reliability score based on the agreement of
the included methods across the sequence. In addition, ZPRED was
used to predict the Z-coordinate (i.e., the distance to the membrane
center) of each amino acid, and the G-scale was used to predict the
free energy of membrane insertion for a window of 21 amino acids
centered around each position in the sequence. (PDF 81 kb) Additional file 2: Figure S2. Protein structure of OoDAD1 as predicted by
ROBETTA, a de novo protein-threading program (http://robetta.bakerlab.org). A,
B, C, D and E are the five predicted models and the fifth model was predicted
to have lowest score (lowest energy) and therefore, considered the
most stable. (PDF 166 kb) After electrophoresis, the proteins on the gel were elec-
trotransferred onto an Amersham Hybond-ECL nitrocel-
lulose membrane (0.45 μm; GE Healthcare) (100 V for
1 h;using a Mini Trans-Blot cell [Bio-Rad Laboratories,
Hercules, CA, USA]). The membrane was blocked with
3 % bovine serum albumin (BSA) dissolved in phosphate-
buffered saline (PBS). It was further incubated for 1 h with
1:3000 dilution of 6X His-antibody conjugated to alkaline
phosphatase (catalogue no. A7058-1VL; Sigma Aldrich
GmbH, Munich, Germany). After repeated washing (3
times) of the membrane with PBS containing 0.05 %
Tween-20, His tagged-OoDAD1 was detected using 5-
bromo-4-chloro-3′-indolyphosphate/nitro-blue tetrazolium
(Sigma Aldrich, St Louis, MO, USA) as substrate. Size
estimation of the developed bands was done using a
pre-stained protein ladder (Fermentas, Hanover, MD,
USA; catalogue # SM0671). Additional file 3: Figure S3. HR response of gall midge resistant rice
injected with recombinant OoDAD1. Plant injection assay showing induction
of HR response in the gall midge resistant rice variety, Jaya, upon injection
with recombinant OoDAD1. Jaya injected with (a) purified protein; (b) protein
elution buffer; (c) BSA dissolved in protein elution buffer. Real-Time PCR and statistical analyses Statistical significance of the difference in OoDAD1
expressions between different samples was determined
using Student’s t-test analysis [36]. The cycling conditions used for Real-Time PCRs were
95 °C for 10 min followed by 40 cycles of 95 °C for 15 s
and 60 °C for 1 min. Real-Time PCR was performed
using SYBR green chemistry and in Applied Biosystems
StepOne Real-Time PCR system. Real-Time PCR mix
(20 μl) contained 1XPower SYBR Green PCR mix (Applied
Biosystems) and 0.5 mM of the primers. Real-Time
PCR was followed by melt curve analysis in order to Table 1 List of primers used for cloning and quantitative Real Time assays of OoDAD1. The ‘Prot’ primers were used for cloning OoDAD1
in pET 28a expression vector
S.No
Name
Primer sequence (5′➔3′)
Annealing Temp. (°C)
Amplicon size (bp)
1
OoDAD-F
ATGACGAATCTAACTACAGTTGTTC
55
339a
2
OoDAD-R
CTAACCGATGAAATTGAATACG
424b
3
RTDAD-F
CATTGTGCTTACCGGTGTCATT
60
177
4
RTDAD-R
CGTTCCGGTGAGATTCCAAT
5
RTActin-F
TGAGACACCATCACCGGAATC
60
149
6
RTActin-R
ATCCAAAGGCCAATCGTGAA
7
ProtDAD-F
GAGACATATGACGAATCTAACTACAGTTGTTC
55
339
8
ProtDAD-R
GAGACTCGAGCTAACCGATGAAATTGAATACG
aAmplicon size when cDNA was used as template
bAmplicon size when genomic DNA was used as template for cloning and quantitative Real Time assays of OoDAD1. The ‘Prot’ primers were used for cloning OoDAD1 Page 10 of 11 Page 10 of 11 Sinha et al. BMC Plant Biology (2015) 15:235 Plant injection assay and staining j
y
g
Fifteen-day-old plants of Jaya, Suraksha and RP2068
were injected with 10 μl (700 ng/μl) of purified recom-
binant OoDAD1 protein, elution buffer or heat-denatured
OoDAD1. Injection of protein was performed using a very Competing interests
h
h
d
l
h Competing interests
The authors declare that they have no competing interests. Competing interests
The authors declare that they have no competing interests. Heterologous expression of OoDAD1 protein and Western
blot analysis The pellet was re-
suspended in 1X SDS PAGE dye (50 mM Tris-Cl pH 6.8;
10 % glycerol; 2 % SDS; 12.5 mM ethylenediaminetetra-
acetic acid; 1 % b-mercaptoethanol; 0.02 % bromophenol
blue) and boiled for 5 mins. The sample was electropho-
resed in a 15 % SDS polyacrylamide gel and the gel was
stained with Coomassie blue dye to visualize the protein
bands. bb e at o s
Hai: Hours after infestation; DAB: 3′3′ diaminobenzidine. Hai: Hours after infestation; DAB: 3′3′ diaminobenzidine. Additional files Black dots indicate
the markings made prior to injection for easy localization of injected regions. (PDF 199 kb) Additional file 3: Figure S3. HR response of gall midge resistant rice
injected with recombinant OoDAD1. Plant injection assay showing induction
of HR response in the gall midge resistant rice variety, Jaya, upon injection
with recombinant OoDAD1. Jaya injected with (a) purified protein; (b) protein
elution buffer; (c) BSA dissolved in protein elution buffer. Black dots indicate
the markings made prior to injection for easy localization of injected regions. (PDF 199 kb) Authors’ contributions SN and JSB designed the research. DKS and IA carried out the experiments. DKS, IA and SN analyzed the data. DKS, IA, JSB and SN wrote the paper. All
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l 20. Mittapalli O, Neal JJ, Shukle RH. Antioxidant defense response in a galling
insect. Proc Natl Acad Sci U S A. 2007;104:1889–94. 1Plant Molecular Biology Group, International Centre for Genetic Engineering
and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110 067, India. 1Plant Molecular Biology Group, International Centre for Genetic Engineering
and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110 067, India. 21. Sinha DK, Lakshmi M, Anuradha G, Rahman SJ, Siddiq EA, Bentur JS, et al. Serine proteases-like genes in the rice gall midge show differential
expression in compatible and incompatible interactions with rice. Int J Mol
Sci. 2011;12:2842–52. 2Directorate of Rice Research, Rajendranagar, Hyderabad 500 030, India. 3Agri
Biotech Foundation, Rajendranagar, Hyderabad 500 030, India. 22. Morel J-B, Dangl JL. The hypersensitive response and the induction of cell
death in plants. Cell Death Differ. 1997;4:671–83. Received: 24 March 2015 Accepted: 16 September 2015 death in plants. Cell Death Differ. 1997;4:671–83. 23. Kelleher DJ, Gilmore R. An evolving view of the eukaryotic 23. Kelleher DJ, Gilmore R. An evolving view of the eukaryotic
oligosaccharyltransferase. Glycobiology. 2006;16:47R–62. oligosaccharyltransferase. Glycobiology. 2006;16:47R–62. 24. Makishima T, Yoshimi M, Komiyama S, Hara N, Nishimoto T. A subunit of the
mammalian oligosaccharyl transferase, DAD1, interacts with Mcl-1, one of
the bcl-2 protein family. J Biochem. 2000;128:399–405. Acknowledgements This research work was partly funded by a grant (number BT/PR8629/PBD/
16/737/2007) from the Department of Biotechnology (DBT), Government of
India, to S.N. IA thanks the University Grants Commission (UGC), Government
of India, for a Senior Research Fellowship. Page 11 of 11 Sinha et al. BMC Plant Biology (2015) 15:235 References Plant J. 1997;11:1187–94. 13. Rawat N, Chiruvuri Naga N, Raman Meenakshi S, Nair S, Bentur JS. A novel
mechanism of gall midge resistance in the rice variety Kavya revealed by
microarray analysis. Funct Integr Genomics. 2012;12:249–64. 14. Rawat N, Himabindu K, Neeraja CN, Nair S, Bentur JS. Suppressive subtraction
hybridization reveals that rice gall midge attack elicits plant-pathogen-like
responses in rice. Plant Physiol Biochem. 2013;63:122–30. 15. Sinha DK, Nagaraju J, Tomar A, Bentur JS, Nair S. Pyrosequencing-based
transcriptome analysis of the Asian Rice Gall Midge reveals differential
response during compatible and incompatible interaction. Int J Mol Sci. 2012;13:13079–103. Submit your next manuscript to BioMed Central
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Submit your manuscript at
www.biomedcentral.com/submit 16. Sinha DK, Atray I, Bentur JS, Nair S. Expression of Orseolia oryzae nucleoside
diphosphate kinase (OoNDPK) is enhanced in rice gall midge feeding on
susceptible rice hosts and its over-expression leads to salt tolerance in
Escherichia coli. Insect Mol Biol. 2012;21:593–603. • Convenient online submission • Thorough peer review 17. Sinha DK, Bentur JS, Nair S. Compatible interaction with its rice host leads
to enhanced expression of gamma subunit of oligosaccharyl transferase
(OoOST) in the Asian rice gall midge (Orseolia oryzae). Insect Mol Biol. 2011;20:567–75. 18. Rawat N, Sinha DK, Rajendrakumar P, Shrivastava P, Neeraja CN, Sundaram RM,
et al. Role of pathogenesis-related genes in rice–gall midge interactions. Curr
Sci. 2010;99:1361–8. 19. Mur LAJ. Hypersensitive response in plants. eLS. 2007. doi:10.1002/
9780470015902.a0020103.
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https://openalex.org/W2468214916
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https://link.springer.com/content/pdf/10.1007%2Fs10955-016-1690-x.pdf
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English
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The Planar Ising Model and Total Positivity
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Journal of statistical physics
| 2,016
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cc-by
| 11,157
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J Stat Phys (2017) 166:72–89
DOI 10.1007/s10955-016-1690-x J Stat Phys (2017) 166:72–89
DOI 10.1007/s10955-016-1690-x The Planar Ising Model and Total Positivity Marcin Lis1 Received: 30 June 2016 / Accepted: 2 December 2016 / Published online: 29 December 2016
© The Author(s) 2016. This article is published with open access at Springerlink.com Abstract A matrix is called totally positive (resp. totally nonnegative) if all its minors are
positive (resp. nonnegative). Consider the Ising model with free boundary conditions and
no external field on a planar graph G. Let a1, . . . , ak, bk, . . . , b1 be vertices placed in a
counterclockwise order on the outer face of G. We show that the k × k matrix of the two-
point spin correlation functions Mi, j = ⟨σai σb j ⟩ is totally nonnegative. Moreover, det M > 0 if and only if there exist k pairwise vertex-
disjoint paths that connect ai with bi. We also compute the scaling limit at criticality of the
probability that there are k parallel and disjoint connections between ai and bi in the double
random current model. Our results are based on a new distributional relation between double
random currents and random alternating flows of Talaska [37]. is totally nonnegative. Moreover, det M > 0 if and only if there exist k pairwise vertex-
disjoint paths that connect ai with bi. We also compute the scaling limit at criticality of the
probability that there are k parallel and disjoint connections between ai and bi in the double
random current model. Our results are based on a new distributional relation between double
random currents and random alternating flows of Talaska [37]. Keywords Ising model · Total positivity · Random currents · Alternating flows Mathematics Subject Classification 82B20 · 60C05 · 05C50 Mathematics Subject Classification 82B20 · 60C05 · 05C50 B Marcin Lis
m.lis@statslab.cam.ac.uk
1
Statistical Laboratory, Centre for Mathematical Sciences, Cambridge University, Wilberforce Road,
Cambridge CB3 0WB, UK B Marcin Lis
m.lis@statslab.cam.ac.uk 1
Statistical Laboratory, Centre for Mathematical Sciences, Cambridge University, Wilberforce Roa
Cambridge CB3 0WB, UK 1 Introduction The Ising model was introduced by Lenz with the intention to describe the behaviour of
ferromagnets, and was first solved in dimension 1 by Ising [19]. Peierls later showed that the
model does undergo a phase transition in dimensions 2 or more [32], and it has been since
the subject of extensive study both in the physics and mathematics literature. Notable results
in the planar case include the exact solution obtained by Onsager [31] and Yang [38], and the 123 The Planar Ising Model and Total Positivity 73 recent breakthrough of Smirnov et al. showing conformal invariance in the critical scaling
limit [7,8,17,18,36]. recent breakthrough of Smirnov et al. showing conformal invariance in the critical scaling
limit [7,8,17,18,36]. A seemingly unrelated notion is that of totally positive matrices characterized by having
all their minors positive. They appear in various areas of mathematics and physics including
oscillations in mechanical systems [12,13] (which was the original motivation to study them),
stochastic processes and statistical mechanics [9,11,22], quantum groups [28–30], and alge-
braic geometry [33,34]. Some of their fundamental properties include the simplicity and
positivity of the spectrum obtained by Gantmacher and Krein [12], and the variation dimin-
ishing property discovered by Shoenberg [35] which says that the number of sign changes
in a vector does not increase after multiplying by a totally positive matrix. In this article we identify total positivity in the planar Ising model. Our results on the Ising
boundary two-point correlation functions are analogous to the results of Fomin [11] on the
walk matrices of random walks on planar graphs. Indeed, the walk matrix can be interpreted
as the two-point correlation function matrix of another very well known ferromagnetic spin
system—the discrete Gaussian free field. Furthermore, Fomin provides an interpretation
of the determinants of walk matrices in terms of probabilities of non-intersection events
involving loop-erased walks of Lawler [25]. In our setting the relevant events concern the
double random current model. The random current model is derived from the power series expansion of the Ising model
partition function. It was introduced by Griffiths, Hurst and Sherman [15], and was later used
by Aizenman et al. [1–3] to obtain a detailed description of the behaviour of the Ising model
on Zd. The model has recently received revived attention from the mathematical community. 2 Main Results Let G = (V, E) be a finite connected planar graph. For positive coupling constants J : E →
(0, ∞), we consider the Ising model on G with free boundary conditions and no external
field, i.e., a probability measure on the space of spin configurations {−1, 1}V given by PIsg(σ) =
1
ZIsg
{u,v}∈E
exp(J{u,v}σuσv),
σ ∈{−1, 1}V , where where ZIsg =
σ∈{−1,1}V
{u,v}∈E
exp(J{u,v}σuσv) is the partition function. Since the coupling constants are positive, the model is ferromagnetic,
i.e., it assigns larger probability to configurations where more pairs of adjacent spins assume
the same value. For u, v ∈V , the two-point spin correlation function is defined to be the
expectation is the partition function. Since the coupling constants are positive, the model is ferromagnetic,
i.e., it assigns larger probability to configurations where more pairs of adjacent spins assume
the same value. For u, v ∈V , the two-point spin correlation function is defined to be the
expectation ⟨σuσv⟩=
σ∈{−1,1}V
σuσvPIsg(σ). A classical argument says that in a ferromagnetic spin system, all two-point functions are
positive (see Lemma 5.1). A classical argument says that in a ferromagnetic spin system, all two-point functions are
positive (see Lemma 5.1). Let W = {w1, w2, . . . , wn} be the set of vertices on the outer face of G listed counterclock-
wise, and let W◦∪W• = W be a partition of W into two sets (one of them possibly empty). We will refer to W as the boundary vertices and for A ⊆W, we will write A◦= A ∩W◦
and A• = A ∩W•. For two natural numbers i, j, we define Ii, j to be the set of numbers
strictly between i and j, e.g. I1,1 = ∅, and I1,3 = I3,1 = {2}. The following result yields
positivity of determinants of matrices whose entries are, up to a sign, the boundary two-point
spin correlation functions. Theorem 2.1 Let A = {wl1, wl2, . . . , wlk} ⊆W with l1 < l2 < · · · < lk. Consider the k ×n
matrix N A
i, j = (−1)s(i, j)⟨σwli σw j ⟩, where s(i, j) = |Ili, j ∩{l1, . . . ,lk}| + 1{w j∈A◦, j<li} + 1{w j∈A•, j>li}. 1 Introduction The interpretation of the determinants of the two-point functions in terms
of random current probabilities is achieved by a new distributional relation between double
random currents and random alternating flows. Postnikov [33]. The interpretation of the determinants of the two-point functions in terms
of random current probabilities is achieved by a new distributional relation between double
random currents and random alternating flows. This article is organized as follows: The next section introduces basic notation and presents
the main results. Section 3 gives a distributional identity for double random currents, and
Sect. 4 provides a closely related identity for random alternating flows. The relationship
between these two, together with the total positivity of alternating flows, is the basis for our
main results, whose proofs are given in Sect. 5. 1 Introduction The most notable example is the result of Aizenman, Duminil-Copin and Sidoravicius [4]
who studied percolation properties of double random currents to show continuity of magneti-
zation for a wide range of Ising models on Zd (including the d = 3 case). Their argument was
later generalized by Björnberg to the setting of quantum Ising models [5]. Also, a new dis-
tributional relation between random currents, Bernoulli percolation and the FK-Ising model
was discovered by Lupu and Werner [27]. One of the main tools used to study the random
current model is the switching lemma [15], and our result may be thought of as its planar
generalization (see Example 2.2). For a recent overview of the applications of the random
current model, see [10]. Since the work of Groeneveld, Boel and Kasteleyn [16], the boundary Ising correlation
functions have been known to satisfy exact Pfaffian relations. We provide an alternative proof
of this fact using the expansion of the Pfaffian as a sum of determinants (see Lemma 5.6). Our
results are hence a refinement of those in [16] in that we relate each of these determinants to
an explicit event in the double random current model. A special property of the (dis-)connection events appearing in our results is that even
though they are non-local, i.e., they depend on a macroscopic (in terms of the size of the
graph) number of edge variables, their probabilities are simple functions of the boundary
measurements which are defined as expectations of local variables. This in particular implies
that these probabilities do not depend on the structure of the graph as long as the boundary
measurements are preserved. This phenomenon also exists in uniform groves and double
dimers as was discovered by Kenyon and Wilson [23,24]. Moreover, the authors proved that
the probability of seeing any type of partition of the boundary vertices induced by cluster
connectivities in these models is a homogenous multi-linear polynomial in the boundary
measurements. We arrive at the total positivity of the Ising boundary two-point functions by representing
them in terms of alternating flows of Talaska which satisfy total positivity [37]. They, in
turn, appear in the combinatorial study of the totally nonnegative Grassmannian initiated by 123 74 M. Lis M. Lis Postnikov [33]. 2 Main Results Then, for any B ⊂W with |B| = k, Then, for any B ⊂W with |B| = k, det N A,B ≥0, 123 75 The Planar Ising Model and Total Positivity where N A,B is the k×k submatrix of N A with columns indexed by B. Moreover, det N A,B > 0
if and only if there exist k pairwise edge-disjoint (possibly empty) directed paths in G such
that each of them starts in A and ends in B, and if two paths meet at a vertex, then their edges
alternate in orientation around it (edge oriented towards or away from the vertex). Here, we
assume that an empty path joining v ∈A◦∩B◦(resp. v ∈A• ∩B•) with itself is represented
by a counterclockwise loop (resp. clockwise loop) attached to v within the outer face. where N A,B is the k×k submatrix of N A with columns indexed by B. Moreover, det N A,B > 0
if and only if there exist k pairwise edge-disjoint (possibly empty) directed paths in G such
that each of them starts in A and ends in B, and if two paths meet at a vertex, then their edges
alternate in orientation around it (edge oriented towards or away from the vertex). Here, we
assume that an empty path joining v ∈A◦∩B◦(resp. v ∈A• ∩B•) with itself is represented
by a counterclockwise loop (resp. clockwise loop) attached to v within the outer face. We note that the choice of W◦and W• determines both the sign factors appearing in the
definition of N A, and the interpretation of the associated determinants in terms of alternating
flows (see Lemma 5.5). This is inherited from the results of [37] through the construction of
Sect. 4 (see Fig. 2). Example 2.1 For any distinct a, b, c ∈W, we have 1 + ⟨σaσb⟩> ⟨σaσc⟩+ ⟨σbσc⟩,
and
1 + ⟨σaσb⟩2 > ⟨σaσc⟩2 + ⟨σbσc⟩2. Indeed, assume that a, b, c are ordered counterclockwise, a, b ∈W◦, c ∈W•, and take
A, B = {a, b, c}. We have Indeed, assume that a, b, c are ordered counterclockwise, a, b ∈W◦, c ∈W•, and take
A, B = {a, b, c}. We have N A,B =
⎡
⎣
1
⟨σaσb⟩
⟨σaσc⟩
−⟨σaσb⟩1
−⟨σbσc⟩
⟨σaσc⟩
−⟨σbσc⟩1
⎤
⎦, and by Theorem 2.1, det N A,B = 1 + ⟨σaσb⟩2 −⟨σaσc⟩2 −⟨σbσc⟩2 > 0, which gives the second inequality. 2 Main Results We define xing(π) to be the number of crossings between the
line segments. It is easy to see that when A, B are as in Corollary 2.2, then xing(π) is the
number of inversions of π treated as a permutation of the index set {1, . . . , k}. Hence, by the
definition of the determinant, det M A,B =
π:A→B
(−1)xing(π)
{a,b}∈π
⟨σaσb⟩. (2.1) (2.1) Moreover, the choice of signs in the definition of the matrix N A yields an analogous expansion
of the determinant for general choices of A and B: Moreover, the choice of signs in the definition of the matrix N A yields an analogous expansion
of the determinant for general choices of A and B: det N A,B =
π:A→B
(−1)xing(π)
{a,b}∈π
⟨σaσb⟩. (2.2) (2.2) (2.2) A verification of (2.2) can be found in [33, Proposition 5.2] or [37, Proposition 2.12]. A verification of (2.2) can be found in [33, Proposition 5.2] or [37, Proposition 2.12]. Our second main result provides an interpretation of these determinants in terms of random
currents. A current on G is a function n : E →{0, 1, 2, . . . }. By ∂n we denote the set of
sources of n, i.e., vertices v such that
u: {u,v}∈E n{u,v} is odd. For A ⊆V , we define
A = {n | ∂n = A}. The weight of a current n is defined by w(n) =
e∈E
(Je)ne
ne! ,
(2.3) (2.3) and the random current probability measure with boundary conditions A is given by and the random current probability measure with boundary conditions A is given by PA
curr(n) = w(n)
Z Acurr
,
n ∈A, PA
curr(n) = w(n)
Z Acurr
,
n ∈A, where Z A
curr =
n∈A w(n) is the partition function. where Z A
curr =
n∈A w(n) is the partition function. where Z A
curr =
n∈A w(n) is the partition function. A
Note that if n1 ∈A and n2 ∈∅, then n1 + n2 ∈A. The double random current
probability measure PA
d-curr with boundary conditions A is defined to be the measure of the
sum of two independent random currents with A and ∅boundary conditions respectively: PA
d-curr(n) = PA
curr ⊗P∅
curr({(n1, n2) ∈A × ∅| n1 + n2 = n}),
n ∈A. 2 Main Results The inequality is strict since we can take empty paths
connecting the vertices with themselves. To obtain the first inequality, we use the second one
together with the Griffiths inequality ⟨σaσb⟩≥⟨σaσc⟩⟨σcσb⟩[14]. which gives the second inequality. The inequality is strict since we can take empty paths
connecting the vertices with themselves. To obtain the first inequality, we use the second one
together with the Griffiths inequality ⟨σaσb⟩≥⟨σaσc⟩⟨σcσb⟩[14]. In the special case when A and B form disjoint contiguous sets of boundary vertices,
we obtain the following total positivity property of the boundary two-point spin correlation
functions with no additional signs. Corollary 2.2 (Total positivity) Let A = {a1, a2, . . . , ak}, B = {b1, b2, . . . bk} be contigu-
ous sets of boundary vertices, i.e., such that a1, . . . , ak, bk, . . . b1 is a counterclockwise order
on A ∪B. Then, the k × k matrix M A,B
i, j
= ⟨σai σb j ⟩ M A,B
i, j
= ⟨σai σb j ⟩ is totally nonnegative. Moreover, M A,B is totally positive if and only if for any A′ ⊂A and
B′ ⊂B such that |A′| = |B′| = l, there exist l pairwise vertex-disjoint paths that connect
A′ and B′. Remark 1 One can prove that if A and B are as above, then det M A,B = det N A,B by showing
that the additional signs in N A,B make the same sign contribution to the determinant as the
reverse order on columns in M A,B. This is evident in the following expansion of these
determinants. Let A, B be any subsets of W of equal cardinality, and let π : A →B be a
bijection. One can interpret π as a pairing of the disjoint union A ⊔B, i.e., a partition of
A ⊔B into pairs {a, π(a)}. One can then think of a diagrammatic representation of π where
points representing A and B are placed in the corresponding order on the boundary of a disk,
and straight line segments connect the points according to π. Here, for each v ∈A◦∩B◦ 123 76 M. Lis (resp. each v ∈A• ∩B•), two copies of v are placed on the circle, and the one corresponding
to a point in A comes immediately after (resp. before) the one corresponding to a point in B
in the counterclockwise order. 2 Main Results For u, v ∈V and a current n, we will write u
n↔v if u and v are connected in G by a
path of edges with non-zero values of n, and we will write u
n↮v otherwise. Let A and B
be contiguous sets of boundary vertices as in Corollary 2.2. We define the event of having
parallel and disjoint connections between A and B (see Fig. 1) by PA,B = {n ∈A∪B | ai
n↔bi for all i, ai
n↮b j for all i ̸= j}. PA,B = {n ∈A∪B | ai
n↔bi for all i, ai
n↮b j for all i ̸= j}. As a consequence of the proof of Theorem 2.1 and Corollary 2.2, we can compute the
probability of PA,B under the double random current measure: Theorem 2.3 Let A and B be contiguous sets of boundary vertices as in Corollary 2.2, and let
v1, v2, . . . , v2k be a counterclockwise order on A ∪B. Consider the 2k ×2k skew-symmetric
matrix satisfying K A∪B
i, j
= ⟨σvi σv j ⟩
for i < j. 123 77 The Planar Ising Model and Total Positivity Fig. 1 The solid edges represent
non-zero values of a current
n ∈PA,B with A = {a1, a2, a3}
and B = {b1, b2, b3}. The black
edges represent odd values and
the grey edges represent even
values
a1
a2
a3
b3
b2
b1
Then,
PA∪B
d-curr(PA,B) = det M A,B
Pf K A∪B ,
where Pf denotes the Pfaffian of a skew-symmetric matrix. Fig. 1 The solid edges represent
non-zero values of a current
n ∈PA,B with A = {a1, a2, a3}
and B = {b1, b2, b3}. The black
edges represent odd values and
the grey edges represent even
values Fig. 1 The solid edges represent
non-zero values of a current
n ∈PA,B with A = {a1, a2, a3}
and B = {b1, b2, b3}. The black
edges represent odd values and
the grey edges represent even
values b2 b1 Then, where Pf denotes the Pfaffian of a skew-symmetric matrix. where Pf denotes the Pfaffian of a skew-symmetric matrix. Example 2.2 We consider the simplest non-trivial case of Theorem 2.3 when k = 2. To
this end, let S = {a, b, c, d} ⊆W be ordered counterclockwise, and let X = {n ∈S |
a
n↔b
n↔c
n↔d}. 2 Main Results Note that by planarity of G, and the fact that each connected component
of n contains an even number of vertices in ∂n, the complement of X in S is P{a,b},{c,d} ∪
P{a,d},{b,c}. We have Pf K S = ⟨σaσb⟩⟨σcσd⟩+ ⟨σaσd⟩⟨σbσc⟩−⟨σaσc⟩⟨σbσd⟩, and hence by
Theorem 2.3, PS
d-curr(P{a,b},{c,d}) = ⟨σaσd⟩⟨σbσc⟩−⟨σaσc⟩⟨σbσd⟩
Pf K S
,
PS
d-curr(P{a,d},{b,c}) = ⟨σaσb⟩⟨σcσd⟩−⟨σaσc⟩⟨σbσd⟩
Pf K S
,
PS
d-curr(X) = ⟨σaσc⟩⟨σbσd⟩
Pf K S
. We note that these formulas follow from the switching lemma of Griffiths, Hurst and Sher-
man [15] (see e.g. [4] for a modern treatment of the lemma). The statement of Theorem 2.3
for k > 2 does not however seem to be a direct consequence of this lemma. Remark 2 For future reference, recall that the Pfaffian of the skew-symmetric matrix K A∪B
is the square root of its determinant, and it is a well known fact that it can be written as Pf K A∪B =
π: pairing of A∪B
(−1)xing(π)
{u,v}∈π
⟨σuσv⟩,
(2.4 Pf K A∪B =
π: pairing of A∪B
(−1)xing(π)
{u,v}∈π
⟨σuσv⟩,
(2.4) (2.4) where the sum is over all pairings of A ∪B, and where xing is defined as in (2.2). where the sum is over all pairings of A ∪B, and where xing is defined as in (2.2). Our last main result concerns the scaling limit at criticality of the parallel connection
probability in double random currents. The proof relies on the computation of Hongler [17]
of the scaling limit of the boundary two-point functions themselves. Analogous and more
general results were obtained by Kenyon and Wilson [24] for the double dimer model,
multichordal loop erased walk, and grove Peano curves. Theorem 2.4 Let D be a bounded finitely-connected domain in the complex plane with a
piecewise C1 boundary. Let ∂sD be the straight part of the boundary, i.e., the part composed 123 123 M. Lis 78 of intervals parallel either to the real or imaginary axis. Let A = {a1, a2, . . . , ak} ⊂∂sD
and B = {b1, b2, . . . bk} ⊂∂sD be such that a1, . . . , ak, bk, . . . , b1 is a counterclockwise
ordering of A ∪B around the outer boundary of D. For ϵ > 0, let Dϵ be the maximal
connected component of the rescaled square nearest-neighbor lattice ϵZ2 contained in D. 2 Main Results Consider the double random current measure PAϵ∪Bϵ
d-curr
on Dϵ with homogeneous critical
coupling constants Je = 1
2 log(
√
2 + 1), where Aϵ and Bϵ are sets of vertices on the outer
face of Dϵ approximating A and B. Then, the following limit exists pA,B(D) := lim
ϵ→0 PAϵ∪Bϵ
d-curr (PAϵ,Bϵ). Moreover, if H is the upper half-plane and ϕ : D →H is a conformal equivalence between
D and H, then pA,B(D) = det ˜M A,B
Pf ˜K A∪B , where ˜M A,B is a k × k matrix, and ˜K A∪B is a 2k × 2k skew-symmetric matrix satisfying ˜M A,B
i, j
=
1
|ϕ(ai) −ϕ(b j)|,
and
˜K A∪B
i, j
=
1
|ϕ(vi) −ϕ(v j)|
for i < j, where v1, v2, . . . , v2k is a counterclockwise order on A ∪B. 3 Random Currents In this section G = (V, E) is a finite (not necessarily planar) graph. Note that the definitions
of the Ising model and the random current model generalize verbatim to arbitrary finite
graphs. g p
Let A ⊆V be a set of even cardinality (possibly empty). With a current n ∈A, we
associate a pair of sets of edges ω(n) = (ω1(n), ω2(n)), where ω1(n) = {e ∈E | ne is odd},
and ω2(n) = {e ∈E | ne is even, ne ̸= 0}. Let EA be the collection of sets of edges for which A is the set of vertices with odd degree in
the induced subgraph. One can see that A := {ω(n) | n ∈A} = {(ω1, ω2) | ω1 ∈EA, ω2 ⊆E \ ω1}. A := {ω(n) | n ∈A} = {(ω1, ω2) | ω1 ∈EA, ω2 ⊆E \ ω1}. We will often identify ω ∈ A with the set ω1 ∪ω2 ⊆E. Note that for u, v ∈V , u
n↔v if
and only if u and v belong to the same connected component of ω(n). A Let xe = tanh Je and ye = (cosh Je)−1. The next result describes the measure P
A
curr on
A induced from the random current measure. Lemma 3.1 The probability of ω ∈ A induced from the random current measure is given
by P
A
curr(ω) =
1
¯Z Acurr
e∈ω1
xe
e∈ω2
(1 −ye)
e∈E\ω
ye, where ¯Z A
curr = Z A
curr
e∈E ye. where ¯Z A
curr = Z A
curr
e∈E ye. 123 79 The Planar Ising Model and Total Positivity P
A
curr(ω) =
n∈A: ω(n)=ω
PA
curr(n)
=
1
Z Acurr
n∈A: ω(n)=ω
e∈E
(Je)ne
ne! =
1
Z Acurr
e∈ω1
∞
k=1
(Je)2k−1
(2k −1)!
e∈ω2
∞
k=1
(Je)2k
(2k)! =
1
Z Acurr
e∈ω1
sinh Je
e∈ω2
(cosh Je −1)
=
1
¯Z Acurr
e∈ω1
xe
e∈ω2
(1 −ye)
e∈E\ω
ye. ⊓⊔ ⊓⊔ ⊓⊔ For ω ⊆E, let E∅(ω) = E∅∩{ω′ | ω′ ⊆ω}. The main result of this section gives a
formula for the probability measure P
A
d-curr on A induced from the double random current
measure. 3 Random Currents Theorem 3.2 The probability of ω ∈ A induced from the double random current measure
is given by is given by P
A
d-curr(ω) =
1
¯Z A
d-curr
|E∅(ω)|
e∈ω1
xe
e∈ω2
x2
e
e∈E\ω
(1 −x2
e ), where ¯Z A
d-curr = Z A
curr Z∅
curr
e∈E(1 −x2
e ). where ¯Z A
d-curr = Z A
curr Z∅
curr
e∈E(1 −x2
e ). Proof Let n1 ∈A, n2 ∈∅, n = n1 + n2, and ω1 = ω(n1), ω2 = ω(n2), ω = ω(n). Note that e ∈ω1 if and only if e ∈ω1
1△ω2
1. Therefore, by Lemma 3.1, each e ∈ω1 carries
a multiplicative weight of xe[(1 −ye) + ye] = xe in the double random current measure. Similarly, e ∈ω2 if and only if e ∈ω1
1 ∩ω2
1 or e ∈(ω1
2 ∪ω2
2) \ (ω1
1 ∪ω2
1). By Lemma 3.1,
the multiplicative contribution of e ∈ω2 is hence x2
e = (1 −ye + ye)2 −y2
e in both cases. The contribution of an edge e ∈E \ ω is then 1 −x2
e . It is now enough to show that there
are exactly |E∅(ω)| choices of ω1
1 and ω2
1 such that ω1
1△ω2
1 = ω1 and ω1
1 ∩ω2
1 ⊂ω2. Indeed,
this is equivalent to freely choosing ω2
1 ∈E∅(ω) and setting ω1
1 = (ω1 \ ω2
1) ∪(ω2
1 ∩ω2)
∈EA. ⊓⊔ ⊓⊔ We note that one can express |E∅(ω)| in terms of the number of vertices, edges, and
connected components of ω (see Lemma 4.2). 4 Random Alternating Flows In this section we assume that G is planar, and we explain how the double random current
measure is related to a measure on alternating flows. To this end, we define a directed
modification ⃗G = (V ∪W + ∪W −, ⃗E) of G as follows. Each edge e ∈E is replaced
by three directed edges ⃗e ∈⃗E: one middle edge ⃗em, and two side edges ⃗es1, ⃗es2 (see Fig. 2). M. Lis 80 wi
wi−1
wi+1
w
w
G 1
wi
wi−1
wi+1
w+
i+1
w−
i+1
w−
i
w+
i
w+
i−1
w−
i−1
G wi
wi−1
wi+1
wi
wi−1
wi+1
w+
i+1
w−
i+1
w−
i
w+
i
w+
i−1
w−
i−1
G
G
Fig. 2 A choice of orientations of edges in a directed modification ⃗G of G. For each boundary vertex w, a
source w+ and a sink w−are added in an order depending on whether w ∈W◦or w ∈W• G Fig. 2 A choice of orientations of edges in a directed modification ⃗G of G. For each boundary vertex w, a
source w+ and a sink w−are added in an order depending on whether w ∈W◦or w ∈W• The side edges lie on opposite sides of ⃗em and have the opposite orientation to ⃗em. The
orientation of each middle edge is chosen arbitrarily, and the edge weights are given by The side edges lie on opposite sides of ⃗em and have the opposite orientation to ⃗em. The
orientation of each middle edge is chosen arbitrarily, and the edge weights are given by x⃗em = sinh 2Je
2
=
xe
1 −x2e
,
x⃗es1 = x⃗es2 = tanh Je
2
= xe
2 . (4.1) (4.1) Moreover, for each w ∈W, two vertices w+ ∈W + and w−∈W −, and two directed edges
(w, w−), (w+, w) ∈⃗E are placed in the external face of G so that w−becomes a sink and
w+ becomes a source. The weights of these edges are set to 1. The vertices are placed in such
a way that w−is comes immediately before (resp. after) w+ if w ∈W◦(resp. if w ∈W•) in
the counterclockwise order around the external face of G (see Fig. 2). ⃗
⃗ A flow on ⃗G is a set of edges F ⊆⃗E such that each vertex has the same number of ingoing
and outgoing edges in F. 4 Random Alternating Flows An alternating flow is a flow such that for each v ∈V , the edges
of the flow alternate in orientation around v (edge oriented towards v or away from v). For
U ⊆W, we will write U + ⊆W + and U −⊆W −for the the corresponding sets of sources
and sinks in ⃗G. For two (possibly intersecting) sets A, B ⊂W of equal cardinality, let FA,B = {alternating flow F on ⃗G | V (F) ∩W + = A+, V (F) ∩W −= B−}, FA,B = {alternating flow F on ⃗G | V (F) ∩W + = A+, V (F) ∩W −= B−}, where V (F) is the set of vertices incident on at least one edge of F. Following Talaska [37], we define the weight of an alternating flow F ∈FA,B by w(F) = 2|A|+|F|−|V (F)|
⃗e∈F
x⃗e,
(4.2) (4.2) and we consider the probability measure on alternating flows with boundary conditions A, B
given by and we consider the probability measure on alternating flows with boundary conditions A, B
given by PA,B
a-flow(F) = w(F)
Z A,B
a-flow
,
F ∈FA,B, where Z A,B
a-flow =
F∈FA,B w(F) is the partition function. Note that in [37] general oriented
planar graphs are considered, and the connection with double random currents described in
this section is realized by our particular choice of ⃗G and its edge weights. where Z A,B
a-flow =
F∈FA,B w(F) is the partition function. Note that in [37] general oriented
planar graphs are considered, and the connection with double random currents described in
this section is realized by our particular choice of ⃗G and its edge weights. There are four different types of local (interior) edge configurations in an alternating flow
F on ⃗G according to the total amount of flow at the endpoints (the number of incoming
edges minus the number of outgoing edges) and the orientation of the edges (see Fig. 3). Note that it is not possible that F contains ⃗eR1 and ⃗eR2 but not ⃗eB since then the alternating 123 The Planar Ising Model and Total Positivity The Planar Ising Model and Total Positivity 81 Fig. 3 Four types of local edge
configurations in alternating
flows on ⃗G
(1a)
(2a)
(2b)
(1b)
condition is violated. 4 Random Alternating Flows Also note that there are three different configurations of type (1a) which
are indistinguishable from the point of view of alternating flows—they can be interchanged
without forcing any other changes in the flow. (1a)
(2a)
(2b)
(1b) Fig. 3 Four types of local edge
configurations in alternating
flows on ⃗G (1b) (1a) condition is violated. Also note that there are three different configurations of type (1a) which
are indistinguishable from the point of view of alternating flows—they can be interchanged
without forcing any other changes in the flow. condition is violated. Also note that there are three different configurations of type (1a) which
are indistinguishable from the point of view of alternating flows—they can be interchanged
without forcing any other changes in the flow. With each F ∈FA,B, as in Sect. 3, we associate a pair of sets ω(F) = (ω1(F), ω2(F)),
where ω1(F) is the set of edges e ∈E such that the local configuration of F at e is of type (1a)
or (1b), and where ω2(F) is the set of edges e ∈E with the local configuration of type (2a) or
(2b). We denote by A,B the image of FA,B under this map. Note that ω1(F) ∈EA△B since,
by the flow condition, one can split ω1(F) into a sourceless part in E∅and a collection of |A|
edge-disjoint directed paths starting at A and ending at B. In particular, every v ∈A ∩B
is the starting and ending point of exactly one such path, and hence, the degree of ω1(F) at
v is even. This means that A,B ⊆ A△B, and hence the map above induces a probability
measure P
A,B
a
flow on A△B, which is supported on A B. The main result of this section casts this measure into a form related to that of the induced
double random current measure from Theorem 3.2. Theorem 4.1 Let A, B ⊂W be such that |A| = |B|, and let ⃗G be one of the directed
modifications of G described above. Let ω ∈ A,B, and let k′(ω) be the number of connected
components of ω that contain a vertex in A ∪B. 4 Random Alternating Flows Then, the probability of ω induced from the
random alternating flow measure is given by P
A,B
a−flow(ω) =
1
¯Z A,B
a-flow
2|A|−k′(ω)|E∅(ω)|
e∈ω1
xe
e∈ω2
x2
e
e∈E\ω
(1 −x2
e ),
where ¯Z A,B
a-flow = Z A,B
a-flow
e∈E(1 −x2
e ). In particular, P
A,B
a-flow = P
B,A
a-flow. P
A,B
a−flow(ω) =
1
¯Z A,B
a-flow
2|A|−k′(ω)|E∅(ω)|
e∈ω1
xe
e∈ω2
x2
e
e∈E\ω
(1 −x2
e ), Before proving the theorem, we need to recall a standard result about the cardinality of
E∅(ω). We give a proof for completeness. For ω ⊆E, we denote by k(ω) the number of
connected components of ω. Lemma 4.2 Let ω ⊆E be such that E∅(ω) is nonempty. Then, Lemma 4.2 Let ω ⊆E be such that E∅(ω) is nonempty. Then, |E∅(ω)| = 2|ω|−|V (ω)|+k(ω). Proof Consider a maximal spanning forest T ⊆ω of ω, and note that |ω\T| = |ω|−|V (ω)|+
k(ω). It is hence enough to construct a bijection between E∅(ω) and the set of subsets of ω\T. To this end, to each e ∈ω \ T we assign the unique cycle Ce ⊆(ω \ T ) ∪{e}. Note that
Ce ∈E∅(ω). The bijection is given by assigning to each ω′ ∈E∅(ω) the set ω′ ∩(ω \ T ), and
its inverse by assigning to each {e1, . . . , el} ⊆ω \ T the set Ce1△· · · △Cel ∈E∅(ω). ⊓⊔ 123 82 M. Lis M. Lis Proof of Theorem 4.1 Let ¯ω be a modification of ω seen as a subset of E where each edge
in ω2 is replaced by two parallel edges. By an alternating orientation of ¯ω we mean an
assignment of orientations to the edges of ¯ω such that for each v ∈V , the edges alternate
in orientation around v. Let F be such that ω(F) = ω, and let ˜F be its equivalence class
under identifying the three local edge configurations of type (1a). Note that such equivalence
classes are in one-to-one correspondence with alternating orientations of ¯ω. We claim that
the number of such alternating orientations is 2k(ω)−k′(ω). (4.3) 2k(ω)−k′(ω). (4.3) Indeed, consider a connected component κ of ¯ω that does not contain a vertex in A ∪B. We claim that κ has exactly 2 different alternating orientations. 4 Random Alternating Flows To see this, consider the
outer boundary ∂outκ of κ, i.e., the set of edges in κ incident on the unbounded face of κ. Because of the alternating condition, the choice of the orientation for one edge determines
the orientation of all other edges in ∂outκ. Moreover, there are no conflicts of orientations
since each vertex of κ has even degree, and hence ∂outκ can be deformed to a cycle by
splitting vertices incident on more than 2 edges from ∂outκ. Then, the orientations of all
edges agree with the clockwise or anticlockwise order on the cycle. After orienting ∂outκ,
one has to consider all connected components of κ \ ∂outκ, and repeat the reasoning with
the only difference being that there is no more freedom of choice of the orientation (if ∂outκ
was oriented clockwise (resp. counterclockwise), then all outer boundaries of the connected
components of κ \∂outκ have to be oriented counterclockwise (resp. clockwise)). One repeats
this procedure until all edges of κ are oriented. On the other hand, there is no freedom of
orientation for components containing a vertex in A ∪B since each such component has to
be oriented from a vertex in A to a vertex in B. Therefore (4.3) holds true. Indeed, consider a connected component κ of ¯ω that does not contain a vertex in A ∪B. We claim that κ has exactly 2 different alternating orientations. To see this, consider the
outer boundary ∂outκ of κ, i.e., the set of edges in κ incident on the unbounded face of κ. Because of the alternating condition, the choice of the orientation for one edge determines
the orientation of all other edges in ∂outκ. Moreover, there are no conflicts of orientations
since each vertex of κ has even degree, and hence ∂outκ can be deformed to a cycle by
splitting vertices incident on more than 2 edges from ∂outκ. Then, the orientations of all
edges agree with the clockwise or anticlockwise order on the cycle. After orienting ∂outκ,
one has to consider all connected components of κ \ ∂outκ, and repeat the reasoning with
the only difference being that there is no more freedom of choice of the orientation (if ∂outκ
was oriented clockwise (resp. counterclockwise), then all outer boundaries of the connected
components of κ \∂outκ have to be oriented counterclockwise (resp. clockwise)). 4 Random Alternating Flows Combining all the previous observations, and using the definition of PA,B
a-flow, we can write 123 83 The Planar Ising Model and Total Positivity P
A,B
a-flow(ω) =
F: ω(F)=ω
PA,B
a-flow(F)
=
1
Z A,B
a-flow
F: ω(F)=ω
w(F)
=
1
Z A,B
a-flow
˜F: ω( ˜F)=ω
w( ˜F)
=
1
Z A,B
a-flow
˜F: ω( ˜F)=ω
2|A|+|ω|−|V (ω)|
e∈ω1
xe
1 −x2e
e∈ω2
x2
e
1 −x2e
=
1
¯Z A,B
a-flow
˜F: ω( ˜F)=ω
2|A|+|ω|−|V (ω)|
e∈ω1
xe
e∈ω2
x2
e
e∈E\ω
(1 −x2
e )
=
1
¯Z A,B
a-flow
2k(ω)−k′(ω)2|A|+|ω|−|V (ω)|
e∈ω1
xe
e∈ω2
x2
e
e∈E\ω
(1 −x2
e )
=
1
¯Z A,B
a-flow
2|A|−k′(ω)|E∅(ω)|
e∈ω1
xe
e∈ω2
x2
e
e∈E\ω
(1 −x2
e ), P
A,B
a-flow(ω) =
F: ω(F)=ω
PA,B
a-flow(F)
=
1
Z A,B
a-flow
F: ω(F)=ω
w(F)
=
1
Z A,B
a-flow
˜F: ω( ˜F)=ω
w( ˜F)
=
1
Z A,B
a-flow
˜F: ω( ˜F)=ω
2|A|+|ω|−|V (ω)|
e∈ω1
xe
1 −x2e
e∈ω2
x2
e
1 −x2e
=
1
¯Z A,B
a-flow
˜F: ω( ˜F)=ω
2|A|+|ω|−|V (ω)|
e∈ω1
xe
e∈ω2
x2
e
e∈E\ω
(1 −x2
e )
=
1
¯Z A,B
a-flow
2k(ω)−k′(ω)2|A|+|ω|−|V (ω)|
e∈ω1
xe
e∈ω2
x2
e
e∈E\ω
(1 −x2
e )
=
1
¯Z A,B
a-flow
2|A|−k′(ω)|E∅(ω)|
e∈ω1
xe
e∈ω2
x2
e
e∈E\ω
(1 −x2
e ), PA,B
a-flow(F) where the second to last equality follows from (4.3), and the last one from Lemma 4.2. ⊓⊔ where the second to last equality follows from (4.3), and the last one from Lemma 4.2. ⊓⊔
In two special cases the measures induced from double random currents and alternating
flows are the same. In two special cases the measures induced from double random currents and alternating
flows are the same. Corollary 4.3 We have that P
∅
d-curr = P
∅,∅
a-flow and P
{a,b}
d-curr = P
{a},{b}
a-flow for any a, b∈W, a ̸= b. Proof By Lemma 5.4, we have that ∅,∅= ∅and {a},{b} = {a,b}, and the statement
follows from Theorems 3.2 and 4.1. ⊓⊔ 4 Random Alternating Flows One repeats
this procedure until all edges of κ are oriented. On the other hand, there is no freedom of
orientation for components containing a vertex in A ∪B since each such component has to
be oriented from a vertex in A to a vertex in B. Therefore (4.3) holds true. We now turn to the total weight w( ˜F) of each equivalence class ˜F, which we define to be
the sum of weights of all flows in ˜F. By (4.1), the total multiplicative contribution to w( ˜F)
of configurations of type (1a) is 23
xe
2
2
xe
1 −x2e
+ 2xe =
2xe
1 −x2e
, where we took the factor 2# edges and did not take the factor 2|A|−# vertices from (4.2) into
account. This contribution is therefore equal to the contribution of a configuration of type
(1b). The contributions of configurations of type (2a) and (2b) also agree and are equal to where we took the factor 2# edges and did not take the factor 2|A|−# vertices from (4.2) into
account. This contribution is therefore equal to the contribution of a configuration of type
(1b). The contributions of configurations of type (2a) and (2b) also agree and are equal to 22
xe
2
xe
1 −x2e
=
2x2
e
1 −x2e
. Hence, by (4.2), we can write Hence, by (4.2), we can write w( ˜F) = 2|A|−|V (ω)|
e∈ω1
2xe
1 −x2e
e∈ω2
2x2
e
1 −x2e
= 2|A|+|ω|−|V (ω)|
e∈ω1
xe
1 −x2e
e∈ω2
x2
e
1 −x2e
. Note that the map ω is constant on each equivalence class ˜F, and hence, with a slight
abuse of notation, we can write ω( ˜F) for the value of ω evaluated at any representative of
˜F. 5 Proofs of Main Results We need to state a few necessary lemmas. The first one goes back to the work of Griffiths,
Hurst and Sherman [15], and we give a proof for completeness. We will write Za,b = Z{a,b},
and we define Za,b
curr = Z∅
curr for a = b. Lemma 5.1 For a, b ∈V , we have ⟨σaσb⟩= Za,b
curr
Z∅curr
. Proof If a = b, then the equality is trivial. Otherwise, for a vertex v and a current n, we
define nv =
u: {u,v}∈E n{u,v}, and we have ⟨σaσb⟩=
σ∈{−1,1}V
σaσb
{u,v}∈E
exp(J{u,v}σuσv)
σ∈{−1,1}V
{u,v}∈E
exp(J{u,v}σuσv) ⟨σaσb⟩=
σ∈{−1,1}V
σaσb
{u,v}∈E
exp(J{u,v}σuσv)
σ∈{−1,1}V
{u,v}∈E
exp(J{u,v}σuσv)
=
σ∈{−1,1}V
σaσb
{u,v}∈E
∞
k=0
(J{u,v})kσ k
u σ k
v /k!
σ∈{−1,1}V
{u,v}∈E
∞
k=0
(J{u,v})kσ ku σ kv /k! =
σ∈{−1,1}V
σaσb
{u,v}∈E
∞
k=0
(J{u,v})kσ k
u σ k
v /k!
σ∈{−1,1}V
{u,v}∈E
∞
k=0
(J{u,v})kσ ku σ kv /k! 123 M. Lis 84 =
σ∈{−1,1}V
σaσb
n∈
w(n)
v∈V
σ nv
v
σ∈{−1,1}V
n∈
w(n)
v∈V
σ nv
v
=
σ∈{−1,1}V
n∈
w(n)
v∈V
σ nv+1{v∈{a,b}}
v
σ∈{−1,1}V
n∈
w(n)
v∈V
σ nv
v
=
2|V |
n∈{a,b}
w(n)
2|V |
n∈∅
w(n)
= Za,b
curr
Z∅curr
. The second to last equality holds true since the only currents that survive the summation
over the symmetric set {−1, 1}|V | are the ones for which the exponent of σv is even at every
vertex. ⊓⊔ The second to last equality holds true since the only currents that survive the summation
over the symmetric set {−1, 1}|V | are the ones for which the exponent of σv is even at every
vertex. ⊓⊔ The next lemma expresses the two-point spin correlation function as a ratio of partition
functions of alternating flows. Recall that we defined A,B ⊆ A△B to be the image of FA,B
under the map ω from Sect. 4. Lemma 5.2 For a, b ∈W, we have Lemma 5.2 For a, b ∈W, we have Lemma 5.2 For a, b ∈W, we have ⟨σaσb⟩= Za,b
a-flow
Z∅
a-flow
. ⟨σaσb⟩= Za,b
a-flow
Z∅
a-flow
. Proof Note that for each F ∈F{a},{b}, the graph ω(F) contains exactly k′ = 1 connected
component that connects a and b. The following is the reverse of the lemma above. Lemma 5.4 Let ω ∈ A△B be such that for each connected component κ of ω, the vertices
in A+ ∪B−that are adjacent to V (κ) alternate between the sources in A+ and the sinks in
B−as one goes around the external face. Then, there exists F ∈FA,B such that ω(F) = ω. Proof Fix a connected component κ of ω. Similarly to the proof of Theorem 4.1, we consider
a graph ¯κ where each edge of κ ∩ω2 is replaced by two parallel edges, and moreover, the
edges connecting each vertex in V (κ) ∩W to the corresponding vertices in A+ ∪B−are
added. It is now enough to show that there exists an alternating orientation of ¯κ where each
vertex in V (¯κ) ∩A+ becomes a source and each vertex in V (¯κ) ∩B−becomes a sink. By
the assumption on κ, we can add (in one of two possible ways) |V (¯κ) ∩(A+ ∪B−)|/2
vertex-disjoint edges connecting in pairs consecutive vertices in V (¯κ) ∩A+ and V (¯κ) ∩B−. We call the resulting graph ¯κ′. It is now enough to construct an alternating orientation of ¯κ′
such that each of the additional edges is directed from a vertex in V (¯κ) ∩B−to a vertex
in V (¯κ) ∩A+, and then restrict the orientation to ¯κ. To this end, note that each vertex of ¯κ′
has even degree. Therefore, as in the proof Theorem 4.1, the outer boundary of ¯κ′ can be
deformed to a cycle, and can be directed clockwise or counterclockwise so that the additional
edges are directed properly. We can then orient the rest of ¯κ′ consistently as in Theorem 4.1,
and the lemma is proved. ⊓⊔ The next lemma is an adaptation of a result of [37]. Lemma 5.5 [37, Corollary 4.3] Let N A,B be as in Theorem 2.1. We have det N A,B = Z A,B
a-flow
Z∅
a-flow
. Proof The proof is a matter of translation of the results of [37] to our setting. To this end,
consider the boundary measurement matrix M from Definition 2.4 of [37] defined for ⃗G with
weights as in Sect. 4. By Corollary 5.3 of [37], the entry corresponding to source a+ and sink
b−is equal to Za,b
a-flow/Z∅
a-flow, and hence, by Lemma 5.2, to ⟨σaσb⟩. Therefore, the boundary
measurement matrix for ⃗G is the matrix of Ising boundary spin correlation functions. 5 Proofs of Main Results Moreover, by Lemma 5.4 we have that {a},{b} = {a}△{b},
and ∅,∅= ∅. Hence, by comparing the formulas in Theorem 3.2 and 4.1, and using
Lemma 5.1, we get Za,b
a-flow
Z∅
a-flow
= 2|{a}|2−k′ Za,b
curr Z∅
curr
Z∅curr Z∅curr
= Za,b
curr
Z∅curr
= ⟨σaσb⟩. ⊓⊔ The next lemma describes an alternating property of sources and sinks in a connected
component of an alternating flow. The next lemma describes an alternating property of sources and sinks in a connected
component of an alternating flow. Lemma 5.3 Let F ∈FA,B, and let κ ⊂F be one of its connected components. Then, the
sources and sinks of κ interlace, i.e., as one goes around the external face, the vertices in
V (κ) ∩(A+ ∪B−) alternate between the sources in A+ and the sinks in B−. Proof Note that κ is an alternating flow itself. Take a source vertex v ∈V (κ) ∩A+, and
traverse the edges of the external face of κ in a counterclockwise order. Since at each vertex
in V that you visit, you take the rightmost possible turn, and since κ is alternating, all the
directed edges are aligned with the direction of traversal until you encounter the consecutive
vertex in v′ ∈V (κ) ∩(A+ ∪B−). Since v′ is either a sink or a source, and since there exists
an edge directed towards v′, it must be a sink. An analogous argument can be made when
starting at v′ but this time the edges are directed against the direction of traversal. Hence, the
next vertex encountered in V (κ) ∩(A+ ∪B−) is again a source, and the lemma is proved. ⊓⊔ 123 123 85 The Planar Ising Model and Total Positivity The following is the reverse of the lemma above. We are now ready to prove our first two main results. The following is the reverse of the lemma above. Indeed, in a flow F ∈FA,B there cannot be a connected component that
contains more than 2 vertices in A ∪B since then the alternating condition from Lemma 5.3
would be violated. On the other hand, if there exists k vertex disjoint paths connecting A and
B, then by Lemma 5.4, there exists a flow in FA,B which maps to these paths under ω. ⊓⊔ Furthermore, by Lemma 5.3 and 5.4, Z A,B
a-flow > 0 if and only if there are k vertex disjoint paths
connecting A and B. Indeed, in a flow F ∈FA,B there cannot be a connected component that
contains more than 2 vertices in A ∪B since then the alternating condition from Lemma 5.3
would be violated. On the other hand, if there exists k vertex disjoint paths connecting A and
B, then by Lemma 5.4, there exists a flow in FA,B which maps to these paths under ω. ⊓⊔ Before proving the rest of our main results, we will need a classical Pfaffian formula
of Groeneveld, Boel and Kasteleyn [16]. We present here a different proof involving the
connection between double random currents and alternating flows. For yet another proof
involving the dimer model, see the recent treatment of the combinatorics of the planar Ising
model [6]. Lemma 5.6 [16, Theorem A] Let K A∪B be as in Theorem 2.3. We have Lemma 5.6 [16, Theorem A] Let K A∪B be as in Theorem 2.3. We have Pf K A∪B = Z A∪B
curr
Z∅curr
. Proof By (2.2), for any A′ ⊂A ∪B with |A′| = |A| and B′ = (A ∪B) \ A′, we have
det N A′,B′ =
π: bijection A′→B′
(−1)xing(π)
{a,b}∈π
⟨σaσb⟩ Recall that a bijection between A′ and B′ can be thought of as a pairing of A ∪B, i.e., a
partition of A ∪B into pairs {a, π(a)}, a ∈A′. Note that each pairing π of A ∪B appears in
a sum as above for exactly 2|A| choices of A′. Indeed, for each of the |A| pairs in π, we can
choose one vertex that will belong to A′. Hence, by (2.4) and by Lemma 5.5, we have Pf K A∪B = 2−|A|
A′,B′
det N A′,B′ = 2−|A|
Z∅
a-flow
A′,B′
Z A′,B′
a-flow. (5.1) (5.1) Recall that A′,B′ ⊂ A∪B is the image of FA′,B′ under the map ω from Sect. The following is the reverse of the lemma above. By Corollary 4.3of [37], it is hence enough to check that the signs in N A agree with those
in the matrix in Corollary 4.3 of [37] where only the columns corresponding to sinks are
considered. In [37], the sign of the entry corresponding to source a+ and sink b−is negative
if the number of sources strictly between a+ and b−in a fixed clockwise order is odd,
and it is positive otherwise. By our choice, for every w ∈W◦(resp. w ∈W•), the sink w−
immediately precedes (resp. succeeds) the source w+ in the counterclockwise order. One can
easily check that as a result, s(i, j) from the definition of N A,B counts the number of sources
strictly between the source w+
li ∈A+ and the sink w−
j ∈B−. Therefore, the signs in the
definition of N A agree with those in [37] (up to changing a clockwise to a counterclockwise
order). Hence, the result follows from Corollary 4.3 of [37]. ⊓⊔ ⊓⊔ Remark 3 The proof of Corollary 4.3 of [37] uses the signed walk interpretation of the
boundary measurement matrix of Postnikov [33]. One can compare this with the Kac–Ward
representation of the planar Ising model [20], where the Ising boundary two-point functions
can be expressed as partition functions of signed non-backtracking walks on the undirected
graph G (see e.g. [21], or [26] for a concise proof of the Kac–Ward formula). 123 123 123 M. Lis 86 M. Lis Proof of Theorem 2.1 It is a direct consequence of Lemma 5.5. The interpretation of empty
paths as counterclockwise or clockwise loops follows from the way the sources and sinks
are placed around the external face (see Sect. 4). ⊓⊔ ⊓⊔ Proof of Corollary 2.2 Note that every square submatrix of M A,B satisfies the assumptions
of Corollary 2.2. Hence, it is enough to prove that det M A,B ≥0, and det M A,B > 0 if and
only if there are k vertex disjoint paths connecting A and B. To this end, note that by (2.1),
(2.2) and Lemma 5.5, det M A,B = det N A,B = Z A,B
a-flow
Z∅
a-flow
≥0. Furthermore, by Lemma 5.3 and 5.4, Z A,B
a-flow > 0 if and only if there are k vertex disjoint paths
connecting A and B. The following is the reverse of the lemma above. Comparing the formulas in
Theorem 3.2 and 4.1, we have PA∪B
d-curr(PA,B) = P
A∪B
d-curr(P A,B)
=
ω∈P A,B
P
A∪B
d-curr(ω)
=
Z A,B
a-flow
Z A∪B
curr Z∅curr
ω∈P A,B
2k′(ω)−|A|P
A,B
a-flow(ω)
=
Z A,B
a-flow
Z A∪B
curr Z∅curr
ω∈P A,B
P
A,B
a-flow(ω)
=
Z A,B
a-flow
Z A∪B
curr Z∅curr
,
( =
Z A,B
a-flow
Z A∪B
curr Z∅curr
ω∈P A,B
2k′(ω)−|A|P
A,B
a-flow(ω) =
Z A,B
a-flow
Z A∪B
curr Z∅curr
ω∈P A,B
P
A,B
a-flow(ω) (5.2) where the second to last equality holds true since, by the definition of PA,B, k′(ω) = |A| for
every ω ∈P A,B. Moreover, since P
A,B
a-flow is supported on A,B, to justify the last equality we
have to show that P A,B = A,B. To this end, note that by Lemma 5.3, for each F ∈FA,B,
we have that ω(F) ∈P A,B, where ω denotes the map from Sect. 4. Indeed, for contiguous
sets A and B, there is only one way of distributing the sources A+ and sinks B−between
the connected components of an alternating flow in such a way that they alternate around the
external face for each component. This means that each connected component of F connects a
single point in A to a single point in B. On the other hand, by Lemma 5.4, for each ω ∈P A,B,
there exists F ∈FA,B such that ω(F) = ω. Therefore, P A,B = A,B and (5.2) holds true. Furthermore, using Lemma 5.5 and 5.6, we get where the second to last equality holds true since, by the definition of PA,B, k′(ω) = |A| for
every ω ∈P A,B. Moreover, since P
A,B
a-flow is supported on A,B, to justify the last equality we
have to show that P A,B = A,B. To this end, note that by Lemma 5.3, for each F ∈FA,B,
we have that ω(F) ∈P A,B, where ω denotes the map from Sect. 4. Indeed, for contiguous
sets A and B, there is only one way of distributing the sources A+ and sinks B−between
the connected components of an alternating flow in such a way that they alternate around the
external face for each component. This means that each connected component of F connects a
single point in A to a single point in B. The following is the reverse of the lemma above. 4. We claim
that each ω ∈ A∪B belongs to A′,B′ for exactly 2k′(ω) choices of A′, where k′(ω) is the
number of connected components of ω containing a vertex in A ∪B. Indeed, by Lemma 5.3
and 5.4, for each connected component κ of ω that intersects A ∪B, there are exactly two
ways of distributing the vertices in V (κ) ∩(A ∪B) between A′ and B′ (choosing one vertex
to be connected to a source or a sink fixes the choices for all other vertices in V (κ)∩(A ∪B) 123 The Planar Ising Model and Total Positivity 87 by the alternating property). Hence, by combining Theorem 3.2 and 4.1, we have (5.1) =
1
Z∅
a-flow
A′,B′
ω∈ A′,B′
2−k′(ω)|E∅(ω)|
e∈ω1
xe
1 −x2e
e∈ω2
x2
e
1 −x2e
=
1
Z∅
a-flow
ω∈ A∪B
2k′(ω)−k′(ω)|E∅(ω)|
e∈ω1
xe
1 −x2e
e∈ω2
x2
e
1 −x2e
= Z A∪B
curr Z∅
curr
Z∅
a-flow
= Z A∪B
curr
Z∅curr
, (5.1) =
1
Z∅
a-flow
A′,B′
ω∈ A′,B′
2−k′(ω)|E∅(ω)|
e∈ω1
xe
1 −x2e
e∈ω2
x2
e
1 −x2e
=
1
Z∅
a-flow
ω∈ A∪B
2k′(ω)−k′(ω)|E∅(ω)|
e∈ω1
xe
1 −x2e
e∈ω2
x2
e
1 −x2e
= Z A∪B
curr Z∅
curr
Z∅
a-flow
= Z A∪B
curr
Z∅curr
, where in the last equality we used Corollary 4.3 to get Z∅
a-flow = (Z∅
curr)2. ⊓⊔ where in the last equality we used Corollary 4.3 to get Z∅
a-flow = (Z∅
curr)2. ⊓⊔ We are now in a position to prove the rest of our main results. We are now in a position to prove the rest of our main results. Proof of Theorem 2.3 Recall that A and B are placed around the outer face in such a way that
allverticesof A comebeforeeveryvertexof B inacounterclockwiseorder.Let P A,B ⊆ A∪B
be the image of the event PA,B under the map from Sect. 3. The following is the reverse of the lemma above. On the other hand, by Lemma 5.4, for each ω ∈P A,B,
there exists F ∈FA,B such that ω(F) = ω. Therefore, P A,B = A,B and (5.2) holds true. Furthermore, using Lemma 5.5 and 5.6, we get det M A,B
Pf K A∪B = Z A,B
a-flowZ∅
curr
Z∅
a-flowZ A∪B
curr
=
Z A,B
a-flow
Z A∪B
curr Z∅curr
, where we used Corollary 4.3 to obtain that Z∅
a-flow = (Z∅
curr)2. Together with (5.2), this
finishes the proof. ⊓⊔ where we used Corollary 4.3 to obtain that Z∅
a-flow = (Z∅
curr)2. Together with (5.2), this
finishes the proof. ⊓⊔ 123 123 88 M. Lis Proof of Theorem 2.4 Hongler in his PhD thesis [17] showed that in the setting of Theo-
rem 2.4, for any a ∈A and b ∈B, the following limit exists Proof of Theorem 2.4 Hongler in his PhD thesis [17] showed that in the setting of Theo-
rem 2.4, for any a ∈A and b ∈B, the following limit exists fa,b(D) := lim
ϵ→0 ϵ−1⟨σaϵσbϵ⟩,
(5.3) (5.3) where aϵ and bϵ are vertices on the outer face of Dϵ approximating a and b. Moreover, the
limit is conformally covariant, i.e., for a domain D′ and a conformal equivalence ϕ : D →D′,
we have where aϵ and bϵ are vertices on the outer face of Dϵ approximating a and b. Moreover, the
limit is conformally covariant, i.e., for a domain D′ and a conformal equivalence ϕ : D →D′,
we have fa,b(D) = fϕ(a),ϕ(b)(D′)|ϕ′(a)ϕ′(b)|
1
2 . (5.4) (5.4) Furthermore, for real numbers a, b, we have fa,b(H) = 2(
√
2 + 1)
π|a −b| . The result now follows from Theorem 2.3 since, by the expansions of the determinant and
Pfaffian (2.1) and (2.4), the normalization factors from (5.3), the constants 2(
√
2 + 1)/π,
and the factors containing derivatives of ϕ from (5.4) cancel out. ⊓⊔ ⊓⊔ Acknowledgements The author thanks the anonymous referees for very useful suggestions. This research
was supported by the Knut and Alice Wallenberg foundation, and was conducted when the author was at
Chalmers University of Technology and the University of Gothenburg. The following is the reverse of the lemma above. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 Interna-
tional License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source,
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(1952) 123
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English
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Agriculture alters the ancestral phenological plasticity to spring warmth in a forest specialist, but not in its generalist sister species
| null | 2,023
|
cc-by
| 19,671
|
Agriculture alters the ancestral phenological plasticity to
1
spring warmth in a forest specialist, but not in its generalis
2
sister species
3
Paul Cuchot1*, Timothée Bonnet2, Olivier Dehorter3, Pierre-Yves Henry3,4† & Céline Teplitsky1†
4
5
1CEFE, Univ Montpellier, CNRS, EPHE, IRD, Montpellier, France
6
2Centre d’Etudes Biologiques de Chizé UMR 7372 Université de la Rochelle-CNRS, Villiers-en-Bois,
7
France.
8
3Centre de Recherches sur la Biologie des Populations d'Oiseaux (CRBPO), Centre d'Ecologie et des
9
Sciences de la Conservation (CESCO UMR 7204), Muséum National d’Histoire Naturelle, Centre
10
National de la Recherche Scientifique, Sorbonne Université, Paris, France.
11
4Mécanismes adaptatifs et évolution (MECADEV UMR 7179), Muséum National d’Histoire Naturell
12
Centre National de la Recherche Scientifique, Brunoy, France.
13
14
* Corresponding author: paul.cuchot@gmail.com
15
† Pierre-Yves Henry and Céline Teplitsky should be considered as joint senior author.
16
17
Keywords: Breeding phenology, plasticity, timing, temperature, global change, anthropization, bi
18
timing
19 Agriculture alters the ancestral phenological plasticity to
1
spring warmth in a forest specialist, but not in its generalis
2
sister species
3
Paul Cuchot1*, Timothée Bonnet2, Olivier Dehorter3, Pierre-Yves Henry3,4† & Céline Teplitsky1†
4
5
1CEFE, Univ Montpellier, CNRS, EPHE, IRD, Montpellier, France
6
2Centre d’Etudes Biologiques de Chizé UMR 7372 Université de la Rochelle-CNRS, Villiers-en-Bois,
7
France. 8
3Centre de Recherches sur la Biologie des Populations d'Oiseaux (CRBPO), Centre d'Ecologie et des
9
Sciences de la Conservation (CESCO UMR 7204), Muséum National d’Histoire Naturelle, Centre
10
National de la Recherche Scientifique, Sorbonne Université, Paris, France. 11
4Mécanismes adaptatifs et évolution (MECADEV UMR 7179), Muséum National d’Histoire Naturell
12
Centre National de la Recherche Scientifique, Brunoy, France. 13
14
* Corresponding author: paul.cuchot@gmail.com
15
† Pierre-Yves Henry and Céline Teplitsky should be considered as joint senior author. 16
17
Keywords: Breeding phenology, plasticity, timing, temperature, global change, anthropization, bi
18
timing
19 Agriculture alters the ancestral phenological plasticity to
1
spring warmth in a forest specialist, but not in its generalist
2
sister species
3
Paul Cuchot1*, Timothée Bonnet2, Olivier Dehorter3, Pierre-Yves Henry3,4† & Céline Teplitsky1†
4
5
1CEFE, Univ Montpellier, CNRS, EPHE, IRD, Montpellier, France
6
2Centre d’Etudes Biologiques de Chizé UMR 7372 Université de la Rochelle-CNRS, Villiers-en-Bois,
7
France. 8
3Centre de Recherches sur la Biologie des Populations d'Oiseaux (CRBPO), Centre d'Ecologie et des
9
Sciences de la Conservation (CESCO UMR 7204), Muséum National d’Histoire Naturelle, Centre
10
National de la Recherche Scientifique, Sorbonne Université, Paris, France. 11
4Mécanismes adaptatifs et évolution (MECADEV UMR 7179), Muséum National d’Histoire Naturelle,
12
Centre National de la Recherche Scientifique, Brunoy, France. 13
14 Agriculture alters the ancestral phenological plasticity to
1
spring warmth in a forest specialist, but not in its generalist
2
sister species
3 3Centre de Recherches sur la Biologie des Populations d'Oiseaux (CRBPO), Centre d'Ecologie et des
9
Sciences de la Conservation (CESCO UMR 7204), Muséum National d’Histoire Naturelle, Centre
10
National de la Recherche Scientifique, Sorbonne Université, Paris, France. 11 3Centre de Recherches sur la Biologie des Populations d'Oiseaux (CRBPO), Centre d'Ecologie et des
9
Sciences de la Conservation (CESCO UMR 7204), Muséum National d’Histoire Naturelle, Centre
10
National de la Recherche Scientifique, Sorbonne Université, Paris, France. 11 4Mécanismes adaptatifs et évolution (MECADEV UMR 7179), Muséum National d’Histoire Naturelle,
12
Centre National de la Recherche Scientifique, Brunoy, France. 13 4Mécanismes adaptatifs et évolution (MECADEV UMR 7179), Muséum National d’Histoire Naturelle,
12
Centre National de la Recherche Scientifique, Brunoy, France. 13 † Pierre-Yves Henry and Céline Teplitsky should be considered as joint senior author. 16 Keywords: Breeding phenology, plasticity, timing, temperature, global change, anthropization, birds,
18
timing
19 1 1 Introduction
43 Theory predicts that the degree of adaptive
56
plasticity in phenological responses to temperature depends on two main parameters: (1) the slope of
57
the optimum phenological response to environmental change, which depends on the temperature-
58
dependence of the timing of the peak in food abundance (Visser & Both, 2005) and (2) environmental
59
predictability (Lande, 2014), i.e., how well the pre-breeding environment predicts the timing of the
60
peak of food abundance. Both can vary within species, according to local environmental conditions,
61
and can differ among species according to their ecological requirements (Visser et al., 2009; Moussus
62
et al., 2011), resulting in differences in phenological plasticity among populations and species. For
63
example, blue and great tit populations inhabiting deciduous forests are more sensitive to temperature
64
in terms of their breeding phenology than populations inhabiting mixed and evergreen forests (Bailey
65
et al 2022). This can be explained by lower peaks of caterpillar abundance in evergreen forests (Blondel
66
et al., 1993), which may require a greater dietary flexibility of nestlings and reduce the reliance of
67 Most seasonal species adjust their breeding phenology in response to temperature (Thackeray et al.,
53
2016; McLean et al., 2022) and there is ample evidence that plasticity is the prevailing mechanism in
54
studied vertebrate models (Gienapp et al., 2006; Charmantier et al., 2008; Canale & Henry, 2010;
55
Charmantier & Gienapp, 2014; Charmantier et al., 2016). Theory predicts that the degree of adaptive
56
plasticity in phenological responses to temperature depends on two main parameters: (1) the slope of
57
the optimum phenological response to environmental change, which depends on the temperature-
58
dependence of the timing of the peak in food abundance (Visser & Both, 2005) and (2) environmental
59
predictability (Lande, 2014), i.e., how well the pre-breeding environment predicts the timing of the
60
peak of food abundance. Both can vary within species, according to local environmental conditions,
61
and can differ among species according to their ecological requirements (Visser et al., 2009; Moussus
62
et al., 2011), resulting in differences in phenological plasticity among populations and species. For
63
example, blue and great tit populations inhabiting deciduous forests are more sensitive to temperature
64
in terms of their breeding phenology than populations inhabiting mixed and evergreen forests (Bailey
65
et al 2022). Introduction
43 Introduction
43 In temperate regions, climate change increases the frequency of warm and early springs (Walther et
44
al., 2002; Parmesan & Yohe, 2003; Lee et al., 2023), so that animals need to advance their reproductive
45
period to match phenological shifts in resource availability (Visser & Both, 2005; Visser & Gienapp,
46
2019). Migratory bird species arrive earlier on their breeding grounds (Inouye et al., 2000; Cotton,
47
2003; Neate-Clegg & Tingley, 2023) and resident species breed earlier (Crick et al., 1997; Dunn &
48
Winkler, 2010) in warm springs. A lack of adjustment can lead to a timing mismatch between preys and
49
predators/consumers ("trophic mismatch hypothesis", Durant et al., 2007; Miller-Rushing et al., 2010;
50
Stenseth & Mysterud, 2002; Visser et al., 1998), ultimately resulting in decreased breeding success for
51
predators/consumer species (Husby et al., 2010; Visser & Gienapp, 2019; Visser et al., 2021). 52 In temperate regions, climate change increases the frequency of warm and early springs (Walther et
44
al., 2002; Parmesan & Yohe, 2003; Lee et al., 2023), so that animals need to advance their reproductive
45
period to match phenological shifts in resource availability (Visser & Both, 2005; Visser & Gienapp,
46
2019). Migratory bird species arrive earlier on their breeding grounds (Inouye et al., 2000; Cotton,
47
2003; Neate-Clegg & Tingley, 2023) and resident species breed earlier (Crick et al., 1997; Dunn &
48
Winkler, 2010) in warm springs. A lack of adjustment can lead to a timing mismatch between preys and
49
predators/consumers ("trophic mismatch hypothesis", Durant et al., 2007; Miller-Rushing et al., 2010;
50
Stenseth & Mysterud, 2002; Visser et al., 1998), ultimately resulting in decreased breeding success for
51
predators/consumer species (Husby et al., 2010; Visser & Gienapp, 2019; Visser et al., 2021). 52 Most seasonal species adjust their breeding phenology in response to temperature (Thackeray et al.,
53
2016; McLean et al., 2022) and there is ample evidence that plasticity is the prevailing mechanism in
54
studied vertebrate models (Gienapp et al., 2006; Charmantier et al., 2008; Canale & Henry, 2010;
55
Charmantier & Gienapp, 2014; Charmantier et al., 2016). Abstract
20 Abstract
20 Phenological adjustment is the first line of adaptive response of vertebrates when ancestral seasonality
21
is disrupted by climate change. The prevailing response is to reproduce earlier in warmer springs, but
22
habitat changes, such as conversion of ancestral (pre-human) habitats into cities and agricultural lands,
23
are expected to affect phenological plasticity, for example due to loss of reliability of environmental
24
cues used by organisms to time reproduction. Relying on two-decade, country level capture-based
25
monitoring of common songbirds’ reproduction, we investigated how the conversion of forest habitat
26
into agricultural lands, and into artificialized surfaces affected the deviation of average phenology and
27
plasticity to local temperature from its presumed ancestral state, i.e., in non-urbanized forests. For
28
this, we built a hierarchical model that simultaneously estimated fledging phenology and its response
29
to spring temperatures based on the changes in the proportion of juveniles captured over the breeding
30
season. Both species fledge earlier in warmer sites (blue tit: 2.94 days/°C, great tit: 3.83 days/°C), in
31
warmer springs (blue tit: 2.49 days/°C, great tit: 2.75 days/°C) and in most urbanized habitats (4 days
32
for blue tit and 2 days for great tit). The reaction norm of fledging phenology to spring temperature
33
varied across sites in both species, but this variation was explained by ancestral habitat only in the
34
deciduous forest specialist, the blue tit. In this species, the responses to spring temperature were
35
shallower in agricultural landscapes. The ancestral reaction norm is preserved in the habitat-generalist
36
species (great tit), as well as along the urbanization gradient and was correlated to mean fledgling
37
phenology (i.e., steeper response in later sites). The effects of habitat change on ancestral adaptive
38
reaction norms provide another way through which combined environmental degradations may
39
threaten populations persistence, to an extent depending on species and changes in their prey
40
phenology and abundance. 41 42 2 2 Introduction
43 This can be explained by lower peaks of caterpillar abundance in evergreen forests (Blondel
66
et al., 1993), which may require a greater dietary flexibility of nestlings and reduce the reliance of
67 3 breeding birds on caterpillars and corresponding temperature cues (Vatka et al., 2011). Moreover,
68
habitats can modulate the effects of pre-breeding temperatures on breeding phenology, for instance
69
by imposing energetic constraints. For example, tree swallows (Tachycineta bicolor) breeding in higher
70
densities display steeper responses to temperature than birds breeding in low density habitats, likely
71
because the latter are poor-quality habitats (Bourret et al, 2015). Differences in phenological
72
responses to environmental cues among species can also be strong (e.g., Radchuk et al., 2019) and
73
these variations can at least partly be explained by ecological characteristics such as the degree of
74
habitat or thermal specialization (Moussus et al., 2011). 75 breeding birds on caterpillars and corresponding temperature cues (Vatka et al., 2011). Moreover,
68
habitats can modulate the effects of pre-breeding temperatures on breeding phenology, for instance
69
by imposing energetic constraints. For example, tree swallows (Tachycineta bicolor) breeding in higher
70
densities display steeper responses to temperature than birds breeding in low density habitats, likely
71
because the latter are poor-quality habitats (Bourret et al, 2015). Differences in phenological
72
responses to environmental cues among species can also be strong (e.g., Radchuk et al., 2019) and
73
these variations can at least partly be explained by ecological characteristics such as the degree of
74
habitat or thermal specialization (Moussus et al., 2011). 75 Habitat anthropization, i.e., human-induced modifications of an ancestral environment (e.g., through
76
urbanization, conversion into agricultural lands, etc.) can also alter bird phenology and ultimately
77
affect their plasticity in response to temperature. Urbanization affects breeding phenology in birds
78
with urban populations laying and singing earlier than their rural conspecifics (Møller et al., 2015;
79
Capilla-Lasheras et al., 2022). Chemical inputs and reduction of habitat heterogeneity caused by the
80
intensification of agricultural practices may also disturb birds breeding phenology by modifying prey
81
phenology and abundance (Vickery et al., 2001; Britschgi et al., 2006; Stanton et al., 2018). Human-
82
induced habitat alterations could also affect the phenological response of birds to temperature. First,
83
in human-altered habitats, the peak of prey abundance may spread over a longer period (Seress et al.,
84
2018). Introduction
43 A broad, flat seasonal peak of prey is poorly predictable, making temperature an irrelevant cue
85
for prey emergence. Moreover, a broader resource peak would reduce the cost of asynchrony, leaving
86
birds less constrained by temperature (Visser & Gienapp, 2019). Second, cues other than temperature
87
may be degraded in anthropized habitats (e.g., blurred information on invertebrate prey phenology
88
and photoperiod change), making the amount of available information poorer and less accurate. Third,
89
anthropized habitats may represent lower-quality habitats, leading to lower plasticity levels (Bourret
90
et al., 2015). However, the effects of anthropization, through urbanization and agriculture, on
91
phenological plasticity in wild bird populations remains poorly studied (Kentie et al., 2018). There is an
92
urgent need for investigating the effects of such human-induced changes on phenological responses
93 4
Habitat anthropization, i.e., human-induced modifications of an ancestral environment (e.g., through
76
urbanization, conversion into agricultural lands, etc.) can also alter bird phenology and ultimately
77
affect their plasticity in response to temperature. Urbanization affects breeding phenology in birds
78
with urban populations laying and singing earlier than their rural conspecifics (Møller et al., 2015;
79
Capilla-Lasheras et al., 2022). Chemical inputs and reduction of habitat heterogeneity caused by the
80
intensification of agricultural practices may also disturb birds breeding phenology by modifying prey
81
phenology and abundance (Vickery et al., 2001; Britschgi et al., 2006; Stanton et al., 2018). Human-
82
induced habitat alterations could also affect the phenological response of birds to temperature. First,
83
in human-altered habitats, the peak of prey abundance may spread over a longer period (Seress et al.,
84
2018). A broad, flat seasonal peak of prey is poorly predictable, making temperature an irrelevant cue
85
for prey emergence. Moreover, a broader resource peak would reduce the cost of asynchrony, leaving
86
birds less constrained by temperature (Visser & Gienapp, 2019). Second, cues other than temperature
87
may be degraded in anthropized habitats (e.g., blurred information on invertebrate prey phenology
88
and photoperiod change), making the amount of available information poorer and less accurate. Third,
89
anthropized habitats may represent lower-quality habitats, leading to lower plasticity levels (Bourret
90
et al., 2015). However, the effects of anthropization, through urbanization and agriculture, on
91
phenological plasticity in wild bird populations remains poorly studied (Kentie et al., 2018). Introduction
43 There is an
92
urgent need for investigating the effects of such human-induced changes on phenological responses
93 4 to global warming as growing urban and agricultural expansions are causing unprecedented declines
94
in wild bird populations (Donald et al., 2001; Reif & Vermouzek, 2019; Rigal et al., 2023) and possibly
95
reducing the conditions for phenological adaptation. 96 to global warming as growing urban and agricultural expansions are causing unprecedented declines
94
in wild bird populations (Donald et al., 2001; Reif & Vermouzek, 2019; Rigal et al., 2023) and possibly
95
reducing the conditions for phenological adaptation. 96 to global warming as growing urban and agricultural expansions are causing unprecedented declines
94
in wild bird populations (Donald et al., 2001; Reif & Vermouzek, 2019; Rigal et al., 2023) and possibly
95
reducing the conditions for phenological adaptation. 96 Using the blue tit (Cyanistes caeruleus) and the great tit (Parus major), two forest species that have
97
been used as models for research on climate-induced predator-prey phenological mismatches (e.g.,
98
Biquet et al., 2022; Nussey et al., 2005), we investigated how the conversion of forest habitats into
99
agricultural lands (forest cover), and into artificialized surfaces (ground imperviousness) affected the
100
deviation of average phenology and plasticity to local temperature from its presumed ancestral state,
101
i.e., in non-urbanized forests. We evaluated the effects of habitat composition at a landscape scale on
102
breeding phenology and plasticity to local temperature using a nationwide (183 sites), long-term (21
103
years) ringing monitoring program of common birds during the reproductive period across mainland
104
France. Such monitoring programs are ideal to investigate the consequences of habitat anthropization
105
on bird breeding phenology as the different ringing stations are spread across most habitats. We
106
considered two gradients of habitat degradation: loss of ancestral habitat, assessed by forest density
107
(both species being primarily forest birds), and artificialization, estimated by the degree of ground
108
imperviousness. We developed an integrative modelling approach based on the changes in the
109
proportion of captured juveniles throughout the breeding seasons for each year and site to infer
110
breeding phenology from the peak of fledging (i.e., when chicks leave the nest). Materials and methods
117 Mist-
140
netting of birds is most efficient in habitats with a low canopy (since mist-nets capture up to 3-to-4 m
141 Study species
118
The blue tit and the great tit are two hole-nesting passerines that have long been used as model to
119
understand the ecology and evolution of breeding phenology in birds (Visser et al., 1998; Blondel,
120
2007; Charmantier et al., 2016; Bonamour, 2021). Both species are forest-dwelling passerines that rely
121
on trees to nest and forage. Yet the great tit has a broader ecological niche (generalist): it occupies
122
urban and disturbed habitats more densely than the blue tit, whereas the blue tit is more specialized
123
on deciduous forests (Gibb, 1954; Snow, 1954; Solonen, 2001; Moussus et al., 2011). They synchronize
124
their reproduction to match the peak of offspring food requirements with the peak of caterpillar
125
abundance (Visser et al. 1998, 2006, Marciniak et al. 2007). Tits breed once to twice per year and start
126
breeding during their first year. Females lay between 5 and 13 eggs per clutch (Gibb., 1950; A. P. Møller
127
et al., 2014). In France, tits initiate breeding in March. Incubation and chick rearing last around 35 days. 128
Once fledged, young individuals are still fed by their parents for about 20 days (Verhulst, 1995). 129 Study species
118
The blue tit and the great tit are two hole-nesting passerines that have long been used as model to
119
understand the ecology and evolution of breeding phenology in birds (Visser et al., 1998; Blondel,
120
2007; Charmantier et al., 2016; Bonamour, 2021). Both species are forest-dwelling passerines that rely
121
on trees to nest and forage. Yet the great tit has a broader ecological niche (generalist): it occupies
122
urban and disturbed habitats more densely than the blue tit, whereas the blue tit is more specialized
123
on deciduous forests (Gibb, 1954; Snow, 1954; Solonen, 2001; Moussus et al., 2011). They synchronize
124
their reproduction to match the peak of offspring food requirements with the peak of caterpillar
125
abundance (Visser et al. 1998, 2006, Marciniak et al. 2007). Tits breed once to twice per year and start
126
breeding during their first year. Females lay between 5 and 13 eggs per clutch (Gibb., 1950; A. P. Møller
127
et al., 2014). In France, tits initiate breeding in March. Materials and methods
117 6
Materials and methods
117
Study species
118
The blue tit and the great tit are two hole-nesting passerines that have long been used as model to
119
understand the ecology and evolution of breeding phenology in birds (Visser et al., 1998; Blondel,
120
2007; Charmantier et al., 2016; Bonamour, 2021). Both species are forest-dwelling passerines that rely
121
on trees to nest and forage. Yet the great tit has a broader ecological niche (generalist): it occupies
122
urban and disturbed habitats more densely than the blue tit, whereas the blue tit is more specialized
123
on deciduous forests (Gibb, 1954; Snow, 1954; Solonen, 2001; Moussus et al., 2011). They synchronize
124
their reproduction to match the peak of offspring food requirements with the peak of caterpillar
125
abundance (Visser et al. 1998, 2006, Marciniak et al. 2007). Tits breed once to twice per year and start
126
breeding during their first year. Females lay between 5 and 13 eggs per clutch (Gibb., 1950; A. P. Møller
127
et al., 2014). In France, tits initiate breeding in March. Incubation and chick rearing last around 35 days. 128
Once fledged, young individuals are still fed by their parents for about 20 days (Verhulst, 1995). 129
130
Capture data
131
Capture data were collected by volunteer bird ringers from 2001 to 2021 following the French Constant
132
ringing Effort Site protocol (Julliard & Jiguet, 2002). Capture sessions start early May (median May 16th,
133
95% range: May 3rd – June 7th) and end early July (July 7th [June 19th – July 24th]), covering most of the
134
incubation and chick fledging periods. The number of capture sessions and locations of mist-nets vary
135
between sites but are held constant within each site among sessions and years. Per spring, on average,
136
there are three capture sessions per site (95% range: 3 to 6 sessions). A capture session lasts from
137
dawn to noon. Captured birds are identified to the species level, ringed with a unique numbered metal
138
ring (or recorded as recapture if already ringed), sexed and aged based on plumage (juvenile for birds
139
born during the ongoing breeding season, or adult if born in previous years; Svensson, 1992). Introduction
43 111 Using the blue tit (Cyanistes caeruleus) and the great tit (Parus major), two forest species that have
97
been used as models for research on climate-induced predator-prey phenological mismatches (e.g.,
98
Biquet et al., 2022; Nussey et al., 2005), we investigated how the conversion of forest habitats into
99
agricultural lands (forest cover), and into artificialized surfaces (ground imperviousness) affected the
100
deviation of average phenology and plasticity to local temperature from its presumed ancestral state,
101
i.e., in non-urbanized forests. We evaluated the effects of habitat composition at a landscape scale on
102
breeding phenology and plasticity to local temperature using a nationwide (183 sites), long-term (21
103
years) ringing monitoring program of common birds during the reproductive period across mainland
104
France. Such monitoring programs are ideal to investigate the consequences of habitat anthropization
105
on bird breeding phenology as the different ringing stations are spread across most habitats. We
106
considered two gradients of habitat degradation: loss of ancestral habitat, assessed by forest density
107
(both species being primarily forest birds), and artificialization, estimated by the degree of ground
108
imperviousness. We developed an integrative modelling approach based on the changes in the
109
proportion of captured juveniles throughout the breeding seasons for each year and site to infer
110
breeding phenology from the peak of fledging (i.e., when chicks leave the nest). 111 We predicted that plasticity to local temperature varied among sampling sites and whether habitat
112
anthropization had an effect on phenology (additive effects) and its response to temperature
113
(interactive effect). We expected phenological plasticity to be more variable and more habitat
114
dependent in the deciduous forest specialist (the blue tit) than in the more generalist species (the great
115
tit). 116 We predicted that plasticity to local temperature varied among sampling sites and whether habitat
112
anthropization had an effect on phenology (additive effects) and its response to temperature
113
(interactive effect). We expected phenological plasticity to be more variable and more habitat
114
dependent in the deciduous forest specialist (the blue tit) than in the more generalist species (the great
115
tit). 116 5 5 Materials and methods
117 173
The date for this inflection point results from the timing of several processes: egg laying (i.e., breeding
174
phenology), eggs incubation, chick rearing, chick mortality in the nest and just after fledging (the few
175
days when recently fledged juveniles remain in the close vicinity of their nest), and occurrence of
176
seconds broods. The duration of egg incubation and chick rearing remains the same and does not vary
177
from year to year or site to site. We thus used xmid as a proxy for breeding phenology. We evaluated
178
the reliability of using fledging as a proxy of breeding (egg-laying) phenology by comparing the
179 The first parameter asymptote corresponds to the upper asymptote of the curve, and describes the
171
proportion of juveniles in the population at the end of the monitoring period. The second parameter
172
xmid is the inflection point of the curve. This parameter corresponds to the peak of juveniles fledging. 173
The date for this inflection point results from the timing of several processes: egg laying (i.e., breeding
174
phenology), eggs incubation, chick rearing, chick mortality in the nest and just after fledging (the few
175
days when recently fledged juveniles remain in the close vicinity of their nest), and occurrence of
176
seconds broods. The duration of egg incubation and chick rearing remains the same and does not vary
177
from year to year or site to site. We thus used xmid as a proxy for breeding phenology. We evaluated
178
the reliability of using fledging as a proxy of breeding (egg-laying) phenology by comparing the
179 The first parameter asymptote corresponds to the upper asymptote of the curve, and describes the
171
proportion of juveniles in the population at the end of the monitoring period. The second parameter
172
xmid is the inflection point of the curve. This parameter corresponds to the peak of juveniles fledging. 173
The date for this inflection point results from the timing of several processes: egg laying (i.e., breeding
174
phenology), eggs incubation, chick rearing, chick mortality in the nest and just after fledging (the few
175
days when recently fledged juveniles remain in the close vicinity of their nest), and occurrence of
176
seconds broods. The duration of egg incubation and chick rearing remains the same and does not vary
177
from year to year or site to site. Materials and methods
117 Incubation and chick rearing last around 35 days. 128
Once fledged, young individuals are still fed by their parents for about 20 days (Verhulst, 1995). 129 6 above the ground). Most CES sites are thus settled in shrublands, woodlands with dense understory,
142
or reedbeds (Eglington et al., 2015). The center of all CES sites is precisely georeferenced. The median
143
spatial coverage is 2 ha (95% range: 1.6 – 4.2 ha), and the median elevation is 91m (95% range: 1m –
144
951m). 145 above the ground). Most CES sites are thus settled in shrublands, woodlands with dense understory,
142
or reedbeds (Eglington et al., 2015). The center of all CES sites is precisely georeferenced. The median
143
spatial coverage is 2 ha (95% range: 1.6 – 4.2 ha), and the median elevation is 91m (95% range: 1m –
144
951m). 145 We selected sites where data were collected during at least 3 consecutive years with at least 3 sessions
146
per spring. We only included sessions lasting a minimum of 5 hours. To secure parameter estimability
147
at site-level, we only kept sites where at least 3 blue tits or 3 great tits were captured on average per
148
year. Only one record per individual per day was used. After data selection according to these criteria,
149
the final data set represented a total of 11489 blue tits (7938 juveniles and 3551 adults) and 23497
150
great tits (16629 juveniles and 6868 adults) for 185 sites over a period of 22 years (Figure 1). On
151
average 9.91 blue tits and 19.2 great tits were captured per site and per year. Maps representing the
152
numbers of mean captured individuals per year for each site and for both species are available in
153
Appendix S9. 154 155
Figure 1: Location of constant ringing effort sites in France for the 2001 - 2021 period and their number of monitored years. 156 155 155 Figure 1: Location of constant ringing effort sites in France for the 2001 - 2021 period and their number of monitored years. 156 Figure 1: Location of constant ringing effort sites in France for the 2001 - 2021 period and their number of monitored years. Materials and methods
117 156 157 7 7 Modelling fledging phenology with capture data
158 We aimed to infer fledging phenology, as a proxy of breeding phenology, from capture data using the
159
progressive increase of the proportion of juveniles among captured birds throughout the breeding
160
season (Figure 2). We modeled the probability for a captured individual to be a juvenile for each
161
species separately in a Bayesian hierarchical framework using Markov Chain Monte Carlo using the
162
program JAGS (Plummer, 2003) via the R package R2jags (Su & Yajima, 2021). 163 We aimed to infer fledging phenology, as a proxy of breeding phenology, from capture data using the
159
progressive increase of the proportion of juveniles among captured birds throughout the breeding
160
season (Figure 2). We modeled the probability for a captured individual to be a juvenile for each
161
species separately in a Bayesian hierarchical framework using Markov Chain Monte Carlo using the
162
program JAGS (Plummer, 2003) via the R package R2jags (Su & Yajima, 2021). 163 The number of juveniles on day t, year j and site k, follows a binomial distribution (eq.1) which is
164
characterized by 2 parameters: the probability that a captured individual is a juvenile pt,j,k and the total
165
number of captured individuals Ntott,j,k. 166 The number of juveniles on day t, year j and site k, follows a binomial distribution (eq.1) which is
164
characterized by 2 parameters: the probability that a captured individual is a juvenile pt,j,k and the total
165
number of captured individuals Ntott,j,k. 166 Njuvt,j,k ∼ Bin(pt,j,k,Ntott,j,k)
(1) Njuvt,j,k ∼ Bin(pt,j,k,Ntott,j,k)
(1)
167 (1) Following Moussus et al. (2011), we assumed that during the breeding period (April to July), pt,j,k follows
168
a sigmoid curve (Figure 2). We thus modeled p separately for each species with a 3-parameter function
169
(eq.2). 170 𝑝
𝑡,𝑗,𝑘=𝑎𝑠𝑦𝑚𝑝𝑡𝑜𝑡𝑒𝑗,𝑘
1+𝑒
𝑥𝑚𝑖𝑑𝑗,𝑘−𝑡
𝑠𝑐𝑎𝑙𝑒𝑗,𝑘
(2) (2) The first parameter asymptote corresponds to the upper asymptote of the curve, and describes the
171
proportion of juveniles in the population at the end of the monitoring period. The second parameter
172
xmid is the inflection point of the curve. This parameter corresponds to the peak of juveniles fledging. Materials and methods
117 We thus used xmid as a proxy for breeding phenology. We evaluated
178
the reliability of using fledging as a proxy of breeding (egg-laying) phenology by comparing the
179 8 estimated breeding phenology with the observed average laying date in five populations of blue and
180
great tits in southern France. Estimated and observed breeding phenology correlated very well ( ≥0.8,
181
Appendix S4). 182 estimated breeding phenology with the observed average laying date in five populations of blue and
180
great tits in southern France. Estimated and observed breeding phenology correlated very well ( ≥0.8,
181
Appendix S4). 182 Appendix S4). 182
183
184
Figure 2 : Modelling the pattern of temporal emergence of juveniles throughout a breeding period. A) In early spring the
185
proportion of juveniles is 0: juveniles are still in the egg or in the nest. A plateau is reached in June, when most chicks have
186
fledged. B) Examples of change of the proportion of juveniles of blue tits in France (102 capture sites) during the breeding
187
season for 3 years (2009 in orange, 2010 in blue and 2011 in purple). Each dot represents a capture session at a site. 188
Phenological changes between years are documented by the change in estimates of xmid. 189 183
184 183 183 184 184 Figure 2 : Modelling the pattern of temporal emergence of juveniles throughout a breeding period. A) In early spring the
185
proportion of juveniles is 0: juveniles are still in the egg or in the nest. A plateau is reached in June, when most chicks have
186
fledged. B) Examples of change of the proportion of juveniles of blue tits in France (102 capture sites) during the breeding
187
season for 3 years (2009 in orange, 2010 in blue and 2011 in purple). Each dot represents a capture session at a site. 188
Phenological changes between years are documented by the change in estimates of xmid. 189 Figure 2 : Modelling the pattern of temporal emergence of juveniles throughout a breeding period. A) In early sprin The last parameter, scale, corresponds to a shape parameter and estimates the slope of the curve at
190
the inflection point. Scale can be interpreted as a measure of the synchrony of fledging. Materials and methods
117 For instance,
191
the more synchronous are egg-laying dates across pairs or sites, the higher the synchrony of chick
192
fledging, and the steeper the curve (high scale value). Conversely, the higher the difference in number
193
of broods across pairs or sites, the shallower the curve. 194 The last parameter, scale, corresponds to a shape parameter and estimates the slope of the curve at
190
the inflection point. Scale can be interpreted as a measure of the synchrony of fledging. For instance,
191
the more synchronous are egg-laying dates across pairs or sites, the higher the synchrony of chick
192
fledging, and the steeper the curve (high scale value). Conversely, the higher the difference in number
193
of broods across pairs or sites, the shallower the curve. 194 The last parameter, scale, corresponds to a shape parameter and estimates the slope of the curve at
190
the inflection point. Scale can be interpreted as a measure of the synchrony of fledging. For instance,
191
the more synchronous are egg-laying dates across pairs or sites, the higher the synchrony of chick
192
fledging, and the steeper the curve (high scale value). Conversely, the higher the difference in number
193
of broods across pairs or sites, the shallower the curve. 194 9 These three parameters were assumed to follow a normal distribution (eq. 3-5). 5 𝑥𝑚𝑖𝑑𝑗,𝑘𝑁(𝜇𝑥𝑚𝑖𝑑𝑗,𝑘, 𝜎𝑥𝑚𝑖𝑑)
(3) 𝑥𝑚𝑖𝑑𝑗,𝑘𝑁(𝜇𝑥𝑚𝑖𝑑𝑗,𝑘, 𝜎𝑥𝑚𝑖𝑑) (3) 𝑥𝑚𝑖𝑑𝑗,𝑘𝑁(𝜇𝑥𝑚𝑖𝑑𝑗,𝑘, 𝜎𝑥𝑚𝑖𝑑) 196 𝑎𝑠𝑦𝑚𝑝𝑡𝑜𝑡𝑒𝑗,𝑘𝑁(𝜇𝑎𝑠𝑦𝑚𝑝𝑡𝑜𝑡𝑒𝑗,𝑘, 𝜎𝑎𝑠𝑦𝑚𝑝𝑡𝑜𝑡𝑒)
(4) (4) 197 (5) 𝑠𝑐𝑎𝑙𝑒𝑗,𝑘𝑁(𝜇𝑠𝑐𝑎𝑙𝑒𝑗,𝑘, 𝜎𝑠𝑐𝑎𝑙𝑒) 198 with µj,k corresponding to the mean of the distribution and σ to the associated variance. For each of
199
these three parameters (𝜇𝑥𝑚𝑖𝑑, 𝜇𝑎𝑠𝑦𝑚𝑝𝑡𝑜𝑡𝑒 and 𝜇𝑠𝑐𝑎𝑙𝑒), we considered two random effects to account
200
respectively for between-year and between-site variation. All priors have been set to be flat (Table
201
S7.1). We ran this Bayesian hierarchical model with three chains of 35000 iterations each and a burn-
202
in of 10000, and no thinning. The Gelman-Rubin convergence diagnostics (Brooks & Gelman, 1998)
203
were satisfied (i.e. < 1.1) for all parameter estimates used for inference in the Results section. We rely
204
on posterior distributions, their medians as point estimates and the associated 95% credible interval
205
(extracted from the highest posterior density) to infer the statistical support for our predictions (i.e . 206
departure of parameter estimates from 0). 207 with µj,k corresponding to the mean of the distribution and σ to the associated variance. Materials and methods
117 (2022) to define the most likely 60-day time windows of highest
214 10 plasticity for each studied site, with a central midpoint (in Julian days) calculated as 1.91*latitude -
215
10.76. In our dataset, average latitude is 47.7, and 95% of the sites are located between 43.7 and 50.6. 216
We then extracted the mean temperature within the defined window for each year and site, which
217
finally allowed us to calculate local temperature anomaly (yearly site temperature minus mean site
218
temperature over the period 2000-2022). 219 plasticity for each studied site, with a central midpoint (in Julian days) calculated as 1.91*latitude -
215
10.76. In our dataset, average latitude is 47.7, and 95% of the sites are located between 43.7 and 50.6. 216
We then extracted the mean temperature within the defined window for each year and site, which
217
finally allowed us to calculate local temperature anomaly (yearly site temperature minus mean site
218
temperature over the period 2000-2022). 219 Because of their associated food availability and their positives effects on their reproductive success
221
(Keller & Van Noordwijk, 1994; Naef-Daenzer et al., 2001; Lambrechts et al., 2004), non-artificialized
222
dense forests are considered as optimal and ancestral habitats for blue and great tits (Amininasab et
223
al., 2016). The proportion of ancestral habitat (i.e., non-artificialized forests) surrounding each site was
224
computed using high-resolution layers provided by the Territory service of the European Earth
225
observation program, Copernicus (https://land.copernicus.eu/pan-european/high-resolution-layers). 226
Data are based on satellite images and combine optical and radar data to characterize forest density
227
(i.e., percentage of tree cover, European Environment Agency & European Environment Agency,
228
2020b; Corsini et al., 2023) and ground imperviousness (i.e., percentage of waterproofed ground; e.g. 229
roads, buildings, European Environment Agency & European Environment Agency, 2020a; Szulkin et
230
al., 2020; Corsini et al., 2021) per 10-m pixel. For each site, we computed the mean value of both
231
variables in a 1-km buffer using the sf R package (Pebesma, 2018). The size of the buffer was chosen
232
according to the study of van Overveld et al. Materials and methods
117 For each of
199
these three parameters (𝜇𝑥𝑚𝑖𝑑, 𝜇𝑎𝑠𝑦𝑚𝑝𝑡𝑜𝑡𝑒 and 𝜇𝑠𝑐𝑎𝑙𝑒), we considered two random effects to account
200
respectively for between-year and between-site variation. All priors have been set to be flat (Table
201
S7.1). We ran this Bayesian hierarchical model with three chains of 35000 iterations each and a burn-
202
in of 10000, and no thinning. The Gelman-Rubin convergence diagnostics (Brooks & Gelman, 1998)
203
were satisfied (i.e. < 1.1) for all parameter estimates used for inference in the Results section. We rely
204
on posterior distributions, their medians as point estimates and the associated 95% credible interval
205
(extracted from the highest posterior density) to infer the statistical support for our predictions (i.e . 206
departure of parameter estimates from 0). 207 Temperature data for each site were extracted from the SAFRAN forecast model provided by the
209
French meteorological agency (Habets et al., 2008; Quintana-Seguí et al., 2008). This model integrates
210
data from meteorological stations and satellite monitoring to estimate climatic variations all over the
211
study area, based on an 8km grid. 212 Temperature data for each site were extracted from the SAFRAN forecast model provided by the
209
French meteorological agency (Habets et al., 2008; Quintana-Seguí et al., 2008). This model integrates
210
data from meteorological stations and satellite monitoring to estimate climatic variations all over the
211
study area, based on an 8km grid. 212 Temperature data for each site were extracted from the SAFRAN forecast model provided by the
209
French meteorological agency (Habets et al., 2008; Quintana-Seguí et al., 2008). This model integrates
210
data from meteorological stations and satellite monitoring to estimate climatic variations all over the
211
study area, based on an 8km grid. 212 The window of temperature driving the initiation of reproduction opens later in the season at higher
213
latitudes. We relied on Bailey et al. (2022) to define the most likely 60-day time windows of highest
214 The window of temperature driving the initiation of reproduction opens later in the season at higher
213
latitudes. We relied on Bailey et al. (2022) to define the most likely 60-day time windows of highest
214 The window of temperature driving the initiation of reproduction opens later in the season at higher
213
latitudes. We relied on Bailey et al. Materials and methods
117 (2017) which showed that the spatial extent of families
233
making excursions outside of their woodlot during the post-fledgling period is equal to 1100 m ± (SE =
234
265, range: 643–2374, n= 6) in blue tits and 666 m (SE = 42, range: 245–1898, n = 64) in great tits. 235 A principal component analysis revealed that forest density is strongly, negatively associated with crop
236
surface area (calculated from Corine Land Cover) in the same buffer area (Figure 3). A decrease in
237
forest density is therefore essentially compensated by an increase of farmland, and conveys
238 A principal component analysis revealed that forest density is strongly, negatively associated with crop
236
surface area (calculated from Corine Land Cover) in the same buffer area (Figure 3). A decrease in
237
forest density is therefore essentially compensated by an increase of farmland, and conveys
238 11 11 information about the degree of habitat anthropization through agriculture. Due to the strong
239
collinearity between forest and farmland cover, only forest cover was used in the models. 240 241 242
Figure 3: Principal component analysis (PCA) plot of all 185 capture sites. Imperviousness (also known as ground-
243
waterproofing) and forest density are averaged in a 1-km buffer around each site and extracted from high resolution layers. 244
Crops proportion is computed in a 1-km buffer too and extracted from Corine Land Cover. Correlations between variables are
245
described in Appendix 1. 246 242 242 Figure 3: Principal component analysis (PCA) plot of all 185 capture sites. Imperviousness (also known as ground-
243
waterproofing) and forest density are averaged in a 1-km buffer around each site and extracted from high resolution layers. 244
Crops proportion is computed in a 1-km buffer too and extracted from Corine Land Cover. Correlations between variables are
245
described in Appendix 1. 246 247 Estimating reaction norms of fledging phenology to temperature
248 We modeled the 𝑥𝑚𝑖𝑑 parameter, the proxy for breeding phenology, according to temperature in
249
order to estimate phenological plasticity to temperature (eq. 6). Models were similar for both species:
250
to account for inter and intra-site variation in phenological response to temperature, we included both
251
mean site temperature (calculated among the 1990-2022 period) and local temperature anomaly
252
(spring temperature deviation from mean site temperature; eq. 6, van de Pol & Wright, 2009). Materials and methods
117 253 We modeled the 𝑥𝑚𝑖𝑑 parameter, the proxy for breeding phenology, according to temperature in
249
order to estimate phenological plasticity to temperature (eq. 6). Models were similar for both species:
250
to account for inter and intra-site variation in phenological response to temperature, we included both
251
mean site temperature (calculated among the 1990-2022 period) and local temperature anomaly
252
(spring temperature deviation from mean site temperature; eq. 6, van de Pol & Wright, 2009). 253 12 12 𝜇𝑥𝑚𝑖𝑑𝑗,𝑘= (𝛼+ 𝜇0𝑘+ 𝜇0𝑗) + (𝛽1 + 𝜇1𝑘) ∗𝑡𝑒𝑚𝑝 𝑎𝑛𝑜𝑚𝑗,𝑘+ 𝛽2 ∗𝑡𝑒𝑚𝑝𝑘
(6) (6) with α corresponding to the between-site intercept (average phenology across sites), u0k to the random
254
site intercept, u0j to the random year intercept, β1 to the mean slope across sites, u1k to the random
255
site slope and β2 to the linear effect of mean site temperature. 256 To evaluate the effects of habitat anthropization on phenological plasticity to local temperature
257
anomalies, we designed a model that integrated the fixed effects of forest density, imperviousness,
258
their interaction with temperature anomaly plus an interaction between mean site temperature and
259
temperature anomaly (eq. 7). 260 𝜇𝑥𝑚𝑖𝑑𝑗,𝑘= 𝛼+ (𝜇0𝑘+ 𝜇0𝑗) + (𝛽1 + 𝜇1𝑘) ∗𝑡𝑒𝑚𝑝𝑒𝑟𝑎𝑡𝑢𝑟𝑒 𝑎𝑛𝑜𝑚𝑎𝑙𝑦𝑗,𝑘+ 𝛽2
∗𝑡𝑒𝑚𝑝𝑒𝑟𝑎𝑡𝑢𝑟𝑒𝑘+ 𝛽3 ∗𝑡𝑒𝑚𝑝𝑒𝑟𝑎𝑡𝑢𝑟𝑒𝑘∗𝑡𝑒𝑚𝑝𝑒𝑟𝑎𝑡𝑢𝑟𝑒 𝑎𝑛𝑜𝑚𝑎𝑙𝑦𝑗,𝑘 𝜇𝑥𝑚𝑖𝑑𝑗,𝑘= 𝛼+ (𝜇0𝑘+ 𝜇0𝑗) + (𝛽1 + 𝜇1𝑘) ∗𝑡𝑒𝑚𝑝𝑒𝑟𝑎𝑡𝑢𝑟𝑒 𝑎𝑛𝑜𝑚𝑎𝑙𝑦𝑗,𝑘+ 𝛽2 (7) + 𝛿1 ∗𝑓𝑜𝑟𝑒𝑠𝑡 𝑑𝑒𝑛𝑠𝑖𝑡𝑦𝑘+ 𝛾1 ∗𝑓𝑜𝑟𝑒𝑠𝑡 𝑑𝑒𝑛𝑠𝑖𝑡𝑦𝑘
(7) ∗𝑡𝑒𝑚𝑝𝑒𝑟𝑎𝑡𝑢𝑟𝑒 𝑎𝑛𝑜𝑚𝑎𝑙𝑦𝑗,𝑘+ 𝛿2 ∗𝑖𝑚𝑝𝑒𝑟𝑣𝑖𝑜𝑢𝑠𝑛𝑒𝑠𝑠𝑘+ 𝛾2 ∗𝑖𝑚𝑝𝑒𝑟𝑣𝑖𝑜𝑢𝑠𝑛𝑒𝑠𝑠𝑘∗𝑡𝑒𝑚𝑝𝑒𝑟𝑎𝑡𝑢𝑟𝑒 𝑎𝑛𝑜𝑚𝑎𝑙𝑦𝑗,𝑘 ∗𝑖𝑚𝑝𝑒𝑟𝑣𝑖𝑜𝑢𝑠𝑛𝑒𝑠𝑠𝑘∗𝑡𝑒𝑚𝑝𝑒𝑟𝑎𝑡𝑢𝑟𝑒 𝑎𝑛𝑜𝑚𝑎𝑙𝑦𝑗,𝑘 We calculated the median and 95% credible intervals for each posterior distributions of the regression
261
parameters β1, β2, β3, δ, δ2, γ1 and γ2. 262
Results
263
Between-site variability in phenological plasticity
264
Juveniles of both species fledged earlier in warmer sites and years (i.e., in sites with higher average
265
temperature and years with high temperature anomaly, Table 1). Average phenology varied strongly
266
among sites in both species (Figure 4; Table 1). Responses to temperature anomaly also varied among
267
sites (Figure 4; Table 1) with larger variance in slopes in great tits than in blue tits (Table 1). Mean
268
fledging phenology differs by only three days between blue and great tit (June 1st for blue tit and May
269 We calculated the median and 95% credible intervals for each posterior distributions of the regression
261
parameters β1, β2, β3, δ, δ2, γ1 and γ2. Materials and methods
117 262 y
p
g
p
y
Juveniles of both species fledged earlier in warmer sites and years (i.e., in sites with higher average
265
temperature and years with high temperature anomaly, Table 1). Average phenology varied strongly
266
among sites in both species (Figure 4; Table 1). Responses to temperature anomaly also varied among
267
sites (Figure 4; Table 1) with larger variance in slopes in great tits than in blue tits (Table 1). Mean
268
fledging phenology differs by only three days between blue and great tit (June 1st for blue tit and May
269 Juveniles of both species fledged earlier in warmer sites and years (i.e., in sites with higher average
265
temperature and years with high temperature anomaly, Table 1). Average phenology varied strongly
266
among sites in both species (Figure 4; Table 1). Responses to temperature anomaly also varied among
267
sites (Figure 4; Table 1) with larger variance in slopes in great tits than in blue tits (Table 1). Mean
268
fledging phenology differs by only three days between blue and great tit (June 1st for blue tit and May
269 13 29th for great tit). The covariance between random intercepts 𝜇0𝑘 and random slopes 𝜇1𝑘 was negative
270
for great tits (Table 1), implying that later breeding populations were more sensitive to local
271
temperature anomaly. In both species, the response to local temperature anomaly appeared to be
272
independent of mean temperature. 273 29th for great tit). The covariance between random intercepts 𝜇0𝑘 and random slopes 𝜇1𝑘 was negative
270
for great tits (Table 1), implying that later breeding populations were more sensitive to local
271
temperature anomaly. In both species, the response to local temperature anomaly appeared to be
272
independent of mean temperature. 273 Table 1: Median and 95% credible intervals of posterior distributions for the estimates of the fledging phenology models (eq. 274
6). Residual variance in mean breeding phenology (intercept) between sites – after accounting for the effect of mean site
275
temperature - corresponds to sigma_int, and between-site variance in phenological response to local temperature anomaly
276
(slope) corresponds to sigma_slope. Covariance between random slopes and random intercepts corresponds to Cov_slope_int. 277 Table 1: Median and 95% credible intervals of posterior distributions for the estimates of the fledging phenology models (eq. 274
6). Materials and methods
117 Residual variance in mean breeding phenology (intercept) between sites – after accounting for the effect of mean site
275
temperature - corresponds to sigma_int, and between-site variance in phenological response to local temperature anomaly
276
(slope) corresponds to sigma_slope. Covariance between random slopes and random intercepts corresponds to Cov_slope_int. 277 Blue tit
Great tit
Median
95% CI
Median
95% CI
Intercept
151.24
[149.72 ; 153.05]
148.19
[146.49 ; 149.83]
Mean
temperature
-2.94
[-3.91 ; -1.97]
-3.83
[-4.7 ; -2.98]
Temperature
anomaly
-2.49
[-3.64 ; -1.21]
-2.75
[-3.75 ; -1.63]
Temperature
anomaly *
mean temperature
-0.39
[-0.99 ; 0.26]
0.05
[-0.44 ; 0.53]
sigma_int
4.66
[3,68 ; 5.72]
4.43
[3.61 ; 5.3]
sigma_slope
1.3
[0,03 ; 3.01]
1.46
[0.45 ; 2.31]
Cov_slope_int
-0.6
[-5.58 ; 2.04]
-2.9
[-6.24 ; -0.01] 278 14 279 280
Figure 4 : Site specific responses of breeding phenology (xmid) to local temperature anomaly. Each grey line represents the
281
estimated phenological response to temperature for a single site, based on the posterior median for parameters in equation
282
(6). The thicker turquoise line represents the predicted mean response to temperature anomaly across all sites. 283
284 280 Figure 4 : Site specific responses of breeding phenology (xmid) to local temperature anomaly. Each grey line represents the
281
estimated phenological response to temperature for a single site, based on the posterior median for parameters in equation
282
(6). The thicker turquoise line represents the predicted mean response to temperature anomaly across all sites. 283 Figure 4 : Site specific responses of breeding phenology (xmid) to local temperature anomaly. Each grey line represents the
281
estimated phenological response to temperature for a single site, based on the posterior median for parameters in equation
282
(6). The thicker turquoise line represents the predicted mean response to temperature anomaly across all sites. 283 Effects of habitat anthropization on phenology and its plasticity to temperature
285
Both species reproduce earlier in more impervious sites (Figure 5; Table 2; Figure S3.1). The slope of
286
the reaction norm of phenology to temperature anomaly was significantly modified by the loss of
287
forest cover (i.e., when moving from the ancestral forest habitat to agricultural landscapes) in blue tits:
288
the lower the forest cover, the flatter the relationship to temperature anomaly (i.e., the lower the
289
dependence; Figure 5; Table 2). Materials and methods
117 Phenological response to local temperature anomaly were similar
290
between most anthropized and ancestral habitats (i.e., non-forest) for both species (Figure 5). Blue tit
291
populations located in average forest sites, tended to be more sensitive to temperature anomaly when
292
artificialization was higher (Figure 5, Table 2). Finally, neither forest cover nor imperviousness explain
293
variations in the slope of the reaction norm to temperature anomaly in great tit, suggesting that this
294
species reacts in the same way, whatever the degree and the type of habitat anthropization. 295 15 15 Table 2: Median and 95% credible intervals of posterior distributions for the estimates of the fledging phenology models which
296
include forest density and imperviousness effects and their interaction with temperature anomaly (eq. 6). 297 Blue tit
Great tit
Median
95% CI
Median
95% CI
Intercept
151.13
[149.54 ; 152.97]
148.22
[146.54 ; 149.81]
Mean
temperature
-2.96
[-3.91 ; -1.97]
-3.66
[-4.56 ; -2.79]
Temperature
anomaly
-2.37
[-3.5 ; -1.04]
-2.75
[-3.76 ; -1.62]
Temperature
anomaly *
mean temperature
-0.35
[-0.9 ; 0.24]
0.06
[-0.46 ; 0.59]
Forest density
-0.23
[-1.16 ; 0.65]
0.11
[-0.67 ; 0.9]
Imperviousness
-0.91
[-1.96 ; 0.12]
-0.96
[-1.86 ; -0.12]
Forest density *
temperature
anomomaly
-0.91
[-1.51 ; -0.35]
-0.29
[-0.78 ; 0.22]
Imperviousness *
temperature
anomaly
-1.12
[-1.77 ; -0.47]
-0.19
[-0.73 ; 0.35]
sigma_int
4.71
[3.7 ; 5.8]
4.41
[3.61 ; 5.26]
sigma_slope
0.87
[0.04 ; 2.35]
1.5
[0.71 ; 2.33]
Cov_slope_int
-1.13
[-6.36 ; 0.96]
-3.05
[-6.36 ; -0.33]
8 298 299 16 300
Figure 5: Effects of both forest density and imperviousness on blue and great tits phenological response to local temperature
301
anomaly. Each line corresponds to a projection of the model with parameters values sampled from the 0.10-0.90 posterior
302
distributions. For both environmental factors,” High” and” Low” correspond respectively to their 0.1 and 0.9 quantile across
303
all capture sites. 304 300 Figure 5: Effects of both forest density and imperviousness on blue and great tits phenological response to local temperature
301
anomaly. Each line corresponds to a projection of the model with parameters values sampled from the 0.10-0.90 posterior
302
distributions. For both environmental factors,” High” and” Low” correspond respectively to their 0.1 and 0.9 quantile across
303
all capture sites. Materials and methods
117 328 Interestingly, the response to mean site temperature and to temperature anomaly was very similar
322
(Table 1), suggesting that differences in fledging phenology across sites could be explained to a large
323
extent by plasticity (Phillimore et al., 2016). If fledging phenology had advanced more in response to
324
the mean site temperature than expected given our estimate of plasticity, we could have suggested
325
that the observed phenological changes were consistent with a response to selection for earlier
326
fledging date, as it is the case in some tits populations (Van Noordwijk et al., 1995; Gienapp et al., 2006;
327
Marrot et al., 2017). 328 Despite the observed phenological variations across sites, the reaction norm of breeding phenology to
329
temperature appeared largely conserved: it was similar in both species, identical across habitats for
330
the generalist great tit, and maintained along the gradient of habitat urbanization for the blue tit. Only
331
forest conversion into farmlands altered the phenological reaction norm of the deciduous forest
332
specialist (the blue tit). A decrease in plasticity when shifting to farmland landscapes can be explained
333
by a change in the abundance invertebrate preys (Vickery et al., 2001; Britschgi et al., 2006; Stanton et
334
al., 2018). For example, a flatter peak of invertebrate prey abundance may require an increased dietary
335
flexibility, decreasing the birds dependence on “ancestral preys” and their associated temperature
336
cues during the breeding season (Vatka et al., 2011). Following the same rationale, Bailey et al. (2022)
337
explained the weaker plasticity of tits in evergreen and mixed forests by the fact that they relied on a
338 Despite the observed phenological variations across sites, the reaction norm of breeding phenology to
329
temperature appeared largely conserved: it was similar in both species, identical across habitats for
330
the generalist great tit, and maintained along the gradient of habitat urbanization for the blue tit. Only
331
forest conversion into farmlands altered the phenological reaction norm of the deciduous forest
332
specialist (the blue tit). A decrease in plasticity when shifting to farmland landscapes can be explained
333
by a change in the abundance invertebrate preys (Vickery et al., 2001; Britschgi et al., 2006; Stanton et
334
al., 2018). Materials and methods
117 304 Figure 5: Effects of both forest density and imperviousness on blue and great tits phenological response to loca
1 Both tit species reproduced earlier in warmer springs. This phenological response of tits to climate
306
warming is already well established (Charmantier et al., 2008; Visser et al., 2009; Phillimore et al., 2016;
307
Bonamour et al., 2019; Shutt et al., 2019; Bailey et al., 2022), and had been proved to be essentially
308
mediated by plastic phenotypic adjustments (Charmantier et al., 2008; Biquet et al., 2022). The
309
advancement of reproduction in warm years is a global phenotypic response, known from many other
310
taxa of seasonal vertebrates (Thackeray et al., 2016; McLean et al., 2022). Nonetheless, our study is
311
the first to establish that this relationship holds over a large variety of habitats (i.e., at country-level). 312
Across study sites, the slope of the reaction norm to temperature ranged from -3.62 to -0.9 day/°C in
313 Both tit species reproduced earlier in warmer springs. This phenological response of tits to climate
306
warming is already well established (Charmantier et al., 2008; Visser et al., 2009; Phillimore et al., 2016;
307
Bonamour et al., 2019; Shutt et al., 2019; Bailey et al., 2022), and had been proved to be essentially
308
mediated by plastic phenotypic adjustments (Charmantier et al., 2008; Biquet et al., 2022). The
309
advancement of reproduction in warm years is a global phenotypic response, known from many other
310
taxa of seasonal vertebrates (Thackeray et al., 2016; McLean et al., 2022). Nonetheless, our study is
311
the first to establish that this relationship holds over a large variety of habitats (i.e., at country-level). 312
Across study sites, the slope of the reaction norm to temperature ranged from -3.62 to -0.9 day/°C in
313 17 17 blue tit (95% range: -3.29 to -1.58) and from -4.81 to -0.16 day/°C in great tit (95% range: -3.68 to -
314
1.50). In most the cited studies on tits, the estimated slope ranged from -4 to -2.5 day/°C. But none of
315
these studies estimated a plasticity close to 0 (i.e., absence of phenotypic response to temperature
316
anomalies). This finding suggests that phenological plasticity varies more across sites, and habitats,
317
than suggested by existing publications. Materials and methods
117 This potentially biased perception of the variability of
318
phenological reaction norms is likely explainable by the fact that the majority of long-term, nest-box
319
based, monitoring programs are located are mainly located in favourable environments for tits, i.e.,
320
forest-dominated habitats (Lambrechts et al., 2010; Culina et al., 2021; Bailey et al., 2022). 321 blue tit (95% range: -3.29 to -1.58) and from -4.81 to -0.16 day/°C in great tit (95% range: -3.68 to -
314
1.50). In most the cited studies on tits, the estimated slope ranged from -4 to -2.5 day/°C. But none of
315
these studies estimated a plasticity close to 0 (i.e., absence of phenotypic response to temperature
316
anomalies). This finding suggests that phenological plasticity varies more across sites, and habitats,
317
than suggested by existing publications. This potentially biased perception of the variability of
318
phenological reaction norms is likely explainable by the fact that the majority of long-term, nest-box
319
based, monitoring programs are located are mainly located in favourable environments for tits, i.e.,
320
forest-dominated habitats (Lambrechts et al., 2010; Culina et al., 2021; Bailey et al., 2022). 321 Interestingly, the response to mean site temperature and to temperature anomaly was very similar
322
(Table 1), suggesting that differences in fledging phenology across sites could be explained to a large
323
extent by plasticity (Phillimore et al., 2016). If fledging phenology had advanced more in response to
324
the mean site temperature than expected given our estimate of plasticity, we could have suggested
325
that the observed phenological changes were consistent with a response to selection for earlier
326
fledging date, as it is the case in some tits populations (Van Noordwijk et al., 1995; Gienapp et al., 2006;
327
Marrot et al., 2017). 328 Interestingly, the response to mean site temperature and to temperature anomaly was very similar
322
(Table 1), suggesting that differences in fledging phenology across sites could be explained to a large
323
extent by plasticity (Phillimore et al., 2016). If fledging phenology had advanced more in response to
324
the mean site temperature than expected given our estimate of plasticity, we could have suggested
325
that the observed phenological changes were consistent with a response to selection for earlier
326
fledging date, as it is the case in some tits populations (Van Noordwijk et al., 1995; Gienapp et al., 2006;
327
Marrot et al., 2017). Materials and methods
117 For example, a flatter peak of invertebrate prey abundance may require an increased dietary
335
flexibility, decreasing the birds dependence on “ancestral preys” and their associated temperature
336
cues during the breeding season (Vatka et al., 2011). Following the same rationale, Bailey et al. (2022)
337
explained the weaker plasticity of tits in evergreen and mixed forests by the fact that they relied on a
338 18 18 broader resource peak in these habitats, which reduced the cost of the trophic phenological mismatch
339
during chick rearing, and then made birds less constrained by spring temperature. Moreover, they
340
predicted that more specialist species, which occupy habitats with a narrow resource peak, should
341
show stronger phenological plasticity. This applies to our results: the most specialist species (blue tit)
342
had a stronger response to temperature anomalies than the generalist species (great tit) but only in its
343
ancestral habitat. In the same environmental context (farmlands vs forests), Bourret et al. (2015)
344
showed in the tree swallow (Tachycineta bicolor) that phenological plasticity was steeper in high
345
density colonies, here populations benefitting from the highest prey abundances (Hussell, 2003). 346
Further research is needed to determine whether such differences in phenological plasticity along the
347
agricultural-artificial gradient are due to changes in resource abundance and phenology, or whether
348
other factors associated with agricultural practices may affect the integration of environmental cues
349
by birds. 350 We detected earlier breeding phenology in more artificialized sites, in line with previous studies
351
showing that urban birds tend to breed earlier than their rural counterparts (Beck & Heinsohn, 2006;
352
Najmanová & Adamík, 2009; Caizergues et al., 2018; Capilla-Lasheras et al., 2022). Our results (up to 2
353
days earlier for blue tits and 4 days earlier for great tits when comparing most anthropized and most
354
ancestral sites, Figure S3.1), are in the range of what was previously observed: 4 to 7 days of lag across
355
different songbirds’ populations located in cities vs in more rural areas (Najmanová & Adamík, 2009). 356
A common explanation for this trend is that springs occur earlier in cities due to the urban heat island
357
effect (Yeh & Price, 2004; Schoech et al., 2008). Materials and methods
117 However, very few sites were located in core urban
358
areas (Figure S1.1), so the heat island effect is unlikely to have a strong impact on breeding phenology
359
across the studied sites. Urbanization may have other effects on breeding phenology, beyond the
360
higher temperatures usually associated to it, such as artificial light and food availability. Several studies
361
suggested that artificial lights can advance bird phenology (Dominoni et al., 2020; Kempenaers et al.,
362
2010; Senzaki et al., 2020). While direct effects of artificial lights on bird reproductive physiology have
363
not been detected (Partecke et al., 2006), indirect effects may enhance reproductive activity, for
364 We detected earlier breeding phenology in more artificialized sites, in line with previous studies
351
showing that urban birds tend to breed earlier than their rural counterparts (Beck & Heinsohn, 2006;
352
Najmanová & Adamík, 2009; Caizergues et al., 2018; Capilla-Lasheras et al., 2022). Our results (up to 2
353
days earlier for blue tits and 4 days earlier for great tits when comparing most anthropized and most
354
ancestral sites, Figure S3.1), are in the range of what was previously observed: 4 to 7 days of lag across
355
different songbirds’ populations located in cities vs in more rural areas (Najmanová & Adamík, 2009). 356
A common explanation for this trend is that springs occur earlier in cities due to the urban heat island
357
effect (Yeh & Price, 2004; Schoech et al., 2008). However, very few sites were located in core urban
358
areas (Figure S1.1), so the heat island effect is unlikely to have a strong impact on breeding phenology
359
across the studied sites. Urbanization may have other effects on breeding phenology, beyond the
360
higher temperatures usually associated to it, such as artificial light and food availability. Several studies
361
suggested that artificial lights can advance bird phenology (Dominoni et al., 2020; Kempenaers et al.,
362
2010; Senzaki et al., 2020). While direct effects of artificial lights on bird reproductive physiology have
363
not been detected (Partecke et al., 2006), indirect effects may enhance reproductive activity, for
364 19 example by allowing increased foraging time in diurnal animals (Deviche & Davies, 2013; Titulaer et al.,
365
2012). Materials and methods
117 Artificial food provisioning (bird tables) could also explain advanced phenology (Bourgault et
366
al., 2009; Harrison et al., 2010; Schoech et al., 2008; Vafidis et al., 2016) in more urbanized populations
367
by facilitating the earlier reach of nutritional thresholds and/or the sequestration of potentially
368
limiting, nutrients required for reproduction (e.g. calcium: Reynolds et al., 2004; protein: Schoech et
369
al., 2004). 370 example by allowing increased foraging time in diurnal animals (Deviche & Davies, 2013; Titulaer et al.,
365
2012). Artificial food provisioning (bird tables) could also explain advanced phenology (Bourgault et
366
al., 2009; Harrison et al., 2010; Schoech et al., 2008; Vafidis et al., 2016) in more urbanized populations
367
by facilitating the earlier reach of nutritional thresholds and/or the sequestration of potentially
368
limiting, nutrients required for reproduction (e.g. calcium: Reynolds et al., 2004; protein: Schoech et
369
al., 2004). 370 Despite earlier breeding in more artificialized areas, the ancestral reaction norm of breeding phenology
371
to temperature anomalies remained unchanged along the gradient of artificialization: blue and great
372
tits adjusted their breeding time to temperature nearly identically in artificialized areas as in core forest
373
(Figure 5). This reaction norm conservatism can be explained by the fact that, although prey abundance
374
is lower in urban areas (lower peak), prey emergences remain synchronized by temperature (Seress et
375
al., 2018). Furthermore, at least in our study design, artificialization tended to occur more in forested
376
areas than in farmlands (Figure 3). It implies that populations from anthropized sites, were indeed
377
surrounded by forest populations (exhibiting the ancestral reaction norm). Since urbanized
378
environments are often ecological traps for passerines (Björklund et al., 2010; Zuñiga-Palacios et al.,
379
2021), we can also assume that forest population act as demographic sources, providing recruits for
380
sinks in artificialized areas. Such an asymmetrical gene flow from ancestral, forest areas towards
381
artificialized areas is expected to prevent the evolution of locally adapted reaction norms (Lenormand,
382
2002). This proximity of forest source populations to sinks in artificialized areas could explain that the
383
reaction norm is conserved along the artificialization gradient for both species. Materials and methods
117 384 Despite earlier breeding in more artificialized areas, the ancestral reaction norm of breeding phenology
371
to temperature anomalies remained unchanged along the gradient of artificialization: blue and great
372
tits adjusted their breeding time to temperature nearly identically in artificialized areas as in core forest
373
(Figure 5). This reaction norm conservatism can be explained by the fact that, although prey abundance
374
is lower in urban areas (lower peak), prey emergences remain synchronized by temperature (Seress et
375
al., 2018). Furthermore, at least in our study design, artificialization tended to occur more in forested
376
areas than in farmlands (Figure 3). It implies that populations from anthropized sites, were indeed
377
surrounded by forest populations (exhibiting the ancestral reaction norm). Since urbanized
378
environments are often ecological traps for passerines (Björklund et al., 2010; Zuñiga-Palacios et al.,
379
2021), we can also assume that forest population act as demographic sources, providing recruits for
380
sinks in artificialized areas. Such an asymmetrical gene flow from ancestral, forest areas towards
381
artificialized areas is expected to prevent the evolution of locally adapted reaction norms (Lenormand,
382
2002). This proximity of forest source populations to sinks in artificialized areas could explain that the
383
reaction norm is conserved along the artificialization gradient for both species. 384 In summary, we showed apparent conservatism of phenological plasticity to local temperature across
385
two gradients of habitat anthropization in two forest songbird species. It suggests that the ancestral
386
reaction norm to temperature remained adaptive – or at least neutral – in most habitats. Only
387
agriculture altered the phenological response to spring warmth in the forest-specialist species. 388
Understanding the causal relationship of alteration of the reaction norm in farmlands will require
389 In summary, we showed apparent conservatism of phenological plasticity to local temperature across
385
two gradients of habitat anthropization in two forest songbird species. It suggests that the ancestral
386
reaction norm to temperature remained adaptive – or at least neutral – in most habitats. Only
387
agriculture altered the phenological response to spring warmth in the forest-specialist species. 388
Understanding the causal relationship of alteration of the reaction norm in farmlands will require
389 20 20 21
further knowledge about prey availability, predictability and species diets along the forest-farmland
390
gradient. Materials and methods
117 We investigated phenology in the blue and great tits because they are common research
391
models in ecology and evolution of phenotypic plasticity, due to their high abundance, widespread
392
distribution and ease to study in artificial nest-box populations (Blondel et al., 2006). But, they have
393
peculiar ecological requirements, usually tied to trees, as other secondary hole-nesting species. We
394
need to broaden the ecological and evolutionary diversity of studied seasonal species (Youngflesh et
395
al., 2023). Our modelling framework allows investigating the plasticity of breeding phenology in any
396
species, provided that juvenile emergence is unimodal and can be documented by the age-structure
397
of repeated samples of individuals throughout the reproductive season. Such a design is common in
398
standardized monitoring schemes of songbirds by capture, covering tens of species at continental scale
399
over decadal time (Robinson et al., 2014; Ahrestani et al., 2017; Morrison et al., 2022). Future studies
400
applying our approach to multispecies dataset will increase the robustness and generality of our
401
understanding of the range - and limits - of plastic compensation of global environmental changes by
402
seasonal organisms (DeWitt et al., 1998). 403
404
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species, provided that juvenile emergence is unimodal and can be documented by the age-structure
397
of repeated samples of individuals throughout the reproductive season. Such a design is common in
398
standardized monitoring schemes of songbirds by capture, covering tens of species at continental scale
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over decadal time (Robinson et al., 2014; Ahrestani et al., 2017; Morrison et al., 2022). Future studies
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applying our approach to multispecies dataset will increase the robustness and generality of our
401
understanding of the range - and limits - of plastic compensation of global environmental changes by
402
seasonal organisms (DeWitt et al., 1998). 403 further knowledge about prey availability, predictability and species diets along the forest-farmland
390
gradient. We investigated phenology in the blue and great tits because they are common research
391
models in ecology and evolution of phenotypic plasticity, due to their high abundance, widespread
392
distribution and ease to study in artificial nest-box populations (Blondel et al., 2006). But, they have
393
peculiar ecological requirements, usually tied to trees, as other secondary hole-nesting species. We
394
need to broaden the ecological and evolutionary diversity of studied seasonal species (Youngflesh et
395
al., 2023). Our modelling framework allows investigating the plasticity of breeding phenology in any
396
species, provided that juvenile emergence is unimodal and can be documented by the age-structure
397
of repeated samples of individuals throughout the reproductive season. Such a design is common in
398
standardized monitoring schemes of songbirds by capture, covering tens of species at continental scale
399
over decadal time (Robinson et al., 2014; Ahrestani et al., 2017; Morrison et al., 2022). Future studies
400
applying our approach to multispecies dataset will increase the robustness and generality of our
401
understanding of the range - and limits - of plastic compensation of global environmental changes by
402
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429
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432
Thirty-Year Study of Phenotypic and Genetic Variation of Blue Tits in Mediterranean Habitat
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434
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breeding phenology in urban birds than in their non-urban neighbours. Ecology Letters, 25(11),
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habitats and the timing of egg laying by Blue Tits. Journal of Field Ornithology, 80(2), 127–134. 442
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443 Bourgault, P., Perret, P., & Lambrechts, M. M. (2009). Food supplementation in distinct Corsican oak
441
habitats and the timing of egg laying by Blue Tits. Journal of Field Ornithology, 80(2), 127–134. 442
https://doi.org/10.1111/j.1557-9263.2009.00214.x
443
Bourret, A., Bélisle, M., Pelletier, F., & Garant, D. (2015). Multidimensional environmental influences
444
on timing of breeding in a tree swallow population facing climate change. Evolutionary
445
Applications, 8(10), 933–944. https://doi.org/10.1111/eva.12315
446
Britschgi, A., Spaar, R., & Arlettaz, R. (2006). Impact of grassland farming intensification on the
447
breeding ecology of an indicator insectivorous passerine, the Whinchat Saxicola rubetra:
448
Lessons for overall Alpine meadowland management. Biological Conservation, 130(2), 193–
449
205. https://doi.org/10.1016/j.biocon.2005.12.013
450 Brooks, S. P., & Gelman, A. (1998). General Methods for Monitoring Convergence of Iterative
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453 Brooks, S. P., & Gelman, A. (1998). General Methods for Monitoring Convergence of Iterative
451
Simulations. Journal of Computational and Graphical Statistics, 7(4), 434–455. 452
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453 Caizergues, A. E., Grégoire, A., & Charmantier, A. (2018). Urban versus forest ecotypes are not
454
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455
Sciences, 285(1882), 20180261. https://doi.org/10.1098/rspb.2018.0261
456 Caizergues, A. E., Grégoire, A., & Charmantier, A. (2018). Urban versus forest ecotypes are not
454
explained by divergent reproductive selection. Proceedings of the Royal Society B: Biological
455
Sciences, 285(1882), 20180261. https://doi.org/10.1098/rspb.2018.0261
456 Canale, C., & Henry, P. (2010). Adaptive phenotypic plasticity and resilience of vertebrates to increasing
457
climatic unpredictability. Climate Research, 43(1), 135–147. https://doi.org/10.3354/cr00897
458
Capilla-Lasheras, P., Thompson, M. J., Sánchez-Tójar, A., Haddou, Y., Branston, C. J., Réale, D.,
459
Charmantier, A., & Dominoni, D. M. (2022). A global meta-analysis reveals higher variation in
460
breeding phenology in urban birds than in their non-urban neighbours. Ecology Letters, 25(11),
461
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717
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meta-analysis. Science
of
The
Total
Environment,
780,
146559. 723
https://doi.org/10.1016/j.scitotenv.2021.146559
724
725
Acknowledgments
726
This study was made possible thanks to the many ringers that voluntarily monitor birds as part of the
727
French Constant ringing Effort Site scheme (Appendix S8), and to the continuous support of Muséum
728
National d’Histoire Naturelle and CNRS. We thank BNP Paribas foundation for funding and supporting
729
this project. A special thanks to M Queroué for her help with data preparation, and JY Barnagaud, LM
730
Chevin, M Van de Pol, V Purushotham and N Rigoudy for useful discussions. Materials and methods
117 Alexis Chaine’s and
731
Montpellier’s tit teams (especially A Charmantier, A Fargevieille, C de Franceschi, A Lucas and P
732
Giovannini) for collecting and transmitting nest boxes monitoring data which allowed us to compare
733
observed and estimated phenology in 5 blue and great tits populations. 734 Youngflesh, C., Montgomery, G. A., Saracco, J. F., Miller, D. A. W., Guralnick, R. P., Hurlbert, A. H.,
717
Siegel, R. B., LaFrance, R., & Tingley, M. W. (2023). Demographic consequences of phenological
718
asynchrony for North American songbirds. Proceedings of the National Academy of Sciences,
719
120(28), e2221961120. https://doi.org/10.1073/pnas.2221961120
720
Zuñiga-Palacios, J., Zuria, I., Castellanos, I., Lara, C., & Sánchez-Rojas, G. (2021). What do we know (and
721
need to know) about the role of urban habitats as ecological traps? Systematic review and
722
meta-analysis. Science
of
The
Total
Environment,
780,
146559. 723
https://doi.org/10.1016/j.scitotenv.2021.146559
724
725 This study was made possible thanks to the many ringers that voluntarily monitor birds as part of the
727
French Constant ringing Effort Site scheme (Appendix S8), and to the continuous support of Muséum
728
National d’Histoire Naturelle and CNRS. We thank BNP Paribas foundation for funding and supporting
729
this project. A special thanks to M Queroué for her help with data preparation, and JY Barnagaud, LM
730
Chevin, M Van de Pol, V Purushotham and N Rigoudy for useful discussions. Alexis Chaine’s and
731
Montpellier’s tit teams (especially A Charmantier, A Fargevieille, C de Franceschi, A Lucas and P
732
Giovannini) for collecting and transmitting nest boxes monitoring data which allowed us to compare
733
observed and estimated phenology in 5 blue and great tits populations. 734 Paul Cuchot led formal analysis and writing of the original draft. Paul Cuchot, Céline Teplitsky and
736
Pierre-Yves Henry shared conceptualization, planning the analysis, and editing drafts. Olivier Dehorter
737
provided the data, and Timothée Bonnet helped with the analysis. All authors commented on the
738
manuscript. 739 Paul Cuchot led formal analysis and writing of the original draft. Paul Cuchot, Céline Teplitsky and
736
Pierre-Yves Henry shared conceptualization, planning the analysis, and editing drafts. Olivier Dehorter
737
provided the data, and Timothée Bonnet helped with the analysis. All authors commented on the
738
manuscript. 739 34 Conflicts of interests
740
The authors declare no competing interests. Materials and methods
117 741
Data availability
742
We confirm that, should the manuscript be accepted, the data supporting the results will be archived
743
in Dryad. 744 Conflicts of interests
740 The authors declare no competing interests. 741 The authors declare no competing interests. 741 Data availability
742 Data availability
742 We confirm that, should the manuscript be accepted, the data supporting the results will be archived
743
in Dryad. 744 We confirm that, should the manuscript be accepted, the data supporting the results will be archived
743
in Dryad. 744 35
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https://openalex.org/W2074323777
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https://oasis.postech.ac.kr/bitstream/2014.oak/14951/1/OAIR003473.pdf
|
English
| null |
Cdk5 Phosphorylates Dopamine D2 Receptor and Attenuates Downstream Signaling
|
PloS one
| 2,013
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cc-by
| 8,261
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Introduction Dopamine signaling is involved in various brain functions
including motor coordination, mood control and reward mecha-
nisms [1]. A major component of dopamine signaling in
vertebrates is exerted by striatal medium spiny neurons (MSNs)
which selectively express a subset of dopamine receptors and
receive dopaminergic input mainly from the ventral tegmental
area (VTA) and substantia nigra (SN) [2]. Dopamine receptors are
G protein-coupled receptors (GPCR) with seven transmembrane
domains and consist of two subtypes, D1-like and D2-like
receptors, that mediate reciprocal actions in dopamine signaling
[1]. For example, dopamine D1-like receptors (D1, D5) activate
adenylyl cyclase through Gas and increase the intracellular level of
cAMP, but dopamine D2-like receptors (D2, D3, D4) inhibit
adenylyl cyclase through Gai and decrease the intracellular level of
cAMP [1,3]. Converging lines of evidence indicate that multiple posttrans-
lational modifications are involved in the fine-tuning of DRD2
activity. Extensive glycosylation of DRD2 was revealed in early
photo-affinity labeling studies [7], and disulfide bond formation
within DRD2 was also identified as an important modification for
ligand binding [8]. Furthermore, phosphorylation sites of DRD2
were initially identified by in vitro assay with radioisotopes,
providing routes for various regulatory pathways mediated by
various kinases [9]. Indeed, protein kinase C (PKC) regulates
DRD2-mediated mobilization of intracellular calcium and mod-
ulates the interaction of DRD2 with cytoskeletal proteins [10]. Phosphorylation by GPCR kinase 2 (GRK2) regulates agonist-
induced resensitization patterns of DRD2 [11]. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/
threonine kinase that has preferential activity due to brain-specific
expression of its essential activators, p35 and p39 [12]. Cdk5 is
involved
in
various
neuronal
processes
including
neuronal
migration and axon guidance, and Cdk5 and p35 null mice show
defects in cortical layering [13]. Recently, it was shown that
phosphorylation of WAVE1 and ephexin by Cdk5 regulates
dendritic spine morphogenesis [14]. Furthermore, Cdk5 also
regulates surface expression levels of the NMDA receptor, NR2B,
and NR2A-mediated NMDA currents [15,16]. It is noteworthy
that multiple pieces of evidence suggest an intimate relationship Among dopamine receptors, the D2 receptor (DRD2) is
implicated in the pathophysiology of multiple major psychiatric
disorders including schizophrenia and drug addiction [4], such
that many antipsychotic drugs at least partially target DRD2. It is
also known that DRD2 activity correlates well with the behavioral
consequences of drugs of abuse in animal models [5]. Abstract The dopamine D2 receptor (DRD2) is a key receptor that mediates dopamine-associated brain functions such as mood,
reward, and emotion. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase whose function has
been implicated in the brain reward circuit. In this study, we revealed that the serine 321 residue (S321) in the third
intracellular loop of DRD2 (D2i3) is a novel regulatory site of Cdk5. Cdk5-dependent phosphorylation of S321 in the D2i3
was observed in in vitro and cell culture systems. We further observed that the phosphorylation of S321 impaired the
agonist-stimulated surface expression of DRD2 and decreased G protein coupling to DRD2. Moreover, the downstream
cAMP pathway was affected in the heterologous system and in primary neuronal cultures from p35 knockout embryos likely
due to the reduced inhibitory activity of DRD2. These results indicate that Cdk5-mediated phosphorylation of S321 inhibits
DRD2 function, providing a novel regulatory mechanism for dopamine signaling. Y-U, Kim D-K, Lee S, Kwak Y, et al. (2013) Cdk5 Phosphorylates Dopamine D2 Receptor and Attenuates Downstream Signaling. PLoS
0.1371/journal.pone.0084482 Editor: James Porter, University of North Dakota, United States of America Editor: James Porter, University of North Dakota, United States of America Editor: James Porter, University of North Dakota, United States of America Received May 20, 2013; Accepted November 14, 2013; Published December 31, 2013 Received May 20, 2013; Accepted November 14, 2013; Published December 31, 2013 Copyright: 2013 Jeong et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the grants (NRF-2012R1A2A2A01012923 and NRF-2012R1A4A1028200) from the Korean government (MSIP) and also
supported under the framework of international cooperation program managed by NRF of Korea (2012K2A1A2033117) and the Korea Brain Research Institute
(KBRI) Basic Research Program of MSIP (2031-415). SKP was a recipient of the 2004 and 2006 National Alliance for Research on Schizophrenia and Depression
(NARSAD) Young Investigator Awards. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: skpark@postech.ac.kr regulatory mechanisms that confer heterogeneity and complexity
to DRD2 properties are not completely understood. Cdk5 Phosphorylates Dopamine D2 Receptor and
Attenuates Downstream Signaling Jaehoon Jeong1, Young-Un Park1, Dae-Kyum Kim1, Saebom Lee1, Yongdo Kwak1, Seol-Ae Lee1,
Haeryun Lee3, Yoo-Hun Suh4, Yong Song Gho1, Daehee Hwang2, Sang Ki Park1* 1 Department of Life Sciences, Pohang University of Science and Technology, Pohang, Republic of Korea, 2 School of Interdisciplinary Bioscience and Bioengineering,
Pohang University of Science and Technology, Pohang, Republic of Korea, 3 Division of Integrative Bioscience and Biotechnology, Pohang University of Science and
Technology, Pohang, Republic of Korea, 4 Korea Brain Research Institute, Daegu, Republic of Korea December 2013 | Volume 8 | Issue 12 | e84482 In Vitro Kinase Assay IP-linked in vitro kinase assay was performed as following. One
whole mouse brain was lysed in 3 mL erythrocytes lysis buffer
(ELB) (50 mM Tris (pH 8.0), 250 mM NaCl, 5 mM EDTA, 0.1%
NP-40)
by
20
strokes
of
a
Dounce
homogenizer
to
get
homogenized brain lysates. The lysates were incubated on ice GST Pull-down Assay 10 mg of purified GST and GST–D2i3 were incubated with rat
brain lysate for 1.5 h at 4uC. 30 mL of glutathione (GSH)-
conjugated Sepharose 4B beads (17-0756-01, GE Healthcare)
equilibrated with lysis buffer was added and incubated for
additional
1 h. Beads
were
collected
by
centrifugation
at
2,0006g and rinsed with lysis buffer 4 times [22,23]. Precipitates
were analyzed by Western blotting using anti-Cdk5 and anti-p35
antibodies. Immunoprecipitation Immunoprecipitation was performed on cell lysates in ELB lysis
buffer. Anti-GFP antibody was added to the lysates and incubated
for 3 h at 4uC. Immunocomplexes were purified using protein-A
agarose. The precipitates were incubated with SDS sample
loading buffer for 30 min at 37uC, and subjected to SDS-PAGE
and Western blots. Plasmid Constructs Human DRD2 long isoform in an EGFP-N1 plasmid vector
and the third intracellular loop of DRD2 (212–373 amino acid
residues including the 29 additional amino acid residues unique to
DRD2 long isoform) in a pFLAG-CMV-2 plasmid vector were
used. Human Cdk5 was inserted in a pCMV-HA plasmid vector
and human p35 was inserted in a pcDNA 3.1 plasmid vector. Human Cdk5 was inserted under a cytomegalovirus (CMV)
promoter along with human p35 in a pcDNA 3.1 vector to make a
dual expression construct (Cdk5/p35) for immunocytochemistry,
receptor internalization assay, [35S]-GTPcS binding assay, radio-
ligand binding assay and cAMP enzyme immunoassay. Immunocytochemistry Transfected HEK 293 cells and striatal neurons cultured on
coverslips were washed once with phosphate buffered saline (PBS)
and fixed by immersion in cold 4% paraformaldehyde/PBS for
30 min. Primary antibody was diluted in the blocking solution (2%
horse serum and 1% Triton X-100 in PBS). Alexafluor-647-
conjugated anti-mouse antibody (A20990, Invitrogen) and Alexa-
fluor-568-conjugated anti-rabbit antibody (A11011, Invitrogen)
were used as secondary antibodies. Hoechst were used for nucleus
staining. Images were obtained by confocal microscopy (Olympus,
FluoView-1000). Materials and Methods The recombinant GST-D2i3 protein was analyzed by LC-MS/
MS following IP-linked in vitro kinase assay. We performed peptide
identification of LC-MS/MS data using X!!Tandem (version Dec-
01-2008). Each RAW data file was first converted to mzXML
using the trans-proteomic pipeline (TPP; version 4.3). MS/MS
scans in the converted mzXMLs were then subjected to search
against the UniProt mouse protein sequence database (release
2010_07) including GST-D2i3 sequence using X!!Tandem. The
tolerance was set to 3 Da for precursor ions and 2 Da for fragment
ions. Enzyme specificity for trypsin was used. Variable modifica-
tion options were used for the carbamidomethylation of cysteine
(57.021 Da),
the
oxidation
of
methionine
(15.995 Da),
the
hydrolysis of asparagine (0.987 Da) and the phosphorylation of
serine (79.966 Da). Antibodies Anti-rabbit serums were raised against peptides containing
phospho-serine 321 (pS321) of the third intracellular loop of
DRD2
(D2i3). Phospho-peptide,
CNPDpSPAKPEK
(PEP-
TRON), was used to make a peptide-conjugated column for
affinity purification (20401, PIERCE). Anti-pS321 antibody was
enriched by an affinity purification system following the manu-
facturer’s instruction. Purified phospho-antibody was stored in
PBS with 0.1% sodium azide and 0.1% gelatin. Anti-mouse anti-
Cdk5 antibody (sc-249) and anti-rabbit anti-p35 antibody (sc-820)
were used for the Western blotting and immunocytochemistry of
Cdk5/p35. Anti-mouse anti-GFP antibody (sc-9996) was used for
the immunoprecipitation and Western blotting of DRD2-GFP. Anti-rabbit anti-FLAG antibody (sc-807), anti-rabbit anti-HA
antibody (sc-805), anti-mouse anti-GST antibody (sc-138), and
anti-mouse anti-GAPDH antibody (sc-32293) were purchased
from Santa Cruz Biotechnologies. Introduction Antidepres-
sants and mood stabilizer efficacy have also been linked to
alterations in the cell surface expression of DRD2 or downstream
intracellular signaling mediated by PKA, ERK and GSK3 [1,4,6]. Despite these critical roles for DRD2 in the brain, the detailed December 2013 | Volume 8 | Issue 12 | e84482 1 PLOS ONE | www.plosone.org DRD2 as a Novel Substrate of Cdk5 between Cdk5 and the dopamine system. Cdk5 phosphorylates
tyrosine hydroxylase (TH), regulating its stability, and thus
maintaining dopaminergic homeostasis [17]. In postsynaptic
neurons, when the T75 residue of dopamine and cyclic-AMP
regulated phosphoprotein-32kD (DARPP-32) is phosphorylated by
Cdk5, it can inhibit PKA activity and thus antagonize dopamine
DRD1-mediated PKA signaling [18]. Interestingly, when cocaine,
an indirect agonist of dopamine receptors, is administrated
chronically in rats, mRNA and protein levels of Cdk5 increase
in medium spiny neurons [19]. Collectively, Cdk5 appears to be
involved in drug-induced synaptic adaptations. In this study, we
show a functional interaction of DRD2 and Cdk5 that further
extends the role of Cdk5 in dopamine signaling. for 30 min, sonicated, and centrifuged at 16,000 6 g for 10 min. The supernatants were immunoprecipitated with anti-rabbit anti-
p35 antibody to obtain an active Cdk5/p35 complex. Cdk5/p35
complex and purified GST fusion protein was mixed with
adenosine 59-triposphate, [c-32P] (NEG-502H, PerkinElmer) and
incubated in kinase buffer (30 mM HEPES (pH 7.2), 10 mM
MgCl2, 0.2 mM DTT) for 1 h at room temperature [18,21]. Purified Cdk5/p25 complex (14–516, Millipore) was also used for
in vitro kinase assay as described above. The 26 sample loading
buffer was added to the reaction mixture and boiled at 100uC. The
samples were then subjected to SDS-PAGE and the dried gel was
assessed by autoradiography. Liquid Chromatography (LC)-Mass Spectrometry (MS)/MS
Analysis Animals The p35 knockout mouse was a kind gift from Dr. Katsuhiko
Mikoshiba at RIKEN Brain Science Institute in Japan and used
for primary neuron culture. Primer sets for genotyping were 59-
GGTCTCCTCTTCTGTCAAGAAG,
59-GCTCTGCTAGA-
CACATACTGTAC and 59- TCCATCT GCACGAGACTAGT
as previously described [20]. ICR mice and Sprague Dawley rats
were used for brain lysate preparation. All animal procedures were
approved by the Pohang University of Science and Technology
Institutional Animal Care and Use Committee. Radioligand Binding Assay Prepared cell membranes were incubated with 0.01 nM [3H]-
spiperone (NET-565, PerkinElmer) and increasing concentrations
of quinpirole (Q102, Sigma) for 30 min in the assay buffer (25 mM
HEPES (pH 7.5), 1.5 mM MgCl2, 100 mM NaCl, 1 mM EDTA). Membranes were filtered on a GF/B filter (Millipore) and washed
3 times with washing buffer (50 mM Tris-HCl (pH 7.4), 100 mM
NaCl). The reaction was terminated by rapid filtration through
GF/C filters. Residual radioactivity was measured using a liquid
scintillation counter [27–29]. [35S]-GTPcS Binding Assay c
Cell membrane fractions were pre-incubated with 1 mM
quinpirole (Q102, Sigma) in the assay buffer (25 mM HEPES
(pH 7.5), 1.5 mM MgCl2, 100 mM NaCl, 1 mM EDTA and
0.01 mM GDP) for 10 min. [35S]-GTPcS (NET-030H, PerkinEl-
mer) was added to the final concentration of 3 nM in 30 mL and
further incubated for 90 min. 170 mL of ice-cold buffer (10 mM
Tris-HCl (pH 8.0), 100 mM NaCl, 10 mM MgCl2, and 0.1 mM
GTP) was added to stop the reaction. Membranes were filtered on
a GF/B filter (Millipore) and washed 3 times with washing buffer
(50 mM Tris-HCl (pH 7.4), 100 mM NaCl). Filters were dried
and radioactivity was measured using the scintillation counter
[25,26]. Cdk5 Phosphorylates Serine 321 in the Third Intracellular
Loop of DRD2 in Cells Cdk5 Phosphorylates Serine 321 in the Third Intracellular
Loop of DRD2 in Cells To identify the phosphorylation of serine 321, we raised
antibody specific for phospho-serine 321 (pS321). Samples from
the IP-linked in vitro kinase assay were analyzed by Western
blotting using anti-pS321 antibody. Blots showed a distinct band
in the kinase reaction that was dependent on GST-D2i3 (Fig. 2A). To verify the potential phosphorylation of serine 321 in DRD2 by
Cdk5 in cells, anti-GFP immunoprecipitates from HEK 293 cells
expressing DRD2-GFP and DRD2 S321A-GFP with or without
HA-Cdk5 and p35 were analyzed by Western blotting using anti-
GFP and anti-pS321 antibodies. Characteristic smeared band
signals by anti-GFP antibody that are known to be due to excessive
glycosylation of DRD2 are observed only in the presence of
DRD2-GFP, and anti-pS321 antibody detected similar DRD2
signals only with Cdk5/p35 co expression (Fig. 2B) [7]. To further
verify the phosphorylation of serine 321 by Cdk5, D2i3 (FLAG-
D2i3) and mutant form of D2i3 (FLAG-D2i3 S321A) were
generated. FLAG-D2i3 and FLAG-D2i3 S321A expressed with or
without HA-Cdk5 and p35 in HEK 293 cells were analyzed by an Cell Membrane Preparation Confluent cells in 100 mm culture-dishes after transfection were
washed with ice-cold PBS and harvested in 1 mL HME buffer
(25 mM
HEPES
(pH 7.5),
2 mM
MgCl2,
1 mM
EDTA). Homogenized lysates were centrifuged with 5006g for 15 min
and
the
supernatants
were
subsequently
centrifuged
with
36,0006g for 30 min. Pellets re-suspended in HME buffer were
used for assays. p
(
g
)
To assess the capacity of Cdk5 to phosphorylate D2i3, we
performed IP-linked in vitro kinase assays using an active Cdk5/
p35 complex enriched from mouse brain lysate by p35 immuno-
precipitation with purified recombinant GST-D2i3 (amino acid
residues 212–373) proteins as the substrates. We observed
phosphorylation signals in the purified GST-D2i3 and GST-
D2i3 S297A proteins, but the signal was significantly diminished
using GST-D2i3 S321A (Fig. 1B). To further verify phosphory-
lation of serine 321 in the GST-D2i3, we performed LC-MS/MS
analysis of the samples from IP-linked in vitro kinase assays using
LTQ XL mass spectrometry. Consistently, phospho-peptides
corresponding to the mass of phospho-serine 321 peptides were
recovered (Fig. 1C). Considering that the data-dependent acqui-
sition during LC-MS/MS analysis tends to detect abundant
proteins in the sample [31], this data suggests that the serine 321
residue is the dominant phosphorylation site of Cdk5 in the D2i3
region. To prove direct phosphorylation of serine 321 in the GST-
D2i3 by Cdk5, in vitro kinase assay using purified Cdk5/GST-p25
complex with purified recombinant GST-D2i3 proteins was
performed. We identified a significant phosphorylation signal in
the GST-D2i3 that was absent in the GST-D2i3 S321A (Fig. 1D). Taken together, these results indicate that the D2i3 S321 residue is
a preferential target for Cdk5-mediated phosphorylation. Cdk5 Phosphorylates Serine 321 in the Third Intracellular
Loop of DRD2 in vitro To identify novel Cdk5 substrates, we performed a systematic
search using (S/T)PX(K/H/R) as the Cdk5 recognition consensus
sequences [30] and identified DRD2 as a candidate substrate. The
consensus sequence, including serine 321, is located in the third
intracellular loop of DRD2 (D2i3) where various regulatory
mechanisms have been implicated [3,10,11]. The sequence is
evolutionarily conserved in DRD2 in vertebrates, implying a
functional importance of the residue (Fig. 1A). cAMP Enzyme Immunoassay Transfected HEK 293 cells were pretreated with 10 mM
rolipram (R6520, Sigma) for 1 h, and then treated with 0.1 mM
forskolin
(F6886,
Sigma)
and
increasing
concentrations
of
quinpirole (Q102, Sigma) for 30 min. Primary cultured striatal
neurons were treated with 10 mM rolipram for 1 h, and then
1 mM dopamine for 1 h [22]. Cell lysates were prepared with 0.1
M HCl and cAMP levels were detected by cAMP enzyme
immunoassay kit (Sapphire Bioscience) following the manufactur-
er’s instruction. Receptor Internalization Assay 24 h after transfection, cells were treated with 1 mM quinpirole
(Q102, Sigma) for 30 min and 90 min at 37uC. Cells were re- PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e84482 2 DRD2 as a Novel Substrate of Cdk5 (MEM) (11095, Invitrogen) containing 0.25% trypsin (T4549-100,
Sigma) and 0.1% DNase I for 6 min at 37uC. Cells were re-
suspened in the plating media (MEM with 0.01 M HEPES
(pH 7.4) and 10% (vol/vol) horse serum (16050-122, GIBCO)). Neurons were cultured for 7 days in vitro (DIV 7) in MEM with
B27 supplement (17504-044, Invitrogen) before being applied to
cAMP enzyme immunoassays. suspended in 2 mL cold PBS and 200 mL aliquots were used for
each reaction. Drug treatments were carried out at room
temperature for 3 h at the following concentrations; 3 nM [3H]-
spiperone (NET-565, PerkinElmer), 3 mM sulpiride (895, TO-
CRIS), 10 mM haloperidol (H1512, Sigma). Hydrophobic [3H]-
spiperone was used to label total expressed receptors and
hydrophilic sulpiride was used to replace membranous receptor-
bound [3H]-spiperone signals. Membrane-associated receptor
signals were calculated by subtracting intracellular receptor values
from the total expressed receptor values. Cells were filtered on a
GF/B (Millipore) filter and washed 3 times with washing buffer
(50 mM Tris-HCl (pH 7.4), 100 mM NaCl). Filters were dried out
and residual radioactivity was measured using a liquid scintillation
counter [24]. Results Cdk5 Phosphorylates Serine 321 in the Third Intracellular
Loop of DRD2 in vitro Cdk5/p35 Complex and DRD2 are Physically Associated Cdk5/p35 Complex and DRD2 are Physically Associated
We investigated the potential physical interaction between the
Cdk5/p35 complex and DRD2 because many Cdk5 substrates are
known to be physically associated with Cdk5/p35 complex
[23,34,35]. First, the GST pull-down experiment was performed. Purified recombinant GST-D2i3 protein was incubated with rat
brain lysate and GST pull-down precipitates were analyzed for Primary Cultured Striatal Neuron (D) In vitro kinase assay with purified Cdk5/GST-p25 complex using GST-D2i3 and GST-D2i3 mutant proteins. Phosphorylated proteins
were shown in an autoradiograph, along with Coomassie brilliant blue staining. Arrowhead indicates radioactive signal corresponding GST-D2i3 and
open arrowhead indicates radioactive signals from GST-p25. doi:10.1371/journal.pone.0084482.g001 ylates serine 321 in the third intracellular loop of DRD2 in vitro. (A) Amino acid sequence alignment showin Figure 1. Cdk5 phosphorylates serine 321 in the third intracellular loop of DRD2 in vitro. (A) Amino acid sequence alignment showing
conserved regions of the DRD2 from various species (shaded). The potential Cdk5 phosphorylation site is indicated by an asterisk. (B) IP-linked in vitro
kinase assay with recombinant GST-D2i3 and GST-D2i3 mutant proteins. Cdk5/p35 complex enriched from mouse brain extract by anti-p35
immunoprecipitation was used for kinase reactions. An autoradiograph of phosphorylated proteins is shown along with Coomassie brilliant blue
staining of the same gel. Arrowhead indicates radioactive signal corresponding to GST-D2i3s and open arrowhead indicates radioactive signals from
p35. (C) MS/MS spectrum of the phosphorylated peptide fragment of D2i3. The theoretical fragmentation patterns are shown below the spectrum. Among all the fragment ions, the detected y- and b-ions are denoted in the spectrum. The y6 and y7 ions strongly indicate the phosphorylation of
serine 321. (D) In vitro kinase assay with purified Cdk5/GST-p25 complex using GST-D2i3 and GST-D2i3 mutant proteins. Phosphorylated proteins
were shown in an autoradiograph, along with Coomassie brilliant blue staining. Arrowhead indicates radioactive signal corresponding GST-D2i3 and
open arrowhead indicates radioactive signals from GST-p25. doi:10.1371/journal.pone.0084482.g001 Western blotting. As shown in Fig. 3A, endogenous Cdk5 and p35
were identified in the pull-down precipitates, indicating a physical
interaction between DRD2 and the Cdk5/p35 complex (Fig. 3A). Moreover, HA-Cdk5 and p35 were detected in the anti-GFP
immunoprecipitates from HEK 293 cell lysates expressing DRD2-
GFP and Cdk5/p35 (Fig. 3B). In addition, we performed immuno-
cytochemicalanalysesandobservedthatDRD2-GFP,HA-Cdk5and
p35 show significant co-localization signals at the membranous area
of HEK 293 cells (Fig. 3C, upper panels). We also investigated co-
localization of DRD2 and Cdk5/p35 in the neuronal context. Consistently, DRD2-GFP also showed significant co-localization
with endogenous Cdk5 and p35 in the cultured striatal neurons
(DIV7), further supporting functional links between DRD2 and
Cdk5/p35 (Fig. 3C, bottom panels). The results indicate that DRD2
and Cdk5/p35 can form a complex and thus, support the notion that
DRD2 is a physiological substrate of Cdk5. Western blotting. As shown in Fig. Primary Cultured Striatal Neuron Striatal area was isolated from the mouse embryonic brain
(E15). Dissected tissue was dissociated in minimal essential media PLOS ONE | www.plosone.org December 2013 | Volume 8 | Issue 12 | e84482 3 DRD2 as a Novel Substrate of Cdk5 Figure 1. Cdk5 phosphorylates serine 321 in the third intracellular loop of DRD2 in vitro. (A) Amino acid sequence alignment showing
conserved regions of the DRD2 from various species (shaded). The potential Cdk5 phosphorylation site is indicated by an asterisk. (B) IP-linked in vitro
kinase assay with recombinant GST-D2i3 and GST-D2i3 mutant proteins. Cdk5/p35 complex enriched from mouse brain extract by anti-p35
immunoprecipitation was used for kinase reactions. An autoradiograph of phosphorylated proteins is shown along with Coomassie brilliant blue
staining of the same gel. Arrowhead indicates radioactive signal corresponding to GST-D2i3s and open arrowhead indicates radioactive signals from
p35. (C) MS/MS spectrum of the phosphorylated peptide fragment of D2i3. The theoretical fragmentation patterns are shown below the spectrum. Among all the fragment ions, the detected y- and b-ions are denoted in the spectrum. The y6 and y7 ions strongly indicate the phosphorylation of
serine 321. (D) In vitro kinase assay with purified Cdk5/GST-p25 complex using GST-D2i3 and GST-D2i3 mutant proteins. Phosphorylated proteins
were shown in an autoradiograph, along with Coomassie brilliant blue staining. Arrowhead indicates radioactive signal corresponding GST-D2i3 and
open arrowhead indicates radioactive signals from GST-p25. doi:10.1371/journal.pone.0084482.g001 igure 1. Cdk5 phosphorylates serine 321 in the third intracellular loop of DRD2 in vitro. (A) Amino acid s Figure 1. Cdk5 phosphorylates serine 321 in the third intracellular loop of DRD2 in vitro. (A) Amino acid sequence alignment showing
conserved regions of the DRD2 from various species (shaded). The potential Cdk5 phosphorylation site is indicated by an asterisk. (B) IP-linked in vitro
kinase assay with recombinant GST-D2i3 and GST-D2i3 mutant proteins. Cdk5/p35 complex enriched from mouse brain extract by anti-p35
immunoprecipitation was used for kinase reactions. An autoradiograph of phosphorylated proteins is shown along with Coomassie brilliant blue
staining of the same gel. Arrowhead indicates radioactive signal corresponding to GST-D2i3s and open arrowhead indicates radioactive signals from
p35. (C) MS/MS spectrum of the phosphorylated peptide fragment of D2i3. The theoretical fragmentation patterns are shown below the spectrum. Among all the fragment ions, the detected y- and b-ions are denoted in the spectrum. The y6 and y7 ions strongly indicate the phosphorylation of
serine 321. Primary Cultured Striatal Neuron 3A, endogenous Cdk5 and p35
were identified in the pull-down precipitates, indicating a physical
interaction between DRD2 and the Cdk5/p35 complex (Fig. 3A). Moreover, HA-Cdk5 and p35 were detected in the anti-GFP
immunoprecipitates from HEK 293 cell lysates expressing DRD2-
GFP and Cdk5/p35 (Fig. 3B). In addition, we performed immuno-
cytochemicalanalysesandobservedthatDRD2-GFP,HA-Cdk5and
p35 show significant co-localization signals at the membranous area
of HEK 293 cells (Fig. 3C, upper panels). We also investigated co-
localization of DRD2 and Cdk5/p35 in the neuronal context. Consistently, DRD2-GFP also showed significant co-localization
with endogenous Cdk5 and p35 in the cultured striatal neurons
(DIV7), further supporting functional links between DRD2 and
Cdk5/p35 (Fig. 3C, bottom panels). The results indicate that DRD2
and Cdk5/p35 can form a complex and thus, support the notion that
DRD2 is a physiological substrate of Cdk5. SDS-gel mobility shift assay. A significant Cdk5-dependent
mobility shift was observed for FLAG-D2i3, but not for FLAG-
D2i3 S321A (Fig. 2C). We also assessed the phosphorylation level
of DRD2 at Ser321 upon agonist stimulation. HEK 293 cells
expressing DRD2-GFP and Cdk5/p35 complex were stimulated
by quinpirole, and anti-GFP immunoprecipitates from the cell
lysates were analyzed by Western blotting using anti-GFP and
anti-pS321 antibodies. We found that Cdk5-mediated phosphor-
ylation of DRD2 at Ser321 was not affected by agonist
stimulation, which appears different from GRK- and PKC-
mediated phosphorylations (Fig. 2D) [32,33]. Together, these
results indicate that Cdk5 can phosphorylate the serine 321
residue of DRD2 in the cellular environment. Cdk5-mediated Phosphorylation of DRD2 Attenuates
Receptor Activity It has been reported that phosphorylation modulates critical
properties of GPCRs such as G protein coupling, receptor December 2013 | Volume 8 | Issue 12 | e84482 PLOS ONE | www.plosone.org 4 DRD2 as a Novel Substrate of Cdk5 Figure 2. Cdk5 phosphorylates serine 321 in the third intracellular loop of DRD2 in cells. Cdk5-mediated phosphorylation of serine 321
was analyzed using anti-pS321 antibody. (A) Samples from IP-linked in vitro kinase assay using GST-D2i3 proteins were analyzed by Western blotting
(WB) with indicated antibodies. Arrowheads indicate GST-D2i3s. (B) DRD2-GFP and DRD2 S321A-GFP was expressed with or without HA-Cdk5 and p35
in HEK 293 cells. Anti-GFP immunoprecipitates were analyzed by Western blotting using anti-GFP and anti-pS321 antibodies. The bracket indicates
DRD2 signals and open arrowhead indicates nonspecific signals from the anti-GFP immunoprecipitates. ‘% input’ is % volume of total lysate for an IP
reaction. Weak endogenous Cdk5 signals were indicated by asterisks. (C) Gel mobility shift assay. HEK 293 cells transfected as indicated were analyzed
by Western blotting. (D) Transfected HEK 293 cells were treated with quinpirole and anti-GFP immunoprecipitates were analyzed by Western blotting
with anti-GFP and anti-pS321 antibodies. Open arrowhead indicates nonspecific signals from anti-GFP immunoprecipitates. doi:10.1371/journal.pone.0084482.g002 Figure 2. Cdk5 phosphorylates serine 321 in the third intracellular loop of DRD2 in cells. Cdk5-mediated phosphorylation of serine 321
was analyzed using anti-pS321 antibody. (A) Samples from IP-linked in vitro kinase assay using GST-D2i3 proteins were analyzed by Western blotting
(WB) with indicated antibodies. Arrowheads indicate GST-D2i3s. (B) DRD2-GFP and DRD2 S321A-GFP was expressed with or without HA-Cdk5 and p35
in HEK 293 cells. Anti-GFP immunoprecipitates were analyzed by Western blotting using anti-GFP and anti-pS321 antibodies. The bracket indicates
DRD2 signals and open arrowhead indicates nonspecific signals from the anti-GFP immunoprecipitates. ‘% input’ is % volume of total lysate for an IP
reaction. Weak endogenous Cdk5 signals were indicated by asterisks. (C) Gel mobility shift assay. HEK 293 cells transfected as indicated were analyzed
by Western blotting. (D) Transfected HEK 293 cells were treated with quinpirole and anti-GFP immunoprecipitates were analyzed by Western blotting
with anti-GFP and anti-pS321 antibodies. Open arrowhead indicates nonspecific signals from anti-GFP immunoprecipitates. doi:10.1371/journal.pone.0084482.g002 Figure 2. Cdk5 phosphorylates serine 321 in the third intracellular loop of DRD2 in cells. Cdk5-mediated Phosphorylation of DRD2 Attenuates
Receptor Activity Cdk5-mediate
l
d
b d
l
f
l k d
k expressing cells were reduced at 30 min and recovered at 90 min,
regardless of the co-expression with Cdk5/p35. Previous studies
have shown that the internalized DRD2 recycles back to the
plasma membrane upon prolonged agonist stimulation [11]. Thus
it appears that Cdk5-mediated phosphorylation of DRD2 is
involved in the resensitization processes following agonist-induced
DRD2 internalization. internalization, intracellular localization, and association with
modulator proteins [9,11,24]. Agonist-induced receptor internal-
ization is a critical regulatory process of signal transduction. We
investigated Cdk5-mediated modulation of DRD2 internalization. HEK 293 cells expressing DRD2-GFP and DRD2 S321A-GFP
with or without Cdk5/p35 were incubated with 1 mM quinpirole
to induce agonist-stimulated DRD2 internalization (Fig. 4A). [3H]-
spiperone signals of DRD2-GFP expressing cells were significantly
reduced at 30 min quinpirole treatment and recovered at 90 min. Interestingly, [3H]-spiperone signals of DRD2-GFP and Cdk5/
p35 expressing cells were also reduced at 30 min quinpirole
treatment but not recovered at 90 min (Fig. 4A, second section). On the other hand, [3H]-spiperone signals of DRD2 S321A-GFP We further evaluated a potential change of agonist-stimulated G
protein coupling
to DRD2 associated with Cdk5-mediated
phosphorylation
using
[35S]-GTPcS
binding
assay
[25,26]. DRD2-GFP and DRD2 S321A-GFP with or without Cdk5/p35
were expressed in HEK 293 cells. Membranes were prepared and
stimulated with 1 mM quinpirole and further allowed [35S]-GTPcS December 2013 | Volume 8 | Issue 12 | e84482 PLOS ONE | www.plosone.org PLOS ONE | www.plosone.org 5 incorporation. DRD2-GFP and Cdk5/p35 expressing cell mem-
Competitive
binding
of
[3H]-spiperone
upon
treatment
of
Figure 3. Cdk5/p35 can form a complex with DRD2. (A) GST pull-down assay using purified recombinant GST-D2i3 protein with rat brain
extract. GST pull-down precipitates were subjected to Western blotting analyses. ‘Bead’ indicates the pull-down precipitate without GST proteins. (B)
Immunoprecipitation of DRD2 and Cdk5/p35 complex. Anti-GFP IP from lysates from transfected cells were subjected to Western blotting analyses. The bracket indicates DRD2 signals and open arrowhead indicates nonspecific signals from the anti-GFP immunoprecipitates. An overexposed blot
for inputs is also shown in the right. (C) Immunocytochemical analyses of DRD2 and Cdk5/p35. HEK 293 cells expressing DRD2-GFP and Cdk5/p35
were stained with anti-Cdk5 and anti-p35 antibodies (Upper panels). DRD2-GFP was expressed alone in the cultured striatal neurons and stained with
anti-Cdk5 and anti-p35 antibodies (Lower panels). Hoechst were used for nucleus staining. The scale bar is 5 mm. Cdk5-mediated Phosphorylation of DRD2 Attenuates
Receptor Activity Error bars represent mean 6 SE (n = 4; **p,0.01; two-tailed t-tests). (E) Cultured striatal neurons from wild type and p35
knockout embryos (DIV 7) were treated with 10 mM rolipram for 1 h followed by 1 mM dopamine for 1 h. Error bars represent mean 6 SE (n = 4;
**p,0.01; two-tailed t-tests). d i 10 1371/j
l
0084482 004 Figure 4. Cdk5-mediated phosphorylation attenuates DRD2 surface expression and downstream
3 Figure 4. Cdk5-mediated phosphorylation attenuates DRD2 surface expression and downstream signaling. (A) DRD2 surface
expression measured by [3H]-spiperone binding assay. Transfected HEK293 cells were stimulated with 1 mM quinpirole for the indicated time and
harvested, followed by 3 nM [3H]-spiperone treatment for 3 h. Radioactivity was measured and surface signals were calculated. Error bars represent
mean 6 SE (n = 8; *p,0.05, **p,0.01; One-way ANOVA with Dunnett post hoc test: compare all columns vs. control column). (B) [35S]-GTPcS binding
assay. Cell membranes were prepared from the cells transfected as indicated. Membrane preparations were incubated with 1 mM quinpirole followed
by 3 nM [35S]-GTPcS for 90 min. Error bars represent mean 6 SE (n = 8; *p,0.05, **p,0.01, ***p,0.001; One-way ANOVA with Bonferroni post hoc
test: compare all pairs of columns). (C) Quinpirole-competing [3H]-spiperone binding assay. Membrane preparations from transfected cells were
incubated with 0.01 nM [3H]-spiperone and increasing concentrations of quinpirole for 30 min. Non-linear regression was obtained by GraphPad. Error bars indicate mean 6 SE (n = 3). (D) cAMP enzyme immunoassays in transfected HEK 293 cells. Transfected cells were pretreated with 10 mM
rolipram for 1 h, and subsequently co-treated with 0.1 mM forskolin and increasing concentrations of quinpirole for 30 min. Non-linear regression was
obtained by GraphPad. Error bars represent mean 6 SE (n = 4; **p,0.01; two-tailed t-tests). (E) Cultured striatal neurons from wild type and p35
knockout embryos (DIV 7) were treated with 10 mM rolipram for 1 h followed by 1 mM dopamine for 1 h. Error bars represent mean 6 SE (n = 4;
**p,0.01; two-tailed t-tests). d i 10 1371/j
l
0084482 004 DRD2 S321A-GFP using cAMP enzyme immunoassay. DRD2-
expressing cells showed decreased cAMP levels in response to
quinpirole in a dose-dependent manner. Remarkably, co-expres-
sion of Cdk5/p35 significantly reduced the maximal inhibition of
cAMP formation (Fig. 4D, left panel). On the other hand, in the
DRD2 S321A-GFP expressing cells, the cAMP formations were Cdk5-mediated Phosphorylation of DRD2 Attenuates
Receptor Activity All images were obtained using
confocal microscopy (Olympus, FluoView-1000). doi:10.1371/journal.pone.0084482.g003
DRD2 as a Novel Substrate of Cdk5 DRD2 as a Novel Substrate of Cdk5 Figure 3. Cdk5/p35 can form a complex with DRD2. (A) GST pull-down assay using purified recombinant GST-D2i3 protein with rat brain
extract. GST pull-down precipitates were subjected to Western blotting analyses. ‘Bead’ indicates the pull-down precipitate without GST proteins. (B)
Immunoprecipitation of DRD2 and Cdk5/p35 complex. Anti-GFP IP from lysates from transfected cells were subjected to Western blotting analyses. The bracket indicates DRD2 signals and open arrowhead indicates nonspecific signals from the anti-GFP immunoprecipitates. An overexposed blot
for inputs is also shown in the right. (C) Immunocytochemical analyses of DRD2 and Cdk5/p35. HEK 293 cells expressing DRD2-GFP and Cdk5/p35
were stained with anti-Cdk5 and anti-p35 antibodies (Upper panels). DRD2-GFP was expressed alone in the cultured striatal neurons and stained with
anti-Cdk5 and anti-p35 antibodies (Lower panels). Hoechst were used for nucleus staining. The scale bar is 5 mm. All images were obtained using
confocal microscopy (Olympus, FluoView-1000). doi:10.1371/journal.pone.0084482.g003 incorporation. DRD2-GFP and Cdk5/p35 expressing cell mem-
brane showed significantly impaired [35S]-GTPcS binding com-
pared to all the other cell membranes (Fig. 4B). These results
indicate that Cdk5-mediated phosphorylation down-regulates
agonist-stimulated G protein binding at DRD2. Competitive
binding
of
[3H]-spiperone
upon
treatment
of
increasing concentrations of quinpirole to the membrane prepa-
ration from transfected was measured. Competing binding of
quinpirole and [3H]-spiperone at DRD2-GFP and DRD2 S321A-
GFP made similar logKi values (29.789 for DRD2-GFP; 29.691
for DRD2 S321A-GFP), indicating that the affinity of ligand to
DRD2 is not significantly affected by Cdk5-mediated phosphor-
ylation at DRD2 (Fig. 4C). Additionally,
quinpirole-competing
[3H]-spiperone
binding
assays were performed to investigate any potential change in
agonist-affinity at DRD2 by Cdk5-mediated phosphorylation. December 2013 | Volume 8 | Issue 12 | e84482 PLOS ONE | www.plosone.org 6 Figure 4. Cdk5-mediated phosphorylation attenuates DRD2 surface expression and downstream signaling. (A) DRD2 surface
expression measured by [3H]-spiperone binding assay. Transfected HEK293 cells were stimulated with 1 mM quinpirole for the indicated time and
harvested, followed by 3 nM [3H]-spiperone treatment for 3 h. Radioactivity was measured and surface signals were calculated. Error bars represent
mean 6 SE (n = 8; *p,0.05, **p,0.01; One-way ANOVA with Dunnett post hoc test: compare all columns vs. control column). (B) [35S]-GTPcS binding
assay. Cell membranes were prepared from the cells transfected as indicated. Cdk5-mediated Phosphorylation of DRD2 Attenuates
Receptor Activity Membrane preparations were incubated with 1 mM quinpirole followed
by 3 nM [35S]-GTPcS for 90 min. Error bars represent mean 6 SE (n = 8; *p,0.05, **p,0.01, ***p,0.001; One-way ANOVA with Bonferroni post hoc
test: compare all pairs of columns). (C) Quinpirole-competing [3H]-spiperone binding assay. Membrane preparations from transfected cells were
incubated with 0.01 nM [3H]-spiperone and increasing concentrations of quinpirole for 30 min. Non-linear regression was obtained by GraphPad. Error bars indicate mean 6 SE (n = 3). (D) cAMP enzyme immunoassays in transfected HEK 293 cells. Transfected cells were pretreated with 10 mM
rolipram for 1 h, and subsequently co-treated with 0.1 mM forskolin and increasing concentrations of quinpirole for 30 min. Non-linear regression was
obtained by GraphPad. Error bars represent mean 6 SE (n = 4; **p,0.01; two-tailed t-tests). (E) Cultured striatal neurons from wild type and p35
knockout embryos (DIV 7) were treated with 10 mM rolipram for 1 h followed by 1 mM dopamine for 1 h. Error bars represent mean 6 SE (n = 4;
**p,0.01; two-tailed t-tests). doi:10.1371/journal.pone.0084482.g004
DRD2 as a Novel Substrate of Cdk5 DRD2 as a Novel Substrate of Cdk5 Figure 4. Cdk5-mediated phosphorylation attenuates DRD2 surface expression and downstream signaling. (A) DRD2 surface
expression measured by [3H]-spiperone binding assay. Transfected HEK293 cells were stimulated with 1 mM quinpirole for the indicated time and
harvested, followed by 3 nM [3H]-spiperone treatment for 3 h. Radioactivity was measured and surface signals were calculated. Error bars represent
mean 6 SE (n = 8; *p,0.05, **p,0.01; One-way ANOVA with Dunnett post hoc test: compare all columns vs. control column). (B) [35S]-GTPcS binding
assay. Cell membranes were prepared from the cells transfected as indicated. Membrane preparations were incubated with 1 mM quinpirole followed
by 3 nM [35S]-GTPcS for 90 min. Error bars represent mean 6 SE (n = 8; *p,0.05, **p,0.01, ***p,0.001; One-way ANOVA with Bonferroni post hoc
test: compare all pairs of columns). (C) Quinpirole-competing [3H]-spiperone binding assay. Membrane preparations from transfected cells were
incubated with 0.01 nM [3H]-spiperone and increasing concentrations of quinpirole for 30 min. Non-linear regression was obtained by GraphPad. Error bars indicate mean 6 SE (n = 3). (D) cAMP enzyme immunoassays in transfected HEK 293 cells. Transfected cells were pretreated with 10 mM
rolipram for 1 h, and subsequently co-treated with 0.1 mM forskolin and increasing concentrations of quinpirole for 30 min. Non-linear regression was
obtained by GraphPad. Cdk5-mediated Phosphorylation Down-regulates the
DRD2-cAMP Signaling Pathway Next, we investigated whether the modification of DRD2 by
Cdk5 affects downstream signaling pathways. We monitored
DRD2-mediated inhibition of forskolin-stimulated cAMP produc-
tion by adenylyl cyclase in the cells expressing DRD2-GFP and December 2013 | Volume 8 | Issue 12 | e84482 PLOS ONE | www.plosone.org 7 DRD2 as a Novel Substrate of Cdk5 effectively inhibited in response to quinpirole treatment regardless
of the expression of Cdk5/p35 (Fig. 4D, right panel). These results
indicate that Cdk5-mediated phosphorylation of DRD2 attenuates
the inhibitory activity of DRD2 on the downstream cAMP
signaling pathway. To further confirm the phenomena in a more
physiologically relevant setting, we made use of primary cultured
neurons from knockout embryos deficient in p35, an essential
Cdk5 activator. Primary cultured striatal neurons were treated
with 1 mM dopamine and analyzed by cAMP enzyme immuno-
assay. Neurons from p35 knockout mice exhibited reduced cAMP
levels compared to wild-type neurons when stimulated with
dopamine (Fig. 4E). Taken together, we concluded that Cdk5-
mediated phosphorylation of DRD2 results in a decrease in the
inhibitory tone on the cAMP pathway exerted by DRD2. the third intracellular loop of DRD2 can be phosphorylated by
Cdk5, which results in a decrease in inhibitory influence of DRD2
on the cAMP pathway. This interaction proposes a novel
regulatory mechanism associated with Cdk5 in dopaminoceptive
neurons that might be linked to the dynamic nature of DRD2
surface availability. y
It should be noted that Cdk5 is known to be a key component in
mediating adaptive changes of the neuronal environment. For
instance, structural and functional alterations of dendritic spines in
the neurons of the limbic circuit are one of the consequences of
repeated psychostimulant exposure [40]. These changes are
accompanied by various molecular changes including the induc-
tion of cAMP response element-binding protein (CREB) and
DFosB, transcription factors that exhibit an enduring up-regula-
tion in response to chronic cocaine administration [41,42]. Importantly, Cdk5 is a target of DFosB [19], and many critical
components involved in dendritic spine dynamics, such as PSD-95,
p21-activated kinase (PAK), b-catenin, and spinophilin, were
reported as Cdk5 substrates [43–46]. Consistently, genetic or
pharmacological manipulations of Cdk5 activity elicit alterations
of dendritic spine morphology and behavioral responses to
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in DRD2 long and short isoforms, the mechanism proposed in this
study may be a general mode of regulation in DRD2-mediated
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mice and primates [37–39]. These findings indicate that the
functionality of DRD2 is highly susceptible to adaptive or
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chronic drug exposure. Our results show that the S321 residue in Cdk5-mediated Phosphorylation Down-regulates the
DRD2-cAMP Signaling Pathway Our results showing that DRD2 is a novel target of Cdk5 provides
additional insight into the adaptive changes of the dopamine
system in response to chronic drug exposures because of the
subsequent DFosB-mediated up-regulation of Cdk5 may induce a
tonic increase in the phosphorylation of DRD2. Moreover, DRD2
is
known
to
affect
numerous
cellular
processes,
including
regulation of cAMP and MAP kinase pathways and downstream
transcriptional events [42,49]. Thus, the findings in this study
might not only depict a direct regulation of DRD2 by Cdk5 but
also provide a novel insight into the adaptive responses of
dopamine system to chronic drug exposure. Author Contributions Conceived and designed the experiments: JJ YUP DH SKP. Performed the
experiments: JJ YUP DKK YK. Analyzed the data: JJ YUP DKK SL YK
SAL HL YSG DH SKP. Contributed reagents/materials/analysis tools:
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49533–49541. 48. Meyer DA, Richer E, Benkovic SA, Hayashi K, Kansy JW, et al. (2008) Striatal
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https://openalex.org/W4283712969
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https://jurnal.ugm.ac.id/ijccs/article/download/65640/33706
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English
| null |
Mobile-based Primate Image Recognition using CNN
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Indonesian Journal of Computing and Cybernetics Systems
| 2,022
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cc-by-sa
| 4,151
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Kata kunci— Pengenalan Gambar, Aplikasi Seluler ,CNN Kata kunci— Pengenalan Gambar, Aplikasi Seluler ,CNN IJCCS (Indonesian Journal of Computing and Cybernetics Systems)
Vol.16, No.2, April 2022, pp. 149~158
ISSN (print): 1978-1520, ISSN (online): 2460-7258
DOI: 10.22146/ijccs.65640 IJCCS (Indonesian Journal of Computing and Cybernetics Systems)
Vol.16, No.2, April 2022, pp. 149~158
ISSN (print): 1978-1520, ISSN (online): 2460-7258
DOI: 10.22146/ijccs.65640 149 ◼ 149 Nuruddin Wiranda*1, Agfianto Eko Putro2
1Department of Computer Education, FKIP, ULM, Banjarmasin, Indonesia
2Department of Computer Science and Electronics, FMIPA UGM, Yogyakarta, Indonesia
e-mail: *1nuruddin.wd@ulm.ac.id, 2agfi@ugm.ac.id Nuruddin Wiranda*1, Agfianto Eko Putro2
1Department of Computer Education, FKIP, ULM, Banjarmasin, Indonesia
2Department of Computer Science and Electronics, FMIPA UGM, Yogyakarta, Indonesia
e-mail: *1nuruddin.wd@ulm.ac.id, 2agfi@ugm.ac.id Nuruddin Wiranda*1, Agfianto Eko Putro2
1Department of Computer Education, FKIP, ULM, Banjarmasin, Indonesia
2Department of Computer Science and Electronics, FMIPA UGM, Yogyakarta, Indonesia
e-mail: *1nuruddin.wd@ulm.ac.id, 2agfi@ugm.ac.id Keywords— Image Recognition, Mobile-based, CNN Abstrak Enam dari 25 spesies primata yang paling terancam punah berada di Indonesia. Primata ini yaitu Orangutan, Lutung, Bekantan, Tarsius tumpara, Kukang, dan Simakobu. Tiga
dari enam primate tersebut kebanyakan hidup di pulau Kalimantan. Salah satu bentuk
pelestarian khazanah primata yang terdapat di Kalimantan adalah dengan melakukan studi
identifikasi primata. Pada penelitian ini dikembangkan aplikasi android dengan metode CNN
untuk mengidentifikasi citra primata di lahan basah Kalimantan. CNN digunakan untuk
mengekstrak fitur spasial dari citra primata sehingga sangat efisien untuk masalah identifikasi
citra. Dataset yang digunakan dalam penelitian ini adalah ImageNets, sedangkan model yang
digunakan adalah MobileNets. Aplikasi diuji menggunakan dua skenario yaitu menggunakan
foto dan rekaman video. Foto diambil secara langsung, kemudian diperkecil menjadi resolusi
256 x 256. Untuk video, video diambil dalam waktu kurang lebih 10 hingga 30 detik dengan
resolusi kamera 2 megapiksel. Hasil pengujian didapatkan rata-rata akurasi 93,6% saat
menggunakan foto dan 79% saat menggunakan rekaman video. Setelah dilakukan perhitungan
akurasinya, selanjutnya dilakukan uji usability dengan menggunakan SUS. Berdasarkan hasil
SUS diketahui bahwa aplikasi yang dikembangkan layak untuk digunakan. Received May 15th,2021; Revised April 23th, 2022; Accepted April 26th, 2022 1. INTRODUCTION Primates play an important role in research to improve human health. Despite being a
small part of animals that are used in the biomedical field, primates share genetic and
physiological similarities with humans, making them invaluable in developing treatments and
vaccines for human diseases [1]–[3]. Primates also have important ecological functions in the
ecosystems they inhabit, such as providing pollination services [4] and recognized as having an
important role as a disperser of plant seeds [5]–[7] particularly in tropical rainforests [8]. Bristol Conservation and Science Foundation (BCSF), International Primatological
Society (IPS), International Union for Conservation of Nature Species Survival Commission
Primate Specialist Group (IUCN / SSC PSG), and Conservation International (CI) have
published as many as 25 of the world’s most endangered primate species [9]. Primates are
threatened with extinction because of their natural ecosystems, mostly for commercial purposes,
such as agriculture, livestock, commercial logging, timber harvesting, and fuelwood production
[10]. Other factors are hunting, disease, climate change, loss, degradation and fragmentation of
their habitats [11]. Of the 25 species found, six of which are found in Indonesia, namely Orangutan [12],
[13], Lutung [14], Tarsius tumpara, Kukang Jawa, Simakobu [15], and Bekantan [11], [16]. The six primate species are scattered in three regions, such as Kalimantan, Sumatra and Bali. Of
the six species, three of them mostly live on the island of Borneo, such as the Orangutan,
Lutung dan Bekantan [16]. One form of preservation of primate treasures found in Kalimantan is by conducting
studies on primate classification. With the primate classification, it is hoped that the public will
be more familiar with protected primate animals. In artificial intelligence, especially machine
learning, classification can be done by classifying primate images using a mathematical
approach. Many methods can be used to classify images, including the Convolutional Neural
Network (CNN). The CNN method can be used to solve problems related to image
classification [17]–[20], recognition [21]–[24], and object detection [25], [26]. CNN is
considered to have excellent accuracy and quality [17], [20], [27]–[29]. CNN uses a multi-layer
neural network consisting of one or more convolutional blocks and sub-sampling layers, after
which there are one or more fully connected layers and an output layer[30]. CNN’s design is
inspired by the structure of the mammalian visual system[31]. The most advantageous aspect of
CNN is reducing the number of parameters in ANN [32]. 1. INTRODUCTION Freytag conducted a study to identify
chimpanzees using Log-Euclidean CNN with an accuracy of 92% [33]. Deb performed facial
recognition of endangered primates, such as golden monkeys, lemurs, and chimpanzees, using
the CNN method [34] with an accuracy of 88%. CNN is also used in the classification of
medical images [35]. Based on the previous explanation, the researchers offer a solution for classifying
primate images using the CNN method on an Android-based application. There are three types
of primate images classified, namely Orangutan, and Bekantan. The application will be tested
using two scenarios to calculate its accuracy. Also, usability testing was carried out using the
System Usability Scale (SUS) to determine how easy it was for users to use the interface of the
application being developed. Abstract Six out of 25 species of primates most endangered are in Indonesia. Six of these
primates are namely Orangutan, Lutung, Bekantan, Tarsius tumpara, Kukang, and Simakobu. Three of the six primates live mostly on the island of Borneo. One form of preservation of
primate treasures found in Kalimantan is by conducting studies on primate identification. In
this study, an android app was developed using the CNN method to identify primate species in
Kalimantan wetlands. CNN is used to extract spatial features from primate images to be very
efficient for image identification problems. The data set used in this study is ImageNets, while
the model used is MobileNets. The application was tested using two scenarios, namely using
photos and video recordings. Photos were taken directly, then reduced to a resolution of 256 x
256. Then, videos were taken in approximately 10 to 30 seconds with two megapixel camera
resolution. The results obtained was an average accuracy of 93.6% when using photos and 79%
when using video recordings. After calculating the accuracy, the usability test using SUS was
performed. Based on the SUS results, it is known that the application developed is feasible to
use Received May 15th,2021; Revised April 23th, 2022; Accepted April 26th, 2022 ◼
150 150 ISSN (print): 1978-1520, ISSN (online): 2460-7258 2. 1.1 Dataset The dataset used in this development is ImageNet. There are two species of animals to
be classified, namely Orangutan (Pongo pygmaeus) and Bekantan (Nasalis larvatus). Samples of
images from each class can be seen in Figure 2. b. Bekantan (Nasalis larvatus) a. Orangutan (Pongo pygmaeus)
b. Bekantan (Nasalis larvatus)
Figure 2. Dataset of Primates a. Orangutan (Pongo pygmaeus) b. Bekantan (Nasalis larvatus) b. Bekantan (Nasalis larvatus) Figure 2. Dataset of Primates 2.1 Application Development Three steps were carried out at the development stage, namely interface design, code
generation, and application functional testing. ISSN (print): 1978-1520, ISSN (online): 2460-7258 Figure 1. Research Method of Primate Wetland Image Recognition. Figure 1. Research Method of Primate Wetland Image Recognition. 2. METHODS The method used in this study consists of two stages, namely application development
and application testing, as shown in Figure 1. IJCCS Vol. 16, No. 2, April 2022 : 149 – 158 IJCCS ISSN (print): 1978-1520, ISSN (online): 2460-7258 ISSN (print): 1978-1520, ISSN (online): 2460-7258 151 2. 1.3 Coding At this stage, code was generated for the application of interface design and image
classification architecture in Android-based applications. The coding was made using android
studio, firebase, and tensorflow [28]. The application was designed to run well on android 4.1
and up. 2. 1.2 Design At this stage, the interface design and the architecture design of primate image
classification were carried out. The application interface consists of three pages, namely a
splash screen, an object detection page, and a page to display the detection results or
descriptions of the objects that have been identified. Image classification architecture design can
be seen in Figure 3. Figure 3. Primates Image Classification Architecture Design
Input :
Primate Image
MobileNet Model
Pre
Processing
Primate Image
Identification
Output: primate
name & description
Wikipedia Input :
Primate Imag Pre
Processing Figure 3. Primates Image Classification Architecture Design Mobile-based Primate Image Recognition using CNN (Nuruddin Wiranda) ◼
152 152 ◼ ISSN (print): 1978-1520, ISSN (online): 2460-7258 Based on Figure 3, the first step in the primate identification process was to receive
input in the form of photos or videos of primates directly. Photos are taken directly, then
reduced to a resolution of 256 x 256. For videos, videos are taken for approximately 10 to 30
seconds with a 2-megapixel camera resolution. The second step was pre-processing to speed up
the identification process. The kind of pre-processing that was done is to reduce the size of the
photo to 256 x 256 pixels. p
p
In the third step, the imaged primate images were compared using the MobileNets
model to determine the primate’s name. MobileNets is one of the Convolutional Neural
Network (CNN) architectures that can be used to overcome the need for excessive computing
resources. Once the name of the primate is known, the application will search for descriptions
on the Wikipedia site based on the primate name. The final step, the output of the application
was to display the name and description of the primate. 3. RESULTS AND DISCUSSION This current study used 300 primate images for testing the first scenario and 30 videos
for testing the second scenario. The purpose of this experiment is to measure the accuracy of the
architecture that we design. After that, we also use SUS to measure how easy it is to use the
interface of the developed application. 2. 2 Application testing There were two types of testing at the application testing stage: testing the accuracy and
testing the application’s usability using the System Usability Scale (SUS) [36]. Application
accuracy testing was carried out to obtain the accuracy of primate recognition from the
application being developed. Testing was carried out using two scenarios as follows: First Scenario: Photos of 3 types of animals were downloaded; each type consisted of
30 photos. The photos were recognized using an application that had been developed, then the
results of the recognition were recorded to calculate accuracy. Second Scenario: The application was tested by classifying using video, the application
was highlighted to the primate video, then the recognition results were recorded to calculate
accuracy. After calculating the accuracy of the application, the next step was to test usability using
SUS, which consists of 10 questions with a ring scale answer, from 1 to 5. Figure 1 shows that
the user strongly disagrees with the test statement, and number 5 states that he strongly agrees
with the test statement [37]. Usability testing is used to see how easy the user uses the
application interface. 2. 1.4 Functional testing The functional testing was carried out using BlackBox testing by testing all functional
features in the application, such as photo and video detection functions and the function of
displaying results. It also ensures that there are no errors during the identification process of
primate animals, resulting in the application not running properly or errors. 3. 1 Application Development Results Figure 3 shows the results of developing an android application using the CNN method. The user will first enter the primate identification interface to identify the desired object, as
shown in Figure 4a. IJCCS Vol. 16, No. 2, April 2022 : 149 – 158 ISSN (print): 1978-1520, ISSN (online): 2460-7258 IJCCS 153 Figure 4. The interface of Primate Animal Identification App: a). display when reading
recognized primates. b) display when displaying detection results. c) display when displaying a
description of the recognized primates
a
b
c b a c b a c Figure 4. The interface of Primate Animal Identification App: a). display when reading
recognized primates. b) display when displaying detection results. c) display when displaying
description of the recognized primates Mobile-based Primate Image Recognition using CNN (Nuruddin Wiranda) 3. 2 Accuracy and Usability Testing Results Testing was carried out using 30 photos from each of the three categories tested with
two scenarios by five application users. The first scenario is done by using photos, and the
second is using direct object detection. The test results can be seen in Table 1. Testing was carried out using 30 photos from each of the three categories tested with
two scenarios by five application users. The first scenario is done by using photos, and the
second is using direct object detection. The test results can be seen in Table 1. Table 1. Accuracy results from 2 scenarios
User to Primates Species
Accuracy (%)
Scenario 1 Scenario 2
1
Bekantan
87
100
Orangutan
83
100
Average
85
100
2
Bekantan
93
40
Orangutan
97
37
Average
95
38.5
3
Bekantan
100
100
Orangutan
100
100
Average
100
100
4
Bekantan
97
100
Orangutan
93
100
Average
95
100
5
Bekantan
93
33
Orangutan
93
80
Average
93
56.5
Grand Total
93.6
79 Mobile-based Primate Image Recognition using CNN (Nuruddin Wiranda) ◼
154 ISSN (print): 1978-1520, ISSN (online): 2460-7258 ◼
4 SUS is a measuring tool that assesses usability of a product. There are some
characteristics of SUS that make it attractive and different from other questionnaires. Although
it cannot help in determining the factors or features that are still problematic in the system, the
system usability scale can help in determining whether the system can be used properly. Table 2. 3. 2 Accuracy and Usability Testing Results SUS Testing Results
Respondents Q1
Q2 Q3 Q4 Q5 Q6 Q7 Q8 Q9 Q10 Total JML * 2.5
1
2
4
4
3
2
4
4
4
3
3
33
83
2
3
2
4
4
3
3
4
3
4
4
34
85
3
2
1
4
3
2
1
3
2
4
4
26
65
4
0
4
4
4
0
0
0
4
4
0
20
50
5
3
1
4
3
2
2
3
3
2
3
26
65
6
2
2
3
4
2
2
3
3
3
3
27
68
7
3
3
4
3
3
1
3
3
3
3
29
73
8
2
3
3
3
3
2
3
2
3
2
26
65
9
3
3
4
3
3
1
4
3
3
3
30
75
10
3
2
3
1
2
2
4
3
2
3
25
63
11
3
2
1
2
1
1
4
3
1
4
22
55
12
2
3
4
2
3
2
4
3
2
3
28
70
13
3
4
4
3
4
2
4
3
3
3
33
83
14
4
4
4
3
4
3
4
3
4
2
35
88
15
2
4
1
4
2
1
4
2
3
4
27
68
16
0
3
4
3
0
4
2
4
0
4
24
60
17
4
3
3
4
4
2
4
4
4
4
36
90
18
2
3
3
3
2
3
3
3
0
4
26
65
19
2
1
2
4
2
3
4
3
0
0
21
53
20
4
4
4
3
4
2
4
3
1
4
33
83
21
2
3
4
4
1
1
3
4
2
3
27
68
22
4
2
4
1
3
3
3
4
4
1
29
73
SUS results
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Factors related to leader implementation of a nationally disseminated community-based exercise program: a cross-sectional study
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The international journal of behavioural nutrition and physical activity
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Published: 4 December 2008 Received: 19 February 2008
Accepted: 4 December 2008 International Journal of Behavioral Nutrition and Physical Activity 2008, 5:62
doi:10.1186/1479-5868-5-62 This article is available from: http://www.ijbnpa.org/content/5/1/62 © 2008 Seguin et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Factors related to leader implementation of a nationally
disseminated community-based exercise program: a cross-sectional
study
Rebecca A Seguin*1,2, Ruth Palombo2,4, Christina D Economos1,2,3,
Raymond Hyatt1,2,4, Julia Kuder1 and Miriam E Nelson1,2,3 Address: 1John Hancock Center for Physical Activity and Nutrition, Tufts University, Boston, MA, USA, 2Friedman School of Nutrition Science and
Policy, Tufts University, Boston, MA, USA, 3Jonathan M. Tisch College of Citizenship and Public Service, Tufts University, Medford, MA, USA and
4Department of Public Health and Family Medicine, School of Medicine, Tufts University; Boston, MA, USA mail: Rebecca A Seguin* - rebecca.seguin@tufts.edu; Ruth Palombo - ruth.palombo@state.ma.us; Email: Rebecca A Seguin* - rebecca.seguin@tufts.edu; Ruth Palombo - ruth.palombo@state.ma.us;
Christina D Economos - christina.economos@tufts.edu; Raymond Hyatt - raymond.hyatt@tufts.edu; Julia Kuder - julia.kuder@tufts.edu;
Miriam E Nelson - miriam nelson@tufts edu * Corresponding author Received: 19 February 2008
Accepted: 4 December 2008 Published: 4 December 2008 International Journal of Behavioral
Nutrition and Physical Activity Open Access Background within a group, and that greater levels of change require
leadership that is more dynamic. It has been suggested
that the most effective leaders are those who rely on not
one but multiple leadership styles and those who are able
to adapt depending on the situation [23,26]. g
An essential component of community-based physical
activity programs are the leaders who implement them [1-
4]. Community program leaders serve many roles in
implementation – from soliciting participation and teach-
ing classes to providing motivation, inspiration, and feed-
back to participants [3,5,6]. Leaders may also carry out a
range of administrative and logistical tasks. Considered succinctly, optimizing leadership is likely a
function of both personal and environmental factors that
facilitate program implementation and sustainability
through separate and collective action [7,13,15,17,21]. Hence, the characteristics of the target group, nature of the
work, type of group structure, and nature of the external
environment may all influence what could be considered
"successful" leadership [13,17,19,27]. Therefore, examin-
ing and understanding the effects of leadership, individ-
ual, and situational characteristics may confer enhanced
success in implementing and sustaining a variety of com-
munity programs [3,15,18,24,28-31]. Leaders often possess a wide variety of backgrounds and
skills, which inevitably influence the programs they
implement and the participants who receive them. In
community-based public health program evaluations,
there are varying types and degrees of success (related to
implementation, participation, biologic/health-related
outcomes, and beyond); it is likely that the characteristics
of the leaders and communities contribute to this variabil-
ity – independently and collectively [7-12]. Currently, there is a gap within the public health literature
by which a comprehensive model could be developed and
utilized to identify, select, and strategically train commu-
nity-based leaders to maximize their skills and capacity. General literature on leadership, however, lends insight to
factors and characteristics worthy of exploration. For
example, it has been proposed that leadership is influ-
enced by the leader's personal characteristics. Research
suggests that such characteristics can be defined on a vari-
ety of levels, such as age, education level, or in a capacity
that is more descriptive of life experiences and personal
habits, such as eating habits or exercise practices. In this
context, a study may seek to determine which factor is
important to program implementation: formal training or
prior program implementation experience? Perhaps nei-
ther; perhaps both. Abstract Background: The benefits of community-based health programs are widely recognized. Howe
examining factors related to community leaders' characteristics and roles in implementation is limi Methods: The purpose of this cross-sectional study was to use a social ecological framework of variables to
explore and describe the relationships between socioeconomic, personal/behavioral, programmatic, leadership,
and community-level social and demographic characteristics as they relate to the implementation of an evidence-
based strength training program by community leaders. Eight-hundred fifty-four trained program leaders in 43
states were invited to participate in either an online or mail survey. Corresponding community-level
characteristics were also collected. Programmatic details were obtained from those who implemented. Four-
hundred eighty-seven program leaders responded to the survey (response rate = 57%), 78% online and 22% by
mail. Results: Of the 487 respondents, 270 implemented the program (55%). One or more factors from Results: Of the 487 respondents, 270 implemented the program (55%). One or more factors from each category
– professional, socioeconomic, personal/behavioral, and leadership characteristics – were significantly different
between implementers and non-implementers, determined by chi square or student's t-tests as appropriate. Implementers reported higher levels of strength training participation, current and lifetime physical activity,
perceived support, and leadership competence (all p < 0.05). Logistic regression analysis revealed a positive
association between implementation and fitness credentials/certification (p = 0.003), program-specific self-efficacy
(p = 0.002), and support-focused leadership (p = 0.006), and a negative association between implementation and
educational attainment (p = 0.002). Conclusion: Among this sample of trained leaders, several factors within the professional, socioeconomic,
personal/behavioral, and leadership categories were related to whether they implemented a community-based
exercise program. It may benefit future community-based physical activity program disseminations to consider
these factors when selecting and training leaders. Page 1 of 12
(page number not for citation purposes) http://www.ijbnpa.org/content/5/1/62 International Journal of Behavioral Nutrition and Physical Activity 2008, 5:62 http://www.ijbnpa.org/content/5/1/62 International Journal of Behavioral Nutrition and Physical Activity 2008, 5:62 Background Also, does it matter whether the lead-
ers themselves model the behavior they are trying to
encourage? Personal commitment and/or experience may
affect the execution of implementation [5,13-20]. There is emerging popularity and demand for evidence-
based health promotion programs. Despite the recog-
nized importance of community program leadership and
an extensive body of literature related to the leadership for
the business sector [14,16,22,32], research in this area as
it relates to public health is limited, in both quantity and
depth. The multilevel influence embedded within com-
munity-based programs implementation and dissemina-
tion efforts invite the social ecological model as a viable
and practical framework for examination [33,34]. The
findings presented here aim to expand understanding of
the role of leadership in community-based program
implementation, and to generate hypotheses for future
studies. Page 2 of 12
(page number not for citation purposes) Study objective
h
f Research
-Clinical
-Epidemiological
-Community-based
-Home-based
Policies
-ACSM/CDC1
-Other national
organizations
- Private entities
Communication
-Internet
-Television/radio
-Books, print media
Knowledge:
Awareness, Beliefs, Attitude
Access:
StrongWomen community program
Behavior Change:
Influence number of women strength training
Contextual concept in which the
StrongWomen Program was disseminated
Changing
Population
Demographic
1 ACSM=American College of Sport Medicine; CDC=Centers for Disease Control and Prevention
Social Ecological Framework
Figure 2
Social Ecological Framework. Social ecological frame-
work describing the leader, participant, and community char-
acteristics examined in this study, and how they may be
related to implementation (leaders) and adherence (partici-
pants). The community-level characteristics also help to
describe the larger contextual landscape of the dissemination
The Participants: Older Women
Infrastructure
•Access to space,
resources, equipment
•Paid versus volunteer
•Institutional support
•Public relation/media
•Barriers, program
logistics
Leader Characteristics
Social
Collective efficacy
-Voter participation
Community cohesion
-Crime status
Program Leaders
Environment
Participant
Characteristics
Age, sex, ethnicity
education, income
marital, health, work status
nutrition knowledge/application
previous/current sports/exercise level
barriers: access, fees, distance, reputability
social/family/physician support; self-efficacy
reported frequency of activity-limiting pain
Personal factors
•Age, ethnicity, income,
education, certifications
•Previous/current strength
training, sport, and general
physical activity experience
•Leadership experience, self-
identification, comfort, style,
self-efficacy
Demographic
Income/SES
-Household income
-Educational attainment
Pop. diversity
-Ethnicity
Community Characteristics Social Ecological Framework
Figure 2
Social Ecological Framework. Social ecological frame-
work describing the leader, participant, and community char-
acteristics examined in this study, and how they may be
related to implementation (leaders) and adherence (partici-
pants). The community-level characteristics also help to
describe the larger contextual landscape of the dissemination
environment. The Participants: Older Women
Infrastructure
•Access to space,
resources, equipment
•Paid versus volunteer
•Institutional support
•Public relation/media
•Barriers, program
logistics
Leader Characteristics
Social
Collective efficacy
-Voter participation
Community cohesion
-Crime status
Program Leaders
Environment
Participant
Characteristics
Age, sex, ethnicity
education, income
marital, health, work status
nutrition knowledge/application
previous/current sports/exercise level
barriers: access, fees, distance, reputability
social/family/physician support; self-efficacy
reported frequency of activity-limiting pain
Personal factors
•Age, ethnicity, income,
education, certifications
•Previous/current strength
training, sport, and general
physical activity experience
•Leadership experience, self-
identification, comfort, style,
self-efficacy
Demographic
Income/SES
-Household income
-Educational attainment
Pop. Dissemina
Figure 1 g
Dissemination Context. Context for development and
dissemination of the StrongWomen Program, a community-
based strength training program targeted to women aged 40
and older. This figure was previously published [6]. Social Ecological Framework
Figure 2
Social Ecological Framework. Social ecological frame-
work describing the leader, participant, and community char-
acteristics examined in this study, and how they may be
related to implementation (leaders) and adherence (partici-
pants). The community-level characteristics also help to
describe the larger contextual landscape of the dissemination
environment. Social Ecological Framework
Figure 2
Social Ecological Framework. Social ecological frame-
work describing the leader, participant, and community char-
acteristics examined in this study, and how they may be
related to implementation (leaders) and adherence (partici-
pants). The community-level characteristics also help to
describe the larger contextual landscape of the dissemination
environment. Social Ecological Framework
Figure 2
Social Ecological Framework. Social ecological frame-
work describing the leader, participant, and community char-
acteristics examined in this study, and how they may be
related to implementation (leaders) and adherence (partici-
pants). The community-level characteristics also help to
describe the larger contextual landscape of the dissemination
environment. ecological framework of variables for this research is
shown in Figure 2. Survey design and development The preliminary survey development involved the synthe-
sis of findings from three sources: approximately 500
post-workshop evaluations completed by leaders; data
from a pilot phone survey conducted in September 2004;
and group program participant interviews previously con-
ducted during program site visits from 2004–2005 [6,35]. Those data were used to compile a working draft concept
and content table, which framed the survey outline. Phone interviews were then conducted with five program Methods The SWP was designed as a community-based program to
be implemented as twice weekly group strength training
classes lasting eight to twelve weeks in community set-
tings. At the time of survey administration (June 2006),
thirty-nine day-long workshops had been conducted from
May 2003–June 2006 – training 854 community leaders
from forty-three states to lead the SWP. Program leaders
are most commonly from nonprofit organizations such as
the USDA Cooperative State Research, Extension, and
Education Service, hospital-based wellness centers, and
community/recreation centers [6]. Contact information
for trained leaders is tracked using a detailed database that
includes phone, email, address, and other professional
information. leaders and five program administrators from a range of
geographic locations to expand upon and revise the out-
line. Drafts of the survey were reviewed and pilot tested
internally among the research team and selected col-
leagues, in both an Internet-based and paper-based for-
mat. leaders and five program administrators from a range of
geographic locations to expand upon and revise the out-
line. Drafts of the survey were reviewed and pilot tested
internally among the research team and selected col-
leagues, in both an Internet-based and paper-based for-
mat. Following the first round of modifications, the survey was
then pilot tested in the online and paper formats with ten
program leaders from six states, including the five leaders
who initially participated in the phone interviews; all
were subsequently excluded from the final survey partici-
pation. Following revisions, the survey along with all
related materials (i.e. cover letter/cover email inviting
leaders to participate, consent form, etc.) were approved
by the Tufts University Human Investigation Review
Board (IRB approval #7049). Study objective
h
f diversity
-Ethnicity
Community Characteristics Contextual concept in which the
StrongWomen Program was disseminated The Participants: Older Women
Infrastructure
•Access to space,
resources, equipment
•Paid versus volunteer
•Institutional support
•Public relation/media
•Barriers, program
logistics
Leader Characteristics
Social
Collective efficacy
-Voter participation
Community cohesion
-Crime status
Program Leaders
Environment
Participant
Characteristics
Age, sex, ethnicity
education, income
marital, health, work status
nutrition knowledge/application
previous/current sports/exercise level
barriers: access, fees, distance, reputability
social/family/physician support; self-efficacy
reported frequency of activity-limiting pain
Personal factors
•Age, ethnicity, income,
education, certifications
•Previous/current strength
training, sport, and general
physical activity experience
•Leadership experience, self-
identification, comfort, style,
self-efficacy
Demographic
Income/SES
-Household income
-Educational attainment
Pop. diversity
-Ethnicity
Community Characteristics Program Leaders Study objective
h
f The purpose of this study was to explore and describe the
relationships between socioeconomic, personal/behavio-
ral, programmatic, leadership, and community-level
social and demographic characteristics as they relate to the
implementation of an evidence-based strength training
program by community leaders. This was a cross-sectional
design that utilized a convenience sample of leaders from
the StrongWomen Program (SWP) – a nationally dissem-
inated community strength training program targeted to
midlife and older women [6]. The primary hypothesis
stated that implementation of the SWP would be posi-
tively associated with a community leader's previous
strength training experience, support, and leadership
characteristics compared to leaders who did not imple-
ment the program. The contextual framework for the dis-
semination of the SWP is shown in Figure 1; an extensive
review of the national dissemination of the SWP – includ-
ing the training workshop, curriculum, and programmatic
details – have been previously published [6]. The social- Other theories on leadership suggest that while personal
characteristics are important, situational factors such as
support, the organization, or environment may interact
with individual factors and modify effectiveness accord-
ingly [7,13,15,17,21-23]. These theories imply that even if
all essential components are in place at the individual
(leader) level, the context or situational characteristics
play an inevitably crucial role. An environment where
appropriate resources are allocated toward development
and where learning is rewarded is also essential to the
development of an effective leader. This concept may be
thought of as quality of institutional or organizational
leadership [14,20,23-26]. What is commonly agreed upon in this area is that appro-
priate leadership is important to bring about change Page 2 of 12
(page number not for citation purposes) Page 2 of 12
(page number not for citation purposes) http://www.ijbnpa.org/content/5/1/62 International Journal of Behavioral Nutrition and Physical Activity 2008, 5:62 ecological framework of variables for this research is
shown in Figure 2. Dissemination Context
Figure 1
Dissemination Context. Context for development and
dissemination of the StrongWomen Program, a community-
based strength training program targeted to women aged 40
and older. This figure was previously published [6]. Leadership factors
d Respondents answered questions about their program-
specific self-efficacy related to their confidence to over-
come potential challenges related to social, physical, and
logistical aspects of program implementation; a com-
bined overall score for program-related self-efficacy was
calculated. The self-efficacy questions were adapted from
the General Self-Efficacy Scale [39]. To characterize lead-
ership competence, respondents were asked whether they
self-identify as a leader and whether they are comfortable
leading people in activities; responses were yes/no [27]. To characterize leadership style, four primary categories
from well-established leadership inventories were uti-
lized: organization, support, communication, and con-
flict resolution [27,40]. Respondents answered eight
questions that were used to characterize their leadership
style, within the aforementioned four categories. The lead-
ership competence and leadership characteristics ques-
tions were informed by the Leadership Practices Inventory Of the 854 leaders surveyed, 487 completed the survey,
yielding a 57% response rate. Of the 487 survey respond-
ents, 381 were online respondents (78%) and 106 were
mail respondents (22%). Analyses were conducted
between online and paper-based respondents in terms of
personal characteristics (i.e. age, sex, race, education,
income, etc.) as well as program-related characteristic (i.e. implementation rates, participant compliance, etc.). Using chi square to compare categorical variables and t
tests to compare continuous variables, no statistically sig-
nificant differences were found between the online and
mail respondents; therefore, all data were analyzed and
are shown together. Of the 854 leaders surveyed, 487 completed the survey,
yielding a 57% response rate. Of the 487 survey respond-
ents, 381 were online respondents (78%) and 106 were
mail respondents (22%). Analyses were conducted
between online and paper-based respondents in terms of
personal characteristics (i.e. age, sex, race, education,
income, etc.) as well as program-related characteristic (i.e. implementation rates, participant compliance, etc.). Using chi square to compare categorical variables and t
tests to compare continuous variables, no statistically sig-
nificant differences were found between the online and
mail respondents; therefore, all data were analyzed and
are shown together. Completed surveys
(N = 487)
Implementers
(N = 270)
Total program leaders surveyed
(N = 854)
106 (22%)
mail
381 (78%)
online
Non-Implementers
(N= 217) Program Leader Survey Response
Figure 3
Program Leader Survey Response. Graphic of overall
survey response rate, percentages of paper and online sur-
veys received, and breakdown of program implementers and
non-implementers. Leadership factors
d Completed surveys
(N = 487)
Implementers
(N = 270)
Total program leaders surveyed
(N = 854)
106 (22%)
mail
381 (78%)
online
Non-Implementers
(N= 217) Program-related personal factors The program-related personal characteristics of respond-
ents asked leaders to categorize their current and previous
activity level, current and previous sports participation,
past and current strength training participation, change in
activity since workshop attendance, and the activity level
of the leader's significant other (if applicable). Physical
activity and nutrition topic areas were derived from the
National Health Interview Survey; specific questions were
developed, pilot tested, and administered for this survey
[38]. All survey data were collected over a three-month period. Paper survey data were entered into SPSS Data Builder
14.0; Internet-based survey data were downloaded to a
Microsoft Excel spreadsheet and subsequently converted
to the SPSS 14.0 format. Data from the paper surveys and
online survey were merged. Data cleaning and recoding as
well as all data analysis were conducted using SPSS 14.0
[36]. Outcome measurements
Implementation Total program leaders surveyed
(N = 854) Survey respondents (herein "respondents") were asked
(no/yes answer format) if they had implemented at least
one program (twice weekly SWP classes) following their
workshop attendance. Individuals who answered yes were
classified as implementers. This was the primary outcome
of interest, and the dependent variable (0 = no, 1 = yes)
for the logistic regression analysis. 381 (78%)
online 106 (22%)
mail Survey data collection Eight-hundred fifty-four leaders were invited to partici-
pate in the survey beginning in June 2006. All leaders who
provided email addresses at workshop trainings in which
they participated received the email invitation, which Page 3 of 12
(page number not for citation purposes) Page 3 of 12
(page number not for citation purposes) http://www.ijbnpa.org/content/5/1/62 International Journal of Behavioral Nutrition and Physical Activity 2008, 5:62 cable); and employer name and type of organization (if
applicable). included a link to the online consent and survey. Those
who did not provide an email address as well as those
who responded asking for a printed version, were mailed
the paper-based version, which included a written consent
form. After the initial email and paper mailing, all nonre-
spondents received both the email-based and paper-based
invitations on two subsequent release dates – each sepa-
rated by approximately three weeks (for a total of three
survey invitations). All respondents were required to give
informed consent to participate. Following survey sub-
mission, all respondents were mailed a thank you letter. Socioeconomic and professional factors Socioeconomic characteristics included the following:
age, sex, race, marital status, educational attainment (i.e. bachelors level), income, and work status; questions were
adapted from the U.S. Census Bureau American Commu-
nity Survey and the Behavioral Risk Factor Surveillance Sys-
tem Survey Questionnaire [37,38]. Professional variables
collected included educational degree concentration (i.e. physical therapy); fitness certification/credential attain-
ment; professional title and job responsibilities (if appli- Non-Implementers
(N= 217) Implementers
(N = 270) Program L
Figure 3 Program Leader Survey Response
Figure 3
Program Leader Survey Response. Graphic of overall
survey response rate, percentages of paper and online sur-
veys received, and breakdown of program implementers and
non-implementers. Program Leader Survey Response
Figure 3
Program Leader Survey Response. Graphic of overall
survey response rate, percentages of paper and online sur-
veys received, and breakdown of program implementers and
non-implementers. http://www.ijbnpa.org/content/5/1/62 International Journal of Behavioral Nutrition and Physical Activity 2008, 5:62 Table 1: Socioeconomic and Professional Characteristics. Non-Implementers
N = 217a
Implementers
N = 270b
P-Value
Age in years, mean (SD)
50 (10)
50 (11)
0.965
Sex, % female
98
98
0.986
Race, % white
93
93
0.953
Married/living with domestic partner, %
73
74
0.465
Education level, %
- Some HS
-
-
1.000
- HS grad
3
3
1.000
- Some college
10
15
0.174
- BS
29
38
0.034
- MS+
58
44
0.003
Household income, %
- <20K
5
3
0.799
- 20-49999
18
26
0.059
- 50-74999
33
30
0.548
- 75-100K
26
21
0.209
- >100K
20
20
0.845
Work status, %
- Full time
69
70
0.862
- Part time
21
21
0.963
- Volunteer only
6
5
0.698
- No work
4
4
1.000
Fitness credential/certification, %
24
35
0.005
Due to the nature of survey data, sample size varies by question. a – sample size range: n = 189–217
b – sample size range: n = 236–270 Table 1: Socioeconomic and Professional Characteristics. bles were compared at the community and the national
level [42,43]. bles were compared at the community and the national
level [42,43]. and other relevant sources, and developed for this survey
[27,40]. Respondents also reported perceived support
from friends, family, and/or their supervisor to imple-
ment the program as well as the reason they attended the
training workshop [41]. Statistical analyses y
The chi-square test was used to compare implementers to
non-implementers on categorical variables, and the t test
was used to compare continuous variables. Logistic regres-
sions examined factors related to implementation. The a
priori hypothesized model was specified as: implementation
= educational attainment + income + program-related self-effi-
cacy + fitness certification/credential + support-focused leader-
ship style + previous strength training experience + age. Additional model details are provided in the Results sec-
tion of this manuscript. The data were analyzed using
SPSS 14.0 [36]. In addition, five community-level varia-
bles were examined that describe specific aspects of com-
munity: education and income (SES); race (population
diversity); crime rates (social cohesion), and voter partici-
pation (collective efficacy) [44]. Page 5 of 12
(page number not for citation purposes) Program logistics and barriers Implementers reported details regarding the SWPs they
implemented. Mean months ± SD between workshop
attendance and program implementation was 5.1 ± 5.5. Other details included duration of program session, the
number of days per week that classes meet, length of class
sessions, number of participants per class, peer leader pro-
gram help, participant attendance rate. They also reported
their reasons for implementing, sustaining, and no longer
leading programs, as applicable, as well as compensation
related to leading the program. Those data are presented
in Table 5. Results
Implementation Of the 487 respondents, 270 (55%) were classified as
implementers; 217 (45%) were classified as non-imple-
menters. See Figure 3. Demographic comparison Individual, community, and national-level comparisons
for education, income, and race as well as community and
national-level voter participation and crime rates for
leader communities are shown in Tables 6. At the individ-
ual-level, respondents had higher levels of education,
higher household income, and less racial diversity than
their respective communities (all p < 0.001), and their
respective communities had higher levels of education,
higher household income, less racial diversity compared
to the national levels (all p < 0.001). In addition, respond-
ents' respective communities had higher voter participa- Leadership characteristics Leadership characteristics
Implementers reported significantly greater perceived
support from their friends, families, and supervisors com-
pared to non-implementers (p < 0.001) as well as greater
levels of program-related self-efficacy (p = 0.003) and
leadership competence (greater comfort leading others, p
= 0.007 and stronger leader self-identification, p = 0.049)
compared to non-implementers. Reasons for workshop
attendance also differed significantly between the groups. A significantly greater percentage of non-implementers
reported "personal health reasons" as their primary rea-
son for attendance (p = 0.002), whereas implementers
reported "to implement a program" and "supervisors sug-
gestion" as their reasons compared to non-implementers,
although the differences were not significant (p = 0.097
and p = 0.074, respectively). Data presented in Table 4. Socioeconomic and professional factors Reported barriers to implementation were different
between implementers and non-implementers. Com-
pared to non-implementers, implementers reported find-
ing participants as a barrier to starting a program (p <
0.001). Compared to implementers, non-implementers
reported being too busy and not having enough support
as their barriers (p = 0.01 and p < 0.001, respectively). Data not shown. The majority of respondents were educated white (93%),
female (98%), and working full- or part-time (≥ 90% col-
lectively). Age, sex, race, marital status, household
income, and work status were not different between non-
implementers and implementers. Non-implementers
reported significantly greater masters-level educational
attainment compared to implementers (p = 0.003);
implementers reported greater bachelor-level attainment
compared to non-implementers (p = 0.034). Data pre-
sented in Table 1. In addition, the occupation distribution
of program leaders is presented in Table 2. International Journal of Behavioral Nutrition and Physical Activity 2008, 5:62 International Journal of Behavioral Nutrition and Physical Activity 2008, 5:62 http://www.ijbnpa.org/content/5/1/62 Table 2: Occupation distribution of program leadersa
Jobs
Percent
Extension Agent
43.0%
Fitness Instructor/Personal Trainer
7.8%
Physician/Nurse
4.1%
Physical Therapist
1.8%
Nutritionist/Dietician
1.6%
Other Healthcare
5.9%
Community Educator/Community Organizer
5.8%
Academic Educator
2.0%
Student
1.2%
Self-employed
1.4%
Other
10.9%
Due to the nature of survey data, sample size varies by question. a – sample size: n = 754 non-implementers and implementers. Data presented in
Table 3. Page 6 of 12
(page number not for citation purposes) Demographic comparisons To assess socioeconomic status (SES), survey respondents
indicated their educational attainment, household
income level, and race (individual level). These variables
were also collected at the community level (respondent
zip codes) and at the national level using 2004 U.S. Cen-
sus data [37]. Means were compared for education,
income, and race between the individual level and com-
munity level, between the individual and national levels,
and between the community and national levels. In addi-
tion, voter participation rates and crime rates were col-
lected at the community level and national level, as
indicators for community participation and collective effi-
cacy, respectively. Statistical means for each of the varia- Page 5 of 12
(page number not for citation purposes) Page 5 of 12
(page number not for citation purposes) International Journal of Behavioral Nutrition and Physical Activity 2008, 5:62 Program-related personal factors Implementers reported significantly lower current physi-
cal inactivity levels, greater current strength training hab-
its, and increased post-workshop physical activity levels
compared to non-implementers (all p < 0.001). Lifetime
physical activity level, current and prior sports participa-
tion, spouse/domestic partner activity level, and prior
strength training experiences were not different between Page 6 of 12
(page number not for citation purposes) Page 6 of 12
(page number not for citation purposes) International Journal of Behavioral Nutrition and Physical Activity 2008, 5:62
http://www.ijbnpa.org/content/5/1/62 http://www.ijbnpa.org/content/5/1/62 International Journal of Behavioral Nutrition and Physical Activity 2008, 5:62 Table 3: Program-Related Personal/Behavioral Characteristics
Non-Implementers
N = 217a
Implementers
N = 270b
P-Value
Current PA level, %
- Not active
10
2
<0.001
- Somewhat active
42
40
0.595
- Active
48
58
0.031
Lifetime PA level, %
- Not active
4
4
1.000
- Somewhat active
54
46
0.113
- Active
42
50
0.136
Current sports participation, %
17
23
0.124
Prior sports participation, %
58
65
0.125
Significant other is active, %c
75
80
0.294
Prior strength training experience, %
42
47
0.263
Currently strength training regularly, %
(at least 1–2 times per week)
59
85
<0.001
Change in activity level since workshop, %
- Less active
6
-
0.001
- About the same
69
54
0.002
- More active
25
46
<0.001
Due to the nature of survey data, sample size varies by question. a – sample size range: n = 212–216
b – sample size range: n = 260–270
c – sample size for non-implementers, n = 157; implementers, n = 183 Table 3: Program-Related Personal/Behavioral Characteristics It is important to note here that the construction of alter-
native models informed by the univariate results occurred
during the analyses of these data. The process involved a
phased approach testing for collinearity among variables
and using step-wise logistic regression. The variables were
first tested for collinearity within their respective catego-
ries (socioeconomic, professional, program-related per-
sonal/behavior, leadership). For example, within the
socioeconomic and professional category, education and
income were highly correlated and thus could not be
included in any regression models together. Using sepa-
rate step-wise logistic regression tests, educational attain-
ment remained in the model while income did not; thus,
education was chosen over income for inclusion. This also
occurred with three variables in the leadership category. Program-related personal factors Program-related self-efficacy, leader self-identification,
and leadership comfort were all correlated, and were sub-
sequently tested in models separately; program-related
self-efficacy was chosen for inclusion based upon the
model's Cox & Snell R-squared value. The final phase
involved testing the remaining variables for potential col-
linearity followed by step-wise logistic regression. The tion rates and higher crime rates compared to the country
overall (p < 0.001 and p = 0.025, respectively). Page 7 of 12
(page number not for citation purposes) Factors related to program implementation: logistic
regression analysis To examine the impact of these measures on program
implementation, a logistic regression model was esti-
mated. The logistic equation presented in Tables 7 was
specified as: implementation = educational attainment + pro-
gram-related self-efficacy + fitness certification/credential +
support-focused leadership style + previous strength training
experience + age. These data revealed that leaders who have
a fitness credential are approximately twice as likely [OR
= 2.3, 95% CI = 1.3–3.9, p = 0.003] to implement the pro-
gram, and that support-focused leadership style and
greater levels of program-related self-efficacy (as meas-
ured by the scores previously described) increase the like-
lihood of implementation (p = 0.006 and p = 0.002,
respectively). Additionally, higher educational attainment
was negatively associated with program implementation
(p = 0.002). Results are shown in Table 7. Page 7 of 12
(page number not for citation purposes) Page 7 of 12
(page number not for citation purposes) nternational Journal of Behavioral Nutrition and Physical Activity 2008, 5:62 International Journal of Behavioral Nutrition and Physical Activity 2008, 5:62 http://www.ijbnpa.org/content/5/1/62 Table 4: Leadership Characteristics: Support, Self-Identification, Comfort, and Self-Efficacy
Non-Implementers
N = 217
Implementers
N = 270
P-Value
Friends/family/supervisor support of program involvement, %a
93
100
<0.001
Reason for workshop attendance, %b
- Supervisor's suggestion
9
14
0.074
- Personal health reasons
32
19
0.002
- To implement a program
41
49
0.097
- Other
18
18
0.741
Program related self-efficacy, mean (SD)c
2.76 (0.60)
2.93 (0.29)
0.003
Self-identify as a "leader", %b
86
94
0.049
Comfortable leading friends or strangers in an activity, %b
95
99
0.007
Due to the nature of survey data, sample size varies by question. Range is noted below. a – sample size for non-implementers, n = 146; implementers, n = 238;
b – sample size range for non-implementers, n = 202–215; implementers, n = 250–265
c – sample size for non-implementers, n = 168; implementers, n = 229 Table 4: Leadership Characteristics: Support, Self-Identification, Comfort, and Self-Efficacy Friends/family/supervisor support of program involvement, %a support, comfort leading groups, and leader self-identifi-
cation were higher among implementers compared to
non-implementers. final model presented (Table 7) is significant (p < 0.001)
with a -2 log likelihood of 379.5 and a Cox & Snell R-
squared value of 0.125, suggesting that this model may
explain approximately 12.5% of the variability in imple-
mentation status. Factors related to program implementation: logistic
regression analysis The social ecological model provided a valuable frame-
work to categorize variables related to leader implementa-
tion. These findings demonstrated that both individual
and interpersonal levels of influence were important. Fit-
ness credentials/certification is an individual leadership
characteristic specific to the implementation of this pro-
gram. For example, leaders in this sample have a variety of
professional expertise – from physical and occupation
therapy to nursing, dietetics, and chiropractics [6]. In the
SWP, fitness credentials are relevant to the program, yet
they may not be for other health-promotion/community-
based programs. However, it may be that, in general, pro-
gram-specific training or experience plays an important
role in leader confidence as well as competence around
the planning, organization, and administration, and/or in
execution of the implementing the program and sustain-
ing it. International Journal of Behavioral Nutrition and Physical Activity 2008, 5:62 International Journal of Behavioral Nutrition and Physical Activity 2008, 5:62 http://www.ijbnpa.org/content/5/1/62 asked in a general context. Respondents were asked to
identify potential leadership strengths and weaknesses of
their own leadership characteristics in terms of organiza-
Table 5: Program Characteristics (Implementers Only)a
Mean (SD)
Duration of program session, weeks
10 (2.5)
Days per week classes meet
2 (0.6)
Length of class sessions, minutes
57 (12)
Number of participants per class
13 (11)
Percent
Peer leader helps with programs, %
72
Attendance rate of participants, %
84
Reason for implementation, %
- To help others
38
- New professional goal
19
- Supervisor's suggestion
12
- Community involvement
11
- Other
20
Reason for continuing to run the program, %b
- To help others
38
- I enjoy strength training
13
- Community involvement
13
- New professional goal
7
- Supervisor request
6
- Other
23
Reason for no longer leading the program, %c
- A volunteer/colleague took over
27
- Due to time/scheduling conflicts
20
- Not enough participant interest
10
- Job no longer supportive
7
- Other
36
Compensation for running the program, %
- It's part of my job/salary
51
- It's 100% volunteer (no pay)
38
- Other
11
Due to the nature of survey data, sample size varies by question. Range is noted below. a – sample size range: n = 246–270 (unless noted otherwise below)
b – sample size: n = 206;
c – sample size: n = 103 Table 5: Program Characteristics (Implementers Only)a three categories of leadership were association with
implementation. As leaders trained in a community-
based health program, support-focused leadership style
was a likely characteristic [31]. Future studies might exam-
ine similar categories among implementers of other
health programs to determine if support-focused leader-
ship remains an important factor. If so, it would be bene-
ficial to include strategies for improving support-focused
leadership during trainings, and to consider this leader-
ship characteristic in selection when necessary. three categories of leadership were association with
implementation. As leaders trained in a community-
based health program, support-focused leadership style
was a likely characteristic [31]. Future studies might exam-
ine similar categories among implementers of other
health programs to determine if support-focused leader-
ship remains an important factor. International Journal of Behavioral Nutrition and Physical Activity 2008, 5:62 If so, it would be bene-
ficial to include strategies for improving support-focused
leadership during trainings, and to consider this leader-
ship characteristic in selection when necessary. In this study, educational attainment was inversely associ-
ated with program implementation. This was an interest-
ing finding and counterintuitive to other findings [3]. However, this is a highly educated cohort, with greater
than 82% of individuals having a bachelors degree or
higher. In some cases, more than one individual from the
same organization was trained. It could be that those in
the masters or higher education level category were at the
director or administrator level in the organization and
therefore their job responsibilities were to oversee pro-
grams but not to actually implement them. While not
"implementers" themselves, these individuals may have
provided mentorship and resource support to leaders who
did implement the program, thus acting as critical pro-
gram advocates who played an essential role in the path-
way to implementation. Qualitative data collected in a
related study may provide clarification in this area (publi-
cation forthcoming). The primary limitation of this study is the convenience
sample and cross-sectional design. This design and sam-
pling structure dictate that all findings are associations in
which causality cannot be inferred or implied, and that
results cannot be extrapolated to other populations or
programs. In future survey research of a similar nature, it
would be optimal to administer the survey prior to and
following a potential implementation timeframe. Specifi-
cally with regard to the sample: they were predominantly
women who were mostly white and of relatively high soci-
oeconomic status (SES). Despite this, there is a range
within SES. Additionally, the national representation of
leaders with varying personal and educational degrees is
beneficial for understanding common factors across pro-
fessions, geographical regions, and urban/rural locations. An additional limitation of this study is response bias. Response bias is a consideration for survey research,
although the response rate was 57%. It is helpful that
response rate does not appear biased by implementation,
as it is similar among implementers and non-implement-
ers. asked in a general context. Respondents were asked to
identify potential leadership strengths and weaknesses of
their own leadership characteristics in terms of organiza-
tion, communications, conflict resolution, and providing
support, which are commonly examined factors in deter-
mining leadership style [17,22,27,40]. Discussion The mission of public health is to prevent disease and pro-
mote health in the greater population through the ongo-
ing collection of health-related data; providing sound
health information, resources, and recommendations;
and supporting the implementation and dissemination of
public health initiatives and programming. While we
understand the importance of physical activity participa-
tion and have data to support the feasibility and benefits
of community-based programming [6,12,29,45-52], there
is limited evidence related to optimizing implementation
rates by leaders. This study sought to describe the charac-
teristics of program leaders from a nationally dissemi-
nated program, and to identify and understand factors
that help community-based leaders apply what they learn
through curricula and trainings to successfully implement
programs. The self-efficacy score was derived from three questions in
which leaders rated their confidence to overcome social,
logistical, and physical challenges related to program
implementation. Program-related self-efficacy was signifi-
cantly higher among implementers, which is consistent
with previous findings [16,31,53], although scores were
quite high among non-implementers as well. Prospective
studies would help clarify whether the act of implementa-
tion itself increased implementers' self-efficacy scores. Of approximately forty distinct factors examined – includ-
ing a range of socioeconomic, professional, personal/
behavioral, leadership, and community variables – these
data revealed that fitness credentials, support-focused
leadership style, and greater levels of program-related self-
efficacy were positively associated with program imple-
mentation, while higher levels of educational attainment
were negatively associated with implementation in a
logistic regression analysis. Additionally, in chi square
group-level comparisons, physical activity level, perceived The leadership assessment was asked separately from pro-
grammatic questions, and this category of questions was Page 8 of 12
(page number not for citation purposes) Page 8 of 12
(page number not for citation purposes) http://www.ijbnpa.org/content/5/1/62 International Journal of Behavioral Nutrition and Physical Activity 2008, 5:62 International Journal of Behavioral Nutrition and Physical Activity 2008, 5:62 In this study, indi-
viduals whose leadership style focused on providing
support were positively associated with implementation
in the logistic regression analysis, while none of the other asked in a general context. Respondents were asked to
identify potential leadership strengths and weaknesses of
their own leadership characteristics in terms of organiza-
tion, communications, conflict resolution, and providing
support, which are commonly examined factors in deter-
mining leadership style [17,22,27,40]. In this study, indi-
viduals whose leadership style focused on providing
support were positively associated with implementation
in the logistic regression analysis, while none of the other Although these data provide valuable insights into initial
program implementation, there are a number of ques-
tions related to program adoption and sustained delivery Page 9 of 12
(page number not for citation purposes) Page 9 of 12
(page number not for citation purposes) International Journal of Behavioral Nutrition and Physical Activity 2008, 5:62 http://www.ijbnpa.org/content/5/1/62 Table 6: Leader Communities: Individual, Community-level, and National Demographic Comparisonsa,b
Individual level
(all trained leaders)
Community-level
(leaders' reported zip code)
National-level
(zip code data, 2004 US Census)
Mean (SD)
Mean (SD)
Mean (SD)
Education levelc
4.3 (0.84)
2.68 (0.42)
2.48 (0.44)
Household incomed
3.33 (1.14)
2.27 (0.55)
2.20 (0.57)
Race (% white)
93.47 (24.70)
78.5 (15.80)
75.1 (22.90)
Voter participation
-
60.73 (8.55)
58.85 (9.88)
Violent crimes per 100,000 people
-
1153 (714)
1070 (837)
a. Individual data as reported on survey; community-level by reported corresponding zip code and national means [37,42,43]. b. All values for each category at all levels are different (p ≤ 0.025). c. Education score corresponds to the five education categories described in the methods section; details shown in manuscript 2. d. Income score corresponds to the five income categories described in the methods section; details shown in manuscript 2. Table 6: Leader Communities: Individual, Community-level, and National Demographic Comparisonsa,b tions, and effective leadership at the community level. Without leadership to initiate, motivate, and sustain phys-
ical activity programs, the causal pathway to behavior
change among community members is interrupted, mini-
mizing the efficacy of programs and reducing the likeli-
hood of community and individual change [4,32,56,57]. (i.e. utilization of the RE-AIM Framework) that would
provide a fruitful area of further investigation [54,55]. The
strengths of these data are that multiple factors from a
broad range of categories were examined in the context of
a nationally disseminated community-based exercise pro-
gram. International Journal of Behavioral Nutrition and Physical Activity 2008, 5:62 The data offer a comprehensive analysis at the
leader level, rather than at the participant outcome-related
level, which is perhaps where participant exposure and
program success begins. Implementation is an important step to increasing access
to community programs, and ultimately, to increasing
participation. Thus, optimizing implementation rates
through leader recruitment and training are viable strate-
gies to achieve that goal. These findings may also help
administrators to improve criteria for leader selection, as
good leaders are difficult to recruit and maintain. Conclusion Studies on other social change issues have identified a
number of factors that are associated with sustained
change. These include policy changes, strategic collabora- Table 7: Logistic Regression Model: Factors Related to Program Implementationa
Variables
Odd Ratio
95% CI
p-value
Educational attainmentb
0.575
0.408
0.810
0.002
Program-related self-efficacyc
2.853
1.483
5.488
0.002
Fitness certification/credentialb
2.265
1.319
3.891
0.003
Support-focused leadership styleb
1.305
1.078
1.580
0.006
Previous strength training experienced
0.758
0.459
1.251
0.278
Age (years)b
1.001
0.979
1.023
0.917
Constant
0.568
0.663
a sample size: n = 314
bmeasured as shown in table 1
cmeasured as shown in table 3
dmeasured as shown in table 2 Table 7: Logistic Regression Model: Factors Related to Program Implementationa
Variables
Odd Ratio
95% CI
p-value
Educational attainmentb
0.575
0.408
0.810
0.002
Program-related self-efficacyc
2.853
1.483
5.488
0.002
Fitness certification/credentialb
2.265
1.319
3.891
0.003
Support-focused leadership styleb
1.305
1.078
1.580
0.006
Previous strength training experienced
0.758
0.459
1.251
0.278
Age (years)b
1.001
0.979
1.023
0.917
Constant
0.568
0.663
a sample size: n = 314
bmeasured as shown in table 1
cmeasured as shown in table 3 e 7: Logistic Regression Model: Factors Related to Program Implementationa nternational Journal of Behavioral Nutrition and Physical Activity 2008, 5:62 http://www.ijbnpa.org/content/5/1/62 community strength training program. Prev Chronic Dis 2008,
5(1):A25. This study's theoretical model offers a contextual frame-
work of factors related to leader implementation, and
contributes knowledge to advance research related to
future community-based programs. Utilizing lessons
learned from this study, future research – particularly pro-
spective studies – examining a similar social ecological
framework that includes personal/behavioral, socioeco-
nomic, professional, leadership, and community factors
in other programs and in other populations are clearly
warranted. Studies including men and individuals with
greater socioeconomic and racial diversity (e.g. low
income and non-white populations) would be beneficial
to the literature and for applications in a variety of public
health programs and settings. community strength training program. Prev Chronic Dis 2008,
5(1):A25. ( )
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scientist can read your work free of charge
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Your research papers will be:
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BioMedcentral Publish with BioMed Central and every
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https://journalofinequalitiesandapplications.springeropen.com/track/pdf/10.1186/s13660-015-0830-1
|
English
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Some extensions for Geragthy type contractive mappings
|
Journal of inequalities and applications
| 2,015
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R ES EA RCH Open Access © 2015 Karapınar et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, pro-
vided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and
indicate if changes were made. Erdal Karapınar1,2*, Hamed H Alsulami2 and Maha Noorwali2 *Correspondence:
erdalkarapinar@yahoo.com
1Department of Mathematics,
Atilim University, ˙Incek, Ankara,
06836, Turkey
2Nonlinear Analysis and Applied
Mathematics Research Group
(NAAM), King Abdulaziz University,
Jeddah, Saudi Arabia Abstract In this paper, we establish some fixed point theorems on some extensions of
Geragthy contractive type mappings in the context of b-metric-like spaces. MSC: 46T99; 46N40; 47H10; 54H25
Keywords: b-metric-like space; fixed point; α-admissible; contractive mapping In this paper, we establish some fixed point theorems on some extensions of
Geragthy contractive type mappings in the context of b-metric-like spaces. MSC: 46T99; 46N40; 47H10; 54H25 In this paper, we establish some fixed point theorems on some extensions of
Geragthy contractive type mappings in the context of b-metric-like spaces. Keywords: b-metric-like space; fixed point; α-admissible; contractive mapping 1 Introduction and preliminaries Karapınar et al. Journal of Inequalities and Applications ( 2015) 2015:303
DOI 10.1186/s13660-015-0830-1 Karapınar et al. Journal of Inequalities and Applications ( 2015) 2015:303
DOI 10.1186/s13660-015-0830-1 Karapınar et al. Journal of Inequalities and Applications ( 2015) 2015:303
DOI 10.1186/s13660-015-0830-1 Some extensions for Geragthy type
contractive mappings Erdal Karapınar1,2*, Hamed H Alsulami2 and Maha Noorwali2 1 Introduction and preliminaries One of the interesting extensions of the notion of a metric space is the dislocated space,
introduced by Hitzler []. This notion was rediscovered by Amini-Harandi [] and given
the name of a metric-like space. Definition .On a nonempty set X we define a function σ : X × X →[,∞) such that Definition .On a nonempty set X we define a function σ : X × X →[,∞) such that
for all x,y,z ∈X: Definition .On a nonempty set X we define a function σ : X × X →[,∞) such that
for all x,y,z ∈X:
(σ) if σ(x,y) = then x = y;
(σ) σ(x,y) = σ(y,x);
(σ) σ(x,y) ≤σ(x,z) + σ(z,y); Definition .On a nonempty set X we define a function σ : X × X →[,∞) such that
for all x,y,z ∈X: for all x,y,z ∈X: (σ) if σ(x,y) = then x = y;
(σ) σ(x,y) = σ(y,x);
(σ) σ(x,y) ≤σ(x,z) + σ(z,y); and the pair (X,σ) is called a dislocated (metric-like) space. and the pair (X,σ) is called a dislocated (metric-like) space. and the pair (X,σ) is called a dislocated (metric-like) space. Throughout this paper, we suppose that N= N ∪{} where N denotes the set of all
positive integers. Further, the symbols R+ and R+
denotes the set of positive reals and the
set of non-negative reals. First, we recall some basic concepts and notations. The concept of a b-metric was introduced by Czerwik [] as a generalization of the met-
ric (see also Bakhtin [] and Bourbaki []) to extend the celebrated Banach contraction
mapping principle. Following this initial paper of Czerwik [], a number of researchers
in nonlinear analysis investigated the topology of the paper and proved several fixed
point theorems in the context of complete b-metric spaces (see e.g. [–] and references
therein). Definition .[] Let X be a nonempty set and s ≥be a given real number. A mapping
d: X × X →[,∞) is said to be a b-metric if for all x,y,z ∈X the following conditions are
satisfied: (bM) d(x,y) = if and only if x = y;
(bM) d(x,y) = d(y,x); (bM) d(x,y) = if and only if x = y; (bM) d(x,y) = if and only if x = y; (bM) d(x,y) = d(y,x); Karapınar et al. Journal of Inequalities and Applications ( 2015) 2015:303 Page 2 of 22 (bM) d(x,z) ≤s[d(x,y) + d(y,z)]. In this case, the pair (X,d) is called a b-metric space (with constant s). In what follows, we recall the notion of b-metric-like space which is an interesting gen-
eralization of both b-metric space and metric-like space. Definition .[] Let X be a nonempty set and s ≥be a given real number. A mapping
d: X × X →[,∞) is said to be b-metric-like if for all x,y,z ∈X the following conditions
are satisfied: (bML) if d(x,y) = then x = y;
(bML) d(x,y) = d(y,x);
(bML) d(x,z) ≤s[d(x,y) + d(y,z)]. (bML) if d(x,y) = then x = y;
(bML) d(x,y) = d(y,x);
(bML) d(x,z) ≤s[d(x,y) + d(y,z)]. In this case, the pair (X,d) is called a b-metric-like space (with constant s). In this case, the pair (X,d) is called a b-metric-like space (with constant s). Example .Let X = C([,T]) be the set of all real continuous functions on the closed
interval [,T]. Let d : X × X →R+
be defined Example .Let X = C([,T]) be the set of all real continuous functions on the closed
interval [,T]. Let d : X × X →R+
be defined Example .Let X = C([,T]) be the set of all real continuous functions on the closed
interval [,T]. Let d : X × X →R+
be defined d(f ,g) = max
f (t) – g(t)
p + a, for all f ,g ∈X, a ∈R+
, and p > . It is easy to see that (X,d) is a complete b-metric-like
space with s = p–. For more examples, see e.g. []. Remark .Let (X,d) be a b-metric-like space with constant s ≥. Then it is clear that
ds(x,y) = |d(x,y) – d(x,x) – d(y,y)| satisfies the following:
(S) ds(x,x) = for all x ∈X. Remark .Let (X,d) be a b-metric-like space with constant s ≥. Then it is clear that
ds(x,y) = |d(x,y) – d(x,x) – d(y,y)| satisfies the following:
(S) ds(x,x) = for all x ∈X. Definition .[] Let (X,d) be a b-metric-like space. Then: Definition .[] Let (X,d) be a b-metric-like space. Then: () a sequence {xn} in X is called convergent to x ∈X if and only if
limn→∞d(xn,x) = d(x,x); () a sequence {xn} in X is called Cauchy sequence if and only if limn,m→∞d(xn,xm)
exists and finite; () (X,d) is complete if and only if every Cauchy sequence {xn} in X converges to x ∈X
so that () (X,d) is complete if and only if every Cauchy sequence {xn} in X converges to x ∈X
so that lim
n→∞d(xn,x) = d(x,x) =
lim
m,n→∞d(xn,xm). Proposition .[] Let (X,d) be a b-metric-like space with constant s and let {xn} be a
sequence in X such that limn→∞d(xn,x) = . Then: Proposition .[] Let (X,d) be a b-metric-like space with constant s and let {xn} be a
sequence in X such that limn→∞d(xn,x) = . Then: () x is unique. ()
sd(x,y) ≤limn→∞d(xn,y) ≤sd(x,y) for all y ∈X. ()
sd(x,y) ≤limn→∞d(xn,y) ≤sd(x,y) for all y ∈X. Lemma .[] Let (X,d) be a b-metric-like space with constant s and {xn} a sequence in
X such that Lemma .[] Let (X,d) be a b-metric-like space with constant s and {xn} a sequence in
X such that X such that d(xn+,xn+) ≤kd(xn,xn+),
n = ,,..., where ≤k and sk < . Then {xn} is a Cauchy sequence in X and limn,m→∞d(xn,xm) = . where ≤k and sk < . Then {xn} is a Cauchy sequence in X and limn,m→∞d(xn,xm) = . Karapınar et al. Journal of Inequalities and Applications ( 2015) 2015:303 Page 3 of 22 Lemma .[] Let (X,d) be a b-metric-like space with constant s and assume that {xn}
and {yn} are sequences in X converging to x and y, respectively. Then
sd(x,y) –
s d(x,x) – d(y,y) ≤liminf
n→∞d(xn,yn) ≤limsup
n→∞
d(xn,yn)
≤sd(x,x) + sd(y,y) + sd(x,y). ≤sd(x,x) + sd(y,y) + sd(x,y). In particular, if d(x,y) = then limn→∞d(xn,yn) = . Moreover, for each z ∈X we have Moreover, for each z ∈X we have
s d(x,z) – d(x,x) ≤liminf
n→∞d(xn,z) ≤limsup
n→∞
d(xn,z) ≤sd(x,z) + sd(x,x). () () In particular, if d(x,x) = , then
s d(x,z) ≤liminf
n→∞d(xn,z) ≤limsup
n→∞
d(xn,z) ≤sd(x,z).
s d(x,z) ≤liminf
n→∞d(xn,z) ≤limsup
n→∞
d(xn,z) ≤sd(x,z). Notice that, in general, a b-metric-like mapping does not need to be continuous. Definition .[] Let (X,d) be a b-metric-like space. Then: The notion of α-admissible and triangular α-admissible mappings were introduced by
Samet et al. [] and Karapınar et al. [], respectively. Definition .Let T : X →X be a mapping and α : X × X →[,∞) be a function. We
say that T is an α-admissible mapping if x,y ∈X,
α(x,y) ≥
⇒
α(Tx,Ty) ≥. Moreover, a self-mapping T is called triangular α-admissible if T is α-admissible and Moreover, a self-mapping T is called triangular α-admissible if T is α-admissible and
x,y,z ∈X,
α(x,z) ≥and α(z,y) ≥
⇒
α(x,y) ≥. For more details on α-admissible and triangular α-admissible mappings, see e.g. [–]. Very recently, Popescu [] refined the notion of triangular α-orbital admissible as fol-
lows. Definition .[] Let T : X →X be a mapping and α : X × X →[,∞) be a function. We say that T is α-orbital admissible if α(x,Tx) ≥
⇒
α
Tx,Tx
≥. α(x,Tx) ≥
⇒
α
Tx,Tx
≥. Furthermore, T is called triangular α-orbital admissible if T is α-orbital admissible and Furthermore, T is called triangular α-orbital admissible if T is α-orbital admissible and α(x,y) ≥and α(y,Ty) ≥
⇒
α(x,Ty) ≥. As mentioned in [] each α-admissible (respectively, triangular α-admissible) mapping
is an α-orbital admissible (respectively, triangular α-orbital admissible) mapping. In the
following example we shall show that the converse is not true. Karapınar et al. Journal of Inequalities and Applications ( 2015) 2015:303 Page 4 of 22 Example .Let X = {a,b,c,d,e,f ,g,h}. We define a self-mapping T : X →X such that
Tx = x, for x = a,d and Example .Let X = {a,b,c,d,e,f ,g,h}. We define a self-mapping T : X →X such that
Tx = x, for x = a,d and Tx = y
for (x,y) ∈
(b,c),(c,b),(e,f ),(f ,e),(g,h),(h,g)
. Moreover, we define α : X × X →R+
, such that α(x,y) =
if (x,y) ∈{(a,b),(a,c),(b,b),(c,c),(b,c),(c,b),(b,d),(c,d),(d,e)},
otherwise. Note that T is α-orbital admissible, since α(b,Tb) = α(b,c) = and α(c,Tc) = α(c,b) = . On the other hand, we have α(d,e) = , but α(Td,Te) = α(d,f ) = . Hence, T is not α-
admissible. Lemma .[] Let T : X →X be a triangular α-orbital admissible mapping. Assume
that there exists x∈X such that α(x,Tx) ≥. Define a sequence {xn} by xn+= Txn for
each n ∈N. We characterize the notion of α-regular in the setting of a b-metric-like space. Definition .(cf. []) Let (X,d) be a b-metric-like space, X is said to be α-regular, if
for every sequence {xn} in X such that α(xn,xn+) ≥(respectively, α(xn+,xn) ≥) for all n
and xn →x ∈X as n →∞, there exists a subsequence {xnk} of {xn} such that α(xnk,x) ≥
(respectively, α(x,xnk) ≥) for all k. In this paper, we shall prove the existence and uniqueness of a fixed point for certain
operators in the setting of b-metric-like spaces. The presented results improve, extend,
and unify a number of existing results in the literature. Definition .[] Let (X,d) be a b-metric-like space. Then: Then we have α(xn,xm) ≥for all m,n ∈N with n < m. Lemma .Let T : X →X be a triangular α-orbital admissible mapping. Assume that
there exists x∈X such that α(Tx,x) ≥. Define a sequence {xn} by xn+= Txn for each
n ∈N. Then we have α(xm,xn) ≥for all m,n ∈N with n < m. Lemma .Let T : X →X be a triangular α-orbital admissible mapping. Assume that
there exists x∈X such that α(Tx,x) ≥. Define a sequence {xn} by xn+= Txn for each
n ∈N. Then we have α(xm,xn) ≥for all m,n ∈N with n < m. We characterize the notion of α-regular in the setting of a b-metric-like space. 2 Main result for b-metric-like spaces In this section, we shall state and prove our main results. First, we recall the following
classes of auxiliary functions. Let be the set of all increasing and continuous functions
ψ : [,∞) →[,∞) with ψ–({}) = {}. Let Fs be the family of all functions β : [,∞) →
[,
s) which satisfy the condition [,
s) which satisfy the condition lim
n→∞β(tn) =
s
implies
lim
n→∞tn = ,
() lim
n→∞β(tn) =
s
implies
lim
n→∞tn = ,
()
≥ lim
n→∞β(tn) =
s
implies
lim
n→∞tn = , () for some s ≥. Definition .Let (X,d) be a b-metric-like space with constant s ≥, and T : X →X be
a map. We say that T is a generalized almost α-ψ-φ-Geraghty contractive type mapping
if there exist a function α : X × X →[,∞), ψ,φ ∈, β ∈Fs, and some L ≥such that () α(x,y)ψ
sd(Tx,Ty)
≤β
ψ
M(x,y)
ψ
M(x,y)
+ Lφ
N(x,y)
,
() α(x,y)ψ
sd(Tx,Ty)
≤β
ψ
M(x,y)
ψ
M(x,y)
+ Lφ
N(x,y)
, alities and Applications ( 2015) 2015:303
Page 5 of 22 Karapınar et al. Journal of Inequalities and Applications ( 2015) 2015:303 Karapınar et al. Journal of Inequalities and Applications ( 2015) 2015:303
Page 5 of 22 Karapınar et al. Journal of Inequalities and Applications ( 2015) 2015:303
Page 5 of 22 Karapınar et al. Journal of Inequalities and Applications ( 2015) 2015:303 Karapınar et al. Journal of Inequalities and Applications ( 2015) 2015:303 Page 5 of 22 all x,y ∈X, where for all x,y ∈X, where M(x,y) = max
d(x,y),d(x,Tx),d(y,Ty), d(x,Ty) + d(y,Tx)
s
and
()
N(x,y) = min
ds(x,Ty),ds(y,Tx),d(x,Tx),d(y,Ty)
. () () Remark .Since the functions belonging to Fs are strictly smaller than
s , for some s ≥,
the expression β(ψ(M(x,y))) in () can be estimated from above as follows: Remark .Since the functions belonging to Fs are strictly smaller than
s , for some s ≥,
the expression β(ψ(M(x,y))) in () can be estimated from above as follows: β
ψ
M(x,y)
<
s
for any x,y ∈X. β
ψ
M(x,y)
<
s
for any x,y ∈X. 2 Main result for b-metric-like spaces Theorem .Let (X,d) be a complete b-metric-like space with constant s ≥and T :
X →X be a generalized almost α-ψ-φ-Geraghty contractive type mapping. We suppose
also that (i) T is triangular α-orbital admissible; (i) T is triangular α-orbital admissible; (i) T is triangular α-orbital admissible; (i) T is triangular α-orbital admissible; (ii) there exists x∈X such that α(x,Tx) ≥; (iii) T is continuous. Then T has a fixed point, u ∈X with d(u,u) = . Proof By (ii) there exists x∈X such that α(x,Tx) ≥. Define a sequence {xn} ⊂X by
xn+= Txn for all n ∈N. As T is triangular α-orbital admissible, by Lemma .we have
α(xn,xn+) ≥for all n ∈N. Throughout the proof, we suppose that xn ̸= xn+for all n ∈
N. Indeed, if there exists nsuch that xn= xn+, then xnbecomes the fixed point of T,
which completes the proof. Since T is a generalized almost α-ψ-φ-Geraghty contractive type mapping we have ψ
sd(xn+,xn+)
≤α(xn,xn+)ψ
sd(Txn,Txn+)
≤β
ψ
M(xn,xn+)
ψ
M(xn,xn+)
+ Lφ
N(xn,xn+)
. () () Thus, we have Thus, we have ψ
sd(xn+,xn+)
<
s ψ
M(xn,xn+)
+ Lφ
N(xn,xn+)
,
() () () where N(xn,xn+) = min{ds(xn,xn+),ds(xn+,xn+),d(xn,xn+),d(xn+,xn+)} = , and M(xn,xn+) = max
d(xn,xn+),d(xn,xn+),d(xn+,xn+), d(xn,xn+) + d(xn+,xn+)
s
. Note that Note that Page 6 of 22 Karapınar et al. Journal of Inequalities and Applications ( 2015) 2015:303 Karapınar et al. Journal of Inequalities and Applications ( 2015) 2015:303 Consequently, we have Consequently, we have M(xn,xn+) = max
d(xn,xn+),d(xn+,xn+)
. If M(xn,xn+) = d(xn+,xn+), then from () we have ψ
sd(xn+,xn+)
<
s ψ
d(xn+,xn+)
≤ψ
d(xn+,xn+)
. ψ
sd(xn+,xn+)
<
s ψ
d(xn+,xn+)
≤ψ
d(xn+,xn+)
. Since ψ is increasing, we derive that sd(xn+,xn+) < d(xn+,xn+), which is a contradiction
as s ≥. Thus, M(xn,xn+) = d(xn,xn+). Again by (), we find ψ
sd(xn+,xn+)
<
s ψ
d(xn,xn+)
≤ψ
d(xn,xn+)
. Hence, we get ψ
sd(xn+,xn+)
<
s ψ
d(xn,xn+)
≤ψ
d(xn,xn+)
. Hence, we get sd(xn+,xn+) ≤d(xn,xn+)
equivalently
d(xn+,xn+) ≤
sd(xn,xn+). () () Case (i): s > . 2 Main result for b-metric-like spaces Since
s> and s
s=
s < , by Lemma ., the sequence {xn} is Cauchy
and lim
n,m→∞d(xn,xm) = . () () lim
n,m→∞d(xn,xm) = . lim
n,m→∞d(xn,xm) = . Case (ii): s = . From (), we have d(xn+,xn+) ≤d(xn,xn+) for all n. Thus, we conclude
that lim
n→∞d(xn,xn+) = r, lim
n→∞d(xn,xn+) = r, () lim
n→∞d(xn,xn+) = r,
() for some r ≥. We shall prove that r = . Suppose, on the contrary, that r > . Note that,
for s = , the inequality () turns into ψ
d(xn+,xn+)
< β
ψ
M(xn,xn+)
ψ
M(xn,xn+)
+ Lφ
N(xn,xn+)
,
() () where N(xn,xn+) = and M(xn,xn+) = d(xn,xn+) as evaluated above. Thus, () yields ψ(d(xn+,xn+))
ψ(d(xn,xn+)) ≤β
ψ
d(xn,xn+)
< . () () By taking the limit as n →∞in () and regarding the continuity of ψ, we get By taking the limit as n →∞in () and regarding the continuity of ψ, we get lim
n→∞β
ψ
d(xn,xn+)
= . lim
n→∞β
ψ
d(xn,xn+)
= . Hence, we have Hence, we have lim
n→∞ψ
d(xn,xn+)
=
and so
lim
n→∞d(xn,xn+) = . lim
n→∞ψ
d(xn,xn+)
=
and so
lim
n→∞d(xn,xn+) = . Karapınar et al. Journal of Inequalities and Applications ( 2015) 2015:303
Page 7 of 22 Karapınar et al. Journal of Inequalities and Applications ( 2015) 2015:303 Page 7 of 22 Consequently r = . In what follows, we shall prove that {xn} is a Cauchy sequence. Indeed
we will prove that limm,n→∞d(xn,xm) = . Suppose, on the contrary, that there exist ε >
and corresponding subsequences {nk} and {mk} of N satisfying nk > mk > k for which () d(xmk,xnk) ≥ε,
() d(xmk,xnk) ≥ε, where nk, mk are chosen as the smallest integers satisfying (), that is, where nk, mk are chosen as the smallest integers satisfying (), that is, d(xmk,xnk–) < ε. () () d(xmk,xnk–) < ε. By (), (), and the triangle inequality, we easily derive that ε ≤d(xmk,xnk) ≤d(xmk,xnk–) + d(xnk–,xnk) < ε + d(xnk–,xnk). () () Using () and the squeeze theorem we get Using () and the squeeze theorem we get lim
k→∞d(xmk,xnk) = ε. () () lim
k→∞d(xmk,xnk) = ε. 2 Main result for b-metric-like spaces In a similar way, we can prove that limk→∞d(xmk,xnk+) = , limk→∞d(xnk,xmk+) = . Regarding that T is a generalized almost α-ψ-φ-Geraghty contractive type mapping, we
have ψ
d(xmk+,xnk+)
≤α(xmk,xnk)ψ
d(Txmk,Txnk)
≤β
ψ
M(xmk,xnk)
ψ
M(xmk,xnk)
+ Lφ
N(xmk,xnk)
,
() () for all x,y ∈X, where M(xmk,xnk) = max
d(xmk,xnk),d(xmk,xmk+),d(xnk,xnk+),
d(xmk,xnk+) + d(xnk,xmk+)
,
() () and N(xmk,xnk) = min
ds(xmk,xnk+),ds(xnk,xmk+),d(xmk,xmk+),d(xnk,xnk+)
. By taking the limit as k →∞in () and taking (), () into account, we get By taking the limit as k →∞in () and taking (), () into account, we get By taking the limit as k →∞in () and taking (), () into account, we get ψ(ε) ≤lim
k→∞β
ψ
M(xmk,xnk)
ψ(ε). () ψ(ε) ≤lim
k→∞β
ψ
M(xmk,xnk)
ψ(ε). () Since β is a Geraghty function, we derive that ψ(M(xmk,xnk)) →. Consequently, we have
d(xmk,xnk) →, which is a contradiction. Hence, we conclude that limm,n→∞d(xn,xm) = ,
and the sequence {xn} is Cauchy for any s ≥. Since β is a Geraghty function, we derive that ψ(M(xmk,xnk)) →. Consequently, we have
d(xmk,xnk) →, which is a contradiction. Hence, we conclude that limm,n→∞d(xn,xm) = ,
and the sequence {xn} is Cauchy for any s ≥. By completeness of (X,d), there exists u ∈X such that By completeness of (X,d), there exists u ∈X such that lim
n→∞d(xn,u) =
lim
n,m→∞d(xn,xm) = d(u,u) = . Page 8 of 22 Karapınar et al. Journal of Inequalities and Applications ( 2015) 2015:303 Karapınar et al. Journal of Inequalities and Applications ( 2015) 2015:303 Since T is continuous, Tu = T
lim
n→∞xn
= lim
n→∞Txn = lim
n→∞xn+= u, □ and u is a fixed point for T. In what follows, we replace the condition of continuity of the operator by the condition
of α-regularity of the space. Theorem .Let (X,d) be a complete b-metric-like space with constant s ≥and T :
X →X be a generalized almost α-ψ-φ-Geraghty contractive type mapping. We suppose
also that: (i) T is triangular α-orbital admissible; (i) T is triangular α-orbital admissible; (i) T is triangular α-orbital admissible; (i) T is triangular α-orbital admissible; (ii) there exists x∈X such that α(x,Tx) ≥; (iii) X is α-regular and d is continuous. (iii) X is α-regular and d is continuous. 2 Main result for b-metric-like spaces Then T has a fixed point, u ∈X with d(u,u) = . Then T has a fixed point, u ∈X with d(u,u) = . Proof Following the lines of the proof of Theorem ., we conclude that there exists u ∈X
such that lim
n→∞d(xn,u) =
lim
n,m→∞d(xn,xm) = d(u,u) = . Since X is α-regular, α(xn,xn+) ≥for all n. Due to the fact that limn→∞xn = u, there
exists a subsequence {xnk} of {xn} such that α(xnk,u) ≥for all k. To prove that u is a fixed
point for T, suppose on the contrary that d(u,Tu) > . Now, by using the properties of ψ and as T is a generalized almost α-ψ-φ-Geraghty
contractive type mapping we have ψ
d(xnk+,Tu)
≤α(xnk,u)ψ
sd(Txnk,Tu)
≤β
ψ
M(xnk,u)
ψ
M(xnk,u)
+ Lφ
N(xnk,u)
. ψ
d(xnk+,Tu)
≤α(xnk,u)ψ
sd(Txnk,Tu)
Thus we have Thus we have ψ
d(xnk+,Tu)
<
s ψ
M(xnk,u)
+ Lφ
N(xnk,u)
,
() () where
N(xnk,u) = min{ds(xnk,Tu),ds(u,xnk+),d(xnk,xnk+),d(u,Tu)},
and
note
that
limk→∞N(xnk,u) = . Moreover, where
N(xnk,u) = min{ds(xnk,Tu),ds(u,xnk+),d(xnk,xnk+),d(u,Tu)},
and
note
that
limk→∞N(xnk,u) = . Moreover, M(xnk,u) = max
d(xnk,u),d(xnk,xnk+),d(u,Tu), d(xnk,Tu) + d(u,xnk+)
s
≤max
d(xnk,u),d(xnk,xnk+),d(u,Tu),
d(xnk,u) + d(u,Tu) + d(u,xnk) + d(xnk,xnk+)
. M(xnk,u) = max
d(xnk,u),d(xnk,xnk+),d(u,Tu), d(xnk,Tu) + d(u,xnk+)
s ≤max
d(xnk,u),d(xnk,xnk+),d(u,Tu), d(xnk,u) + d(u,Tu) + d(u,xnk) + d(xnk,xnk+)
. alities and Applications ( 2015) 2015:303
Page 9 of 22 Karapınar et al. Journal of Inequalities and Applications ( 2015) 2015:303 Page 9 of 22 Hence Hence lim
k→∞M(xnk,u) ≤max
,d(u,Tu), d(u,Tu)
= d(u,Tu), and by the definition of M(xnk,u) we have limk→∞M(xnk,u) = d(u,Tu). and by the definition of M(xnk,u) we have limk→∞M(xnk,u) = d(u,Tu). By the continuity of ψ and the b-metric-like d, taking the limit as k goes to ∞on both
sides of () we have ψ
d(u,Tu)
≤
s ψ
d(u,Tu)
. Thus = ψ(d(u,Tu))
ψ(d(u,Tu)) ≤
s , which is a contradiction in the case s > . Hence d(u,Tu) = ;
therefore Tu = u. In the case s = we take the limit as k goes to ∞on both sides of ψ
d(xnk+,Tu)
≤β
ψ
M(xnk,u)
ψ
M(xnk,u)
and get limk→∞β(ψ(M(xnk,u))) = and as β ∈Fso we have limk→∞ψ(M(xnk,u)) = . Thus we have d(u,Tu) = ; therefore Tu = u. □ For the uniqueness of a fixed point of a generalized α-ψ-φ contractive mapping, we will
consider the following hypothesis. (H) For all x,y ∈Fix(T), either α(x,y) ≥or α(y,x) ≥. (H) For all x,y ∈Fix(T), either α(x,y) ≥or α(y,x) ≥. Here, Fix(T) denotes the set of fixed points of T. Here, Fix(T) denotes the set of fixed points of T. Theorem .Adding condition (H) to the hypotheses of Theorem .(or Theorem .), we
obtain the uniqueness of the fixed point of T. Proof Suppose that x∗and y∗are two fixed points of T. Then it is obvious that M(x∗,y∗) =
d(x∗,y∗) and N(x∗,y∗) = . Thus we have So, we have ψ
d
x∗,y∗
≤ψ
sd
Tx∗,Ty∗
≤α
x∗,y∗
ψ
sd
Tx∗,Ty∗
≤β
ψ
d
x∗,y∗
ψ
d
x∗,y∗
<
s ψ
d
x∗,y∗
, which is a contradiction. □ which is a contradiction. □ □ which is a contradiction. which is a contradiction. Definition .Let (X,d) be a b-metric-like space with constant s ≥, T : X →X be a
map, we say that T is a generalized rational α-ψ-φ-Geraghty contractive mapping of type
(I) if there exist a function α : X ×X →[,∞), ψ,φ ∈, β ∈Fs, and some L ≥such that α(x,y)ψ
sd(Tx,Ty)
≤β
ψ
K(x,y)
ψ
K(x,y)
+ Lφ
N(x,y)
,
() () for all x,y ∈X, where N(x,y) is defined as in () and for all x,y ∈X, where N(x,y) is defined as in () and K(x,y) = max
d(x,y), d(x,Tx)d(y,Ty)
+ d(x,y)
, d(x,Tx)d(y,Ty)
+ d(Tx,Ty)
. () () Karapınar et al. Journal of Inequalities and Applications ( 2015) 2015:303 Page 10 of 22 Definition .Let (X,d) be a b-metric-like space with constant s ≥, T : X →X be a map,
we say that T is a generalized rational α-ψ-φ-Geraghty contractive of type (II) mapping
if there exist a function α : X × X →[,∞), ψ,φ ∈, β ∈Fs and some L ≥such that α(x,y)ψ
sd(Tx,Ty)
≤β
ψ
Q(x,y)
ψ
Q(x,y)
+ Lφ
N(x,y)
,
() () for all x,y ∈X, where N(x,y) is defined as in () and for all x,y ∈X, where N(x,y) is defined as in () and Q(x,y) = max
d(x,y), d(x,Tx)d(y,Ty) + ds(x,Ty)ds(y,Tx)
+ s(d(x,Tx) + d(y,Ty))
. () () Theorem .Let (X,d) be a complete b-metric-like space with constant s ≥and T : X →
X be a generalized rational α-ψ-φ-Geraghty contractive mapping of type (I) such that:
(i) T is triangular α orbital admissible; Theorem .Let (X,d) be a complete b-metric-like space with constant s ≥and T : X →
X be a generalized rational α-ψ-φ-Geraghty contractive mapping of type (I) such that: (i) T is triangular α-orbital admissible; (i) T is triangular α-orbital admissible; (ii) there exists x∈X such that α(x,Tx) ≥; (ii) there exists x∈X such that α(x,Tx) ≥; (iii) T is continuous. Thus we have Then T has a fixed point, u ∈X with d(u,u) = . Proof We shall use the same techniques as in the proof of Theorem .. First of all, we shall
construct a sequence {xn} ⊂X where xn+= Txn for which α(xn,xn+) ≥and xn ̸= xn+for
all n ∈N. Proof We shall use the same techniques as in the proof of Theorem .. First of all, we shall
construct a sequence {xn} ⊂X where xn+= Txn for which α(xn,xn+) ≥and xn ̸= xn+for
all n ∈N. Since T is generalized rational α-ψ-φ-Geraghty contractive of type (I) we have ψ
sd(xn+,xn+)
≤α(xn,xn+)ψ
sd(Txn,Txn+)
≤β
ψ
K(xn,xn+)
ψ
K(xn,xn+)
+ Lφ
N(xn,xn+)
. () () Since N(xn,xn+) = , the above inequality implies that Since N(xn,xn+) = , the above inequality implies that Since N(xn,xn+) = , the above inequality implies that ψ
sd(xn+,xn+)
≤β
ψ
K(xn,xn+)
ψ
K(xn,xn+)
,
() () where where K(xn,xn+) = max
d(xn,xn+), d(xn,xn+)d(xn+,xn+)
+ d(xn,xn+)
, d(xn,xn+)d(xn+,xn+)
+ d(xn+,xn+)
. On the other hand, we have d(xn,xn+)d(xn+,xn+)
+ d(xn+,xn+)
≤d(xn,xn+)d(xn+,xn+)
d(xn+,xn+)
= d(xn,xn+) and and d(xn,xn+)d(xn+,xn+)
+ d(xn,xn+)
≤d(xn,xn+)d(xn+,xn+)
d(xn,xn+)
= d(xn+,xn+). Consequently, we get K(xn,xn+) ≤max{d(xn,xn+),d(xn+,xn+)}. Consequently, we get K(xn,xn+) ≤max{d(xn,xn+),d(xn+,xn+)}. Consequently, we get K(xn,xn+) ≤max{d(xn,xn+),d(xn+,xn+)}. If max{d(x x
) d(x
x
)} = d(x
x
) then from () together with Remark If max{d(xn,xn+),d(xn+,xn+)} = d(xn+,xn+), then from () together with Remark .,
we have ψ
sd(xn+,xn+)
<
s ψ
d(xn+,xn+)
. alities and Applications ( 2015) 2015:303
Page 11 of 22 Karapınar et al. Journal of Inequalities and Applications ( 2015) 2015:303
Page 11 of 22 Karapınar et al. Journal of Inequalities and Applications ( 2015) 2015:303 Page 11 of 22 This is a contradiction since ψ is increasing. Thus, we have max{d(xn,xn+),d(xn+,xn+)} =
d(xn,xn+) and by the definition of K(xn,xn+) we shall have K(xn,xn+) = d(xn,xn+). Con-
sequently, the inequality () turns into ψ
d(xn+,xn+)
≤ψ
sd(xn+,xn+)
≤β
ψ
K(xn,xn+)
ψ
d(xn,xn+)
. () () By Remark ., we get By Remark ., we get ψ
sd(xn+,xn+)
< ψ
d(xn,xn+)
and hence,d(xn+,xn+) <
sd(xn,xn+). () ψ
sd(xn+,xn+)
< ψ
d(xn,xn+)
and hence,d(xn+,xn+) <
sd(xn,xn+). Thus we have () () Case (i): s > . Since
s> and s
s=
s < , by Lemma ., the sequence {xn} is Cauchy
and lim
n,m→∞d(xn,xm) = . () lim
n,m→∞d(xn,xm) = . lim
n,m→∞d(xn,xm) = . () Case (ii): s = . Since {d(xn,xn+)} is a decreasing sequence, there exists r ≥such that lim
n→∞d(xn,xn+) = r,
() () for some r ≥. We shall prove that r = . Suppose, on the contrary, that r > . By letting
n →∞in () we find ψ(r) ≤lim
n→∞β
ψ
K(xn,xn+)
ψ(r). ψ(r) ≤lim
n→∞β
ψ
K(xn,xn+)
ψ(r). It yields = limn→∞β(ψ(K(xn,xn+))). Since β ∈F, we get ψ(K(xn,xn+)) →, which im-
plies that d(xn,xn+) →, that is, r = . It yields = limn→∞β(ψ(K(xn,xn+))). Since β ∈F, we get ψ(K(xn,xn+)) →, which im-
plies that d(xn,xn+) →, that is, r = . In what follows, we shall prove that {xn} is a Cauchy sequence. Indeed we will prove that
limm,n→∞d(xn,xm) = . Suppose, on the contrary, that there exist ε > and corresponding
subsequences {nk} and {mk} of N satisfying nk > mk > k for which d(xmk,xnk) ≥ε,
() () d(xmk,xnk) ≥ε, ere nk, mk are chosen as the smallest integers satisfying (), that is, where nk, mk are chosen as the smallest integers satisfying (), that is, d(xmk,xnk–) < ε. () () d(xmk,xnk–) < ε. By (), (), and the triangle inequality, we easily derive that By (), (), and the triangle inequality, we easily derive that By (), (), and the triangle inequality, we easily derive that ε ≤d(xmk,xnk) ≤d(xmk,xnk–) + d(xnk–,xnk)
< ε + d(xnk–,xnk). () ε ≤d(xmk,xnk) ≤d(xmk,xnk–) + d(xnk–,xnk) () Using () and the squeeze theorem we get Using () and the squeeze theorem we get Using () and the squeeze theorem we get lim
k→∞d(xmk,xnk) = ε. () () lim
k→∞d(xmk,xnk) = ε. ies and Applications ( 2015) 2015:303
Page 12 of 22 Karapınar et al. Journal of Inequalities and Applications ( 2015) 2015:303 Karapınar et al. Thus we have Journal of Inequalities and Applications ( 2015) 2015:303 Page 12 of 22 Regarding that T is generalized rational α-ψ-φ-Geraghty contractive mapping of
type (I), we have ψ
d(xmk+,xnk+)
≤α(xmk,xnk)ψ
d(Txmk,Txnk)
≤β
ψ
K(xmk,xnk)
ψ
K(xmk,xnk)
+ Lφ
N(xmk,xnk)
,
() () for all x,y ∈X, where K(xmk,xnk) = max
d(xmk,xnk), d(xmk,xmk+)d(xnk,xnk+)
+ d(xmk,xnk)
,
d(xmk,xmk+)d(xnk,xnk+)
+ d(xmk+,xnk+)
() K(xmk,xnk) = max
d(xmk,xnk), d(xmk,xmk+)d(xnk,xnk+)
+ d(xmk,xnk)
, d(xmk,xmk+)d(xnk,xnk+)
+ d(xmk+,xnk+) d(xmk,xmk+)d(xnk,xnk+)
+ d(xmk+,xnk+)
() () and N(xmk,xnk) = min
ds(xmk,xnk+),ds(xnk,xmk+),d(xmk,xmk+),d(xnk,xnk+)
. N(xmk,xnk) = min
ds(xmk,xnk+),ds(xnk,xmk+),d(xmk,xmk+),d(xnk,xnk+)
. It is clear that lim
k→∞K(xmk,xnk) = ε
and
lim
k→∞N(xmk,xnk) = . By taking the limit as k →∞in () and taking (), () into account, we get By taking the limit as k →∞in () and taking (), () into account, we get ψ(ε) ≤lim
k→∞β
ψ
K(xmk,xnk)
lim
k→∞ψ
K(xmk,xnk)
≤lim
k→∞β
ψ
K(xmk,xnk)
ψ(ε). ( () Since β is a Geraghty function, we derive that ψ(K(xmk,xnk)) →. Consequently, we have
d(xmk,xnk) →, which is a contradiction. Hence limm,n→∞d(xn,xm) = , and the sequence
{xn} is Cauchy for any s ≥. Since β is a Geraghty function, we derive that ψ(K(xmk,xnk)) →. Consequently, we have
d(xmk,xnk) →, which is a contradiction. Hence limm,n→∞d(xn,xm) = , and the sequence
{xn} is Cauchy for any s ≥. By completeness of (X,d), there exists u ∈X such that By completeness of (X,d), there exists u ∈X such that lim
n→∞d(xn,u) = d(u,u) =
lim
n,m→∞d(xn,xm) = . Now, if T is continuous, then Tu = T
lim
n→∞xn
= lim
n→∞Txn = lim
n→∞xn+= u, Tu = T
lim
n→∞xn
= lim
n→∞Txn = lim
n→∞xn+= u, and u is a fixed point for T. □ and u is a fixed point for T. Thus we have Theorem .Let (X,d) be a complete b-metric-like space with constant s ≥and T : X →
X be a generalized rational α-ψ-φ-Geraghty contractive of mapping type (I) such that:
( )
l
b
l d
bl Theorem .Let (X,d) be a complete b-metric-like space with constant s ≥and T : X →
X be a generalized rational α-ψ-φ-Geraghty contractive of mapping type (I) such that: (i) T is triangular α-orbital admissible; (ii) there exists x∈X such that α(x,Tx) ≥; (iii) X is α-regular and d is continuous. Then T has a fixed point, u ∈X with d(u,u) = . Then T has a fixed point, u ∈X with d(u,u) = . alities and Applications ( 2015) 2015:303
Page 13 of 22 Karapınar et al. Journal of Inequalities and Applications ( 2015) 2015:303
Page 13 of 22 Karapınar et al. Journal of Inequalities and Applications ( 2015) 2015:303 Page 13 of 22 Proof Verbatim of the proof of Theorem ., we conclude that the iterative sequence {xn}
is Cauchy and converges to u ∈X. Since X is α-regular, then, as in the proof of Theo-
rem ., there exists a subsequence {xnk} of {xn} such that ψ
d(xnk+,Tu)
<
s ψ
K(xnk,u)
+ Lφ
N(xnk,u)
,
() () where K(xnk,u) = max{d(xnk,u),
d(xnk ,xnk+)d(u,Tu)
+d(xnk ,u)
,
d(xnk ,xnk+)d(u,Tu)
+d(xnk+,Tu)
}. where K(xnk,u) = max{d(xnk,u),
d(xnk ,xnk+)d(u,Tu)
+d(xnk ,u)
,
d(xnk ,xnk+)d(u,Tu)
+d(xnk+,Tu)
}. ( nk , )
( nk+,
)
Hence limk→∞K(xnk,u) = and as in the proof of Theorem .limk→∞N(xnk,u) = . ( nk
)
( nk+
)
Hence limk→∞K(xnk,u) = and as in the proof of Theorem . Thus taking the limit as k →∞on both sides of () and keeping in mind that ψ and d
are continuous we have ψ(d(u,Tu)) ≤. Hence d(u,Tu) = ; therefore Tu = u. □ Theorem .Adding condition (H) to the hypotheses of Theorem .(or Theorem .),
we obtain uniqueness of the fixed point of T. Theorem .Adding condition (H) to the hypotheses of Theorem .(or Theorem .),
we obtain uniqueness of the fixed point of T. Proof As in the proof of Theorem ., we suppose that x∗and y∗are two fixed points of T. Then, clearly, we have K(x∗,y∗) = d(x∗,y∗) and N(x∗,y∗) = . Thus we have So, we have Proof As in the proof of Theorem ., we suppose that x∗and y∗are two fixed points of T. Then, clearly, we have K(x∗,y∗) = d(x∗,y∗) and N(x∗,y∗) = . So, we have ψ
d
x∗,y∗
≤ψ
sd
Tx∗,Ty∗
≤α
x∗,y∗
ψ
sd
Tx∗,Ty∗
≤β
ψ
d
x∗,y∗
ψ
d
x∗,y∗
<
s ψ
d
x∗,y∗
, ψ
d
x∗,y∗
≤ψ
sd
Tx∗,Ty∗
≤α
x∗,y∗
ψ
sd
Tx∗,Ty∗
≤β
ψ
d
x∗,y∗
ψ
d
x∗,y∗
<
s ψ
d
x∗,y∗
, ψ
d
x∗,y∗
≤ψ
sd
Tx∗,Ty∗
≤α
x∗,y∗
ψ
sd
Tx∗,Ty∗
≤β
ψ
d
x∗,y∗
ψ
d
x∗,y∗
<
s ψ
d
x∗,y∗
, which is a contradiction. □ which is a contradiction. □ □ which is a contradiction. which is a contradiction. Theorem .Let (X,d) be a complete b-metric-like space with constant s ≥and T :
X →X be a generalized rational α-ψ-φ-Geraghty contractive mapping of type (II) such
that: (i) T is triangular α-orbital admissible; (i) T is triangular α-orbital admissible; (i) T is triangular α-orbital admissible; (ii) there exists x∈X such that α(x,Tx) ≥; (iii) T is continuous. Then T has a fixed point, u ∈X with d(u,u) = . Then T has a fixed point, u ∈X with d(u,u) = . Proof Verbatim of the lines in the proof of Theorem ., we construct a sequence {xn} ⊂X
where xn+= Txn for which α(xn,xn+) ≥and xn ̸= xn+for all n ∈N. Moreover, by using
the fact that T is a generalized rational α-ψ-φ-Geraghty contractive mapping of type (II)
and the property of ψ we have ψ
sd(xn+,xn+)
≤α(xn,xn+)ψ
sd(Txn,Txn+)
≤β
ψ
Q(xn,xn+)
ψ
Q(xn,xn+)
+ Lφ
N(xn,xn+)
. () () By using the same arguments as in the proof of Theorem ., we derive that ψ
sd(xn+,xn+)
<
s ψ
Q(xn,xn+)
,
() ψ
sd(xn+,xn+)
<
s ψ
Q(xn,xn+)
, () Karapınar et al. Thus we have Journal of Inequalities and Applications ( 2015) 2015:303 Page 14 of 22 where Q(xn,xn+) = max
d(xn,xn+), d(xn,xn+)d(xn+,xn+) + ds(xn,xn+)ds(xn+,xn+)
+ s[d(xn,xn+) + d(xn+,xn+)]
. Since d(xn,xn+) ≤s[d(xn,xn+) + d(xn+,xn+)], we have Since d(xn,xn+) ≤s[d(xn,xn+) + d(xn+,xn+)], we have d(xn,xn+) ≥d(xn,xn+)d(xn,xn+)
+ d(xn,xn+)
≥
d(xn,xn+)d(xn,xn+)
+ s[d(xn,xn+) + d(xn+,xn+)]. d(xn,xn+) ≥d(xn,xn+)d(xn,xn+)
+ d(xn,xn+) ≥
d(xn,xn+)d(xn,xn+)
+ s[d(xn,xn+) + d(xn+,xn+)]. Hence, we get Q(xn,xn+) = d(xn,xn+). By using () we get ψ(sd(xn+,xn+)) ≤ψ(d(xn,xn+)). Since ψ is increasing we
have d(xn+,xn+) <
sd(xn,xn+). If s > then, as in the proof of Theorem ., by using
Lemma ., we conclude that {xn} is a Cauchy sequence and limn,m→∞d(xn,xm) = . If
s = , by verbatim of the proof of Theorem ., we deduce that {xn} is a Cauchy sequence. Since (X,d) is complete, there exists u ∈X such that = limn,m→∞d(xn,xm) =
limn→∞d(xn,u) = d(u,u). Now, since T is continuous, Tu = T(limn→∞xn) = limn→∞Txn = By using () we get ψ(sd(xn+,xn+)) ≤ψ(d(xn,xn+)). Since ψ is increasing we
have d(xn+,xn+) <
sd(xn,xn+). If s > then, as in the proof of Theorem ., by using
Lemma ., we conclude that {xn} is a Cauchy sequence and limn,m→∞d(xn,xm) = . If
s = , by verbatim of the proof of Theorem ., we deduce that {xn} is a Cauchy sequence. Since (X,d) is complete, there exists u ∈X such that = limn,m→∞d(xn,xm) =
limn→∞d(xn,u) = d(u,u). Now, since T is continuous, Tu = T(limn→∞xn) = limn→∞Txn =
limn→∞xn+= u and u is a fixed point for T. □ Theorem .Let (X,d) be a complete b-metric-like space with constant s ≥and T :
X →X be a generalized rational α-ψ-φ-Geraghty contractive mapping of type (II) such
that: Theorem .Let (X,d) be a complete b-metric-like space with constant s ≥and T :
X →X be a generalized rational α-ψ-φ-Geraghty contractive mapping of type (II) such
that: (i) T is triangular α-orbital admissible; (i) T is triangular α-orbital admissible; (i) T is triangular α-orbital admissible; (ii) there exists x∈X such that α(x,Tx) ≥; (iii) X is α-regular and d is continuous. Then T has a fixed point, u ∈X with d(u,u) = . Proof By following the proof of Theorem .line by line, we see that {xn} converges to
u ∈X. Due to the fact that X is α-regular and by following the lines of the proof of Theo-
rem .there exists a subsequence {xnk} of {xn} such that ψ
d(xnk+,Tu)
<
s ψ
Q(xnk,u)
+ Lφ
N(xnk,u)
,
() () where where Q(xnk,u) = max
d(xnk,u), d(xnk,xnk+)d(u,Tu) + ds(xnk,Tu)ds(u,xnk+)
+ s[d(xnk,xnk+) + d(u,Tu)]
. Note that limk→∞Q(xnk,u) = and limk→∞N(xnk,u) = . Thus taking the limit as n →
∞on both sides of () and keeping in mind that ψ and d are continuous we have
ψ(d(u,Tu)) = , and so d(u,Tu) = . Thus u = Tu. □ Theorem .Let (X,d) be a complete b-metric-like space with constant s ≥and T :
X →X be a mapping. Suppose that there exist a function α : X × X →[,∞), ψ ∈,
β ∈Fs such that α(x,y)ψ
sd(Tx,Ty)
≤β
ψ
d(x,y)
ψ
d(x,y)
,
() () Karapınar et al. Journal of Inequalities and Applications ( 2015) 2015:303 Page 15 of 22 for all x,y ∈X. Suppose also that:
(i) T is triangular α-orbital admissible;
(ii) there exists x∈X such that α(x,Tx) ≥;
(iii) T is continuous. Then T has a fixed point, u ∈X with d(u,u) = . Then T has a fixed point, u ∈X with d(u,u) = . Proof By (ii) there exists x∈X such that α(x,Tx) ≥. Define a sequence {xn} ⊂X by
xn+= Txn for all n ∈N. As T is triangular α-orbital admissible, by Lemma .we have
α(xn,xn+) ≥for all n ∈N. Notice that if there exists a natural number nsuch that
xn= xn+, then the proof is complete. To avoid this trivial case, from now on, we assume
that xn ̸= xn+for all n ∈N. Theorem .Let (X,d) be a complete b-metric-like space with constant s ≥and T :
X →X be a generalized rational α-ψ-φ-Geraghty contractive mapping of type (II) such
that: Remark .Notice that we get several corollaries by replacing the auxiliary functions
ψ and β in a proper way. In particular, by taking ψ(t) = t we find the extended version of
several existing results. Theorem .Let (X,d) be a complete b-metric-like space with constant s ≥and T :
X →X be a generalized rational α-ψ-φ-Geraghty contractive mapping of type (II) such
that: Since T satisfies () we have Since T satisfies () we have ψ
sd(xn+,xn+)
≤α(xn,xn+)ψ
sd(Txn,Txn+)
≤β
ψ
d(xn,xn+)
ψ
d(xn,xn+)
. Thus, we have ψ
sd(xn+,xn+)
<
s ψ
d(xn,xn+)
< ψ
d(xn,xn+)
. () () Since ψ is increasing, we have d(xn+,xn+) <
sd(xn,xn+). Since ψ is increasing, we have d(xn+,xn+) <
sd(xn,xn+). Since ψ is increasing, we have d(xn+,xn+) <
sd(xn,xn+). Case (i): If s > , then, since
s> and s
s=
s < , by Lemma ., {xn} is a Cauchy se-
quence and limn,m→∞d(xn,xm) = . Case (ii): If s = , then as d(xn+,xn+) < d(xn,xn+), there exists r ≥such that
limn→∞d(xn,xn+) = r. If r > then taking the limit as n →∞on both sides of ψ
d(xn+,xn+)
≤β
ψ
d(xn,xn+)
ψ
d(xn,xn+)
we get limn→∞β(ψ(d(xn,xn+))) = and as β is a Geraghty function, we derive that we get limn→∞β(ψ(d(xn,xn+))) = and as β is a Geraghty function, we derive that lim
n→∞d(xn,xn+) = . () () lim
n→∞d(xn,xn+) = . lim
n→∞d(xn,xn+) = . That is, r = . In what follows we shall prove that {xn} is a Cauchy sequence. Indeed we
will prove that limm,n→∞d(xn,xm) = . Suppose, on the contrary, that there exist ε > and
corresponding subsequences {nk} and {mk} of N satisfying nk > mk > k for which d(xmk,xnk) ≥ε,
() () d(xmk,xnk) ≥ε, where nk, mk are chosen as the smallest integers satisfying (), that is, where nk, mk are chosen as the smallest integers satisfying (), that is, where nk, mk are chosen as the smallest integers satisfying (), that is, d(xmk,xnk–) < ε. () () d(xmk,xnk–) < ε. By (), (), and the triangle inequality, we easily derive that By (), (), and the triangle inequality, we easily derive that () ε ≤d(xmk,xnk) ≤d(xmk,xnk–) + d(xnk–,xnk) < ε + d(xnk–,xnk). () Karapınar et al. Journal of Inequalities and Applications ( 2015) 2015:303 Page 16 of 22 Using () and the squeeze theorem we get () lim
k→∞d(xmk,xnk) = ε. As T satisfies (), we have ψ
d(xmk+,xnk+)
≤α(xmk,xnk)ψ
d(Txmk,Txnk)
≤β
ψ
d(xmk,xnk)
ψ
d(xmk,xnk)
. Theorem .Let (X,d) be a complete b-metric-like space with constant s ≥and T :
X →X be a generalized rational α-ψ-φ-Geraghty contractive mapping of type (II) such
that: () () Taking the limit as k →∞for () we get limk→∞β(ψ(d(xmk,xnk))) = . Thus lim
k→∞d(xmk,xnk) = . lim
k→∞d(xmk,xnk) = . Therefore, {xn} is a Cauchy sequence for s ≥. By completeness of (X,d), there exists u ∈X
such that = limn,m→∞d(xn,xm) = limn→∞d(xn,u) = d(u,u). Since T is continuous, Tu = T(limn→∞xn) = limn→∞Txn = limn→∞xn+= u and u is a
fixed point for T. □
Theorem .Let (X,d) be a complete b-metric-like space with constant s ≥and T :
X →X be a mapping. Suppose that there exist a function α : X × X →[,∞), ψ ∈, and
β ∈Fs such that Therefore, {xn} is a Cauchy sequence for s ≥. By completeness of (X,d), there exists u ∈X
such that = limn,m→∞d(xn,xm) = limn→∞d(xn,u) = d(u,u). Since T is continuous, Tu = T(limn→∞xn) = limn→∞Txn = limn→∞xn+= u and u is a
fixed point for T. □ Theorem .Let (X,d) be a complete b-metric-like space with constant s ≥and T :
X →X be a mapping. Suppose that there exist a function α : X × X →[,∞), ψ ∈, and
β ∈Fs such that () for all x,y ∈X. Suppose also that:
(i) T is triangular α-orbital admissible;
(ii) there exists x∈X such that α(x,Tx) ≥;
(iii) X is α-regular and d is continuous. Th
T h
fi
d
i t
∈X
ith d(
)
for all x,y ∈X. Suppose also that:
(i) T is triangular α-orbital admissible;
(ii) there exists x∈X such that α(x,Tx) ≥;
(iii) X is α-regular and d is continuous. Th
T h
fi
d
i t
X
ith d(
)
for all x,y ∈X. Suppose also that:
(i) T is triangular α-orbital admissible;
(ii) there exists x∈X such that α(x,Tx) ≥;
(iii) X is α-regular and d is continuous. Th
T h
fi
d
i t
X
ith d(
)
Then T has a fixed point, u ∈X with d(u,u) = . Then T has a fixed point, u ∈X with d(u,u) = . Theorem .Adding condition (H) to the hypotheses of Theorem .(or Theorem .),
we obtain the uniqueness of the fixed point of T. Remark .Notice that we get several corollaries by replacing the auxiliary functions
ψ and β in a proper way. In particular, by taking ψ(t) = t we find the extended version of
several existing results. 3 Expected consequences In this section, we shall consider some immediate consequences of our main results. In this section, we shall consider some immediate consequences of our main results. The following result is obtained by letting L = in Theorem .or .. The following result is obtained by letting L = in Theorem .or .. Corollary .Let (X,d) be a complete b-metric-like space with constant s ≥and T : X →
X be a mapping. Suppose that there exist α : X × X →[,∞), ψ ∈, β ∈Fs such that α(x,y)ψ
sd(Tx,Ty)
≤β
ψ
M(x,y)
ψ
M(x,y)
,
() () Karapınar et al. Journal of Inequalities and Applications ( 2015) 2015:303 Page 17 of 22 Karapınar et al. Journal of Inequalities and Applications ( 2015) 2015:303 for all x,y ∈X, where M(x,y) = max
d(x,y),d(x,Tx),d(y,Ty), d(x,Ty) + d(y,Tx)
s
. () () () Suppose also that: Suppose also that: (i) T is triangular α-orbital admissible; (i) T is triangular α-orbital admissible; (ii) there exists x∈X such that α(x,Tx) ≥; (ii) there exists x∈X such that α(x,Tx) ≥; (iii) T is continuous or (iii)′ X is α-regular and d is continuous. (iii) T is continuous or (iii)′ X is α-regular and d is continuous. Then T has a fixed point. Then T has a fixed point. Adding condition (H) to the hypothesis of Corollary ., we guarantee the uniqueness
of the fixed point. Again by letting L = in Theorem .and Theorem .we get two more corollaries as
Corollary .. We skip the details regarding the volume of the paper. Corollary .Let (X,d) be a complete b-metric-like space with constant s ≥, T : X →X
be a map and α : X × X →[,∞) be a function. Suppose that T satisfies at least one of the
following conditions: Corollary .Let (X,d) be a complete b-metric-like space with constant s ≥, T : X →X
be a map and α : X × X →[,∞) be a function. Suppose that T satisfies at least one of the
following conditions: (a) α(x,y)d(Tx,Ty) ≤
sM(x,y); (a) α(x,y)d(Tx,Ty) ≤
sM(x,y); (b) α(x,y)d(Tx,Ty) ≤
sK(x,y); (b) α(x,y)d(Tx,Ty) ≤
sK(x,y); where M(x,y), K(x,y) are defined as in (), (). Suppose also that: where M(x,y), K(x,y) are defined as in (), (). Suppose also that: (i) T is triangular α-orbital admissible; (i) T is triangular α-orbital admissible; (ii) there exists x∈X such that α(x,Tx) ≥; (iii) T is continuous or (iii)′ X is α-regular and d is continuous. (iii) T is continuous or (iii)′ X is α-regular and d is continuous. Then T has a unique fixed point u ∈X with d(u u) = Then T has a unique fixed point u ∈X with d(u,u) = . Then T has a unique fixed point u ∈X with d(u,u) = . Proof It is sufficient to take L = , ψ(t) = t, and β(t) =
s in Theorem .and Theorem .
(and thus, Theorem .or Theorem ., Theorem .or Theorem ., respectively). □ Proof It is sufficient to take L = , ψ(t) = t, and β(t) =
s in Theorem .and Theorem .
(and thus, Theorem .or Theorem ., Theorem .or Theorem ., respectively). (iii) T is continuous or (iii)′ X is α-regular and d is continuous. 3.1 For standard b-metric-like spaces If we set α(x,y) = for all x,y ∈X in Theorem ., then we derive the following results. Corollary .Let (X,d) be a complete b-metric-like space with constant s ≥such that d
is continuous and T : X →X be a mapping. Suppose that there exist ψ,φ ∈, β ∈Fs, and
some L ≥such that ψ
sd(Tx,Ty)
≤β
ψ
M(x,y)
ψ
M(x,y)
+ Lφ
N(x,y)
,
() ψ
sd(Tx,Ty)
≤β
ψ
M(x,y)
ψ
M(x,y)
+ Lφ
N(x,y)
,
() () y ∈X, where for all x,y ∈X, where for all x,y ∈X, where M(x,y) = max
d(x,y),d(x,Tx),d(y,Ty), d(x,Ty) + d(y,Tx)
s
and
()
N(x,y) = min
ds(x,Ty),ds(y,Tx),d(x,Tx),d(y,Ty)
. () () () Then T has a unique fixed point u ∈X with d(u,u) = . If we set α(x,y) = for all x,y ∈X in Theorem ., then we derive the following results. Corollary .Let (X,d) be a complete b-metric-like space with constant s ≥such that d
is continuous and T : X →X be a mapping. Suppose that there exist ψ,φ ∈, β ∈Fs, and
some L ≥such that Corollary .Let (X,d) be a complete b-metric-like space with constant s ≥such that d
is continuous and T : X →X be a mapping. Suppose that there exist ψ,φ ∈, β ∈Fs, and
some L ≥such that ψ
sd(Tx,Ty)
≤β
ψ
K(x,y)
ψ
K(x,y)
+ Lφ
N(x,y)
,
() () ll x,y ∈X, where for all x,y ∈X, where for all x,y ∈X, where K(x,y) = max
d(x,y), d(x,Tx)d(y,Ty)
+ d(x,y)
, d(x,Tx)d(y,Ty)
+ d(Tx,Ty)
and
()
N(x,y) = min
ds(x,Ty),ds(y,Tx),d(x,Tx),d(y,Ty)
. () () () Then T has a unique fixed point u ∈X with d(u,u) = . If we set α(x,y) = for all x,y ∈X in Theorem ., then we derive the following results. Corollary .Let (X,d) be a complete b-metric-like space with constant s ≥such that d
is continuous and T : X →X be a mapping. Suppose that there exist ψ,φ ∈, β ∈Fs, and
some L ≥such that Corollary .Let (X,d) be a complete b-metric-like space with constant s ≥such that d
is continuous and T : X →X be a mapping. Suppose also that: □ Adding condition (H) to the hypothesis of Corollary ., we guarantee the uniqueness
of the fixed point. Corollary .Let (X,d) be a complete b-metric-like space with constant s ≥, T : X →X
be a map, and α : X × X →[,∞) be a function. Suppose that T satisfies at least one of the
following conditions: Corollary .Let (X,d) be a complete b-metric-like space with constant s ≥, T : X →X
be a map, and α : X × X →[,∞) be a function. Suppose that T satisfies at least one of the
following conditions: (c) α(x,y)d(Tx,Ty) ≤
sQ(x,y),
where M(x,y), K(x,y), Q(x,y) are defined as in (). Suppose also that: (i) T is triangular α-orbital admissible; (i) T is triangular α-orbital admissible; (ii) there exists x∈X such that α(x,Tx) ≥; (ii) there exists x∈X such that α(x,Tx) ≥; (iii) T is continuous or (iii)′ X is α-regular and d is continuous. Then T has a fixed point u ∈X with d(u,u) = . Then T has a fixed point u ∈X with d(u,u) = . Proof It is sufficient to take L = , ψ(t) = t, and β(t) =
s in Theorem .or Theorem .,
respectively. □ Proof It is sufficient to take L = , ψ(t) = t, and β(t) =
s in Theorem .or Theorem .,
respectively. □ Karapınar et al. Journal of Inequalities and Applications ( 2015) 2015:303 Page 18 of 22 3.1 For standard b-metric-like spaces 3.1 For standard b-metric-like spaces Suppose that there exist ψ,φ ∈, β ∈Fs, and
some L ≥such that ψ
sd(Tx,Ty)
≤β
ψ
Q(x,y)
ψ
Q(x,y)
+ Lφ
N(x,y)
,
() () x,y ∈X, where for all x,y ∈X, where Q(x,y) = max
d(x,y), d(x,Tx)d(y,Ty) + ds(x,Ty)ds(y,Tx)
+ s(d(x,Tx) + d(y,Ty))
and
()
N(x,y) = min
ds(x,Ty),ds(y,Tx),d(x,Tx),d(y,Ty)
. () () () Then T has a fixed point u ∈X with d(u,u) = . Karapınar et al. Journal of Inequalities and Applications ( 2015) 2015:303 Karapınar et al. Journal of Inequalities and Applications ( 2015) 2015:303 Page 19 of 22 If take L = in Corollaries .-., we get three more consequences. Regarding the vol-
ume of the paper, we skip the details. Corollary .Let (X,d) be a complete b-metric-like space with constant s ≥such that d
is continuous and T : X →X be mapping. Suppose that there exist ψ ∈ and β ∈Fs such
that () for all x,y ∈X. Then T has a unique fixed point u ∈X with d(u,u) = . □ Proof It follows from Theorem .by α(x,y) = for all x,y ∈X. □ 3.2 For b-metric-like spaces endowed with a partial order In this section, from our main results, we shall derive easily various fixed point results on
a b-metric-like space endowed with a partial order. We, first, recall some notions. Definition .Let (X,⪯) be a partially ordered set and T : X →X be a given mapping. We say that T is nondecreasing with respect to ⪯if x,y ∈X,
x ⪯y
⇒
Tx ⪯Ty. x,y ∈X,
x ⪯y
⇒
Tx ⪯Ty. Definition .Let (X,⪯) be a partially ordered set. A sequence {xn} ⊂X is said to be
nondecreasing (respectively, nonincreasing) with respect to ⪯if xn ⪯xn+(respectively,
xn+⪯xn for all n). Definition .Let (X,⪯) be a partially ordered set and d be a b-metric-like on X. We say
that (X,⪯,d) is regular if for every nondecreasing (respectively, nonincreasing) sequence
{xn} ⊂X such that xn →x ∈X as n →∞, there exists a subsequence {xnk} of {xn} such
that xnk ⪯x (respectively, xnk ⪰x) for all k. (ii) T is continuous or (ii)′ (X,⪯,d) is regular and d is continuous.
Th
T h
fi
d
i t
X
ith d(
)
We have the following result. We have the following result. Corollary .Let (X,⪯) be a partially ordered set (which does not contain an infinite
totally unordered subset) and d be a b-metric-like on X with constant s ≥such that (X,d)
is complete. Let T : X →X be a nondecreasing mapping with respect to ⪯. Suppose that
there exist ψ ∈, β ∈Fs such that ψ
sd(Tx,Ty)
≤β
ψ
M(x,y)
ψ
M(x,y)
,
() ψ
sd(Tx,Ty)
≤β
ψ
M(x,y)
ψ
M(x,y)
, () for all x,y ∈X with x ⪰y or y ⪰x where M(x,y) is defined as in (). Suppose also that the
following conditions hold: (ii) T is continuous or (ii)′ (X,⪯,d) is regular and d is continuous. Karapınar et al. Journal of Inequalities and Applications ( 2015) 2015:303 Page 20 of 22 Proof Define the mapping α : X × X →[,∞) by Proof Define the mapping α : X × X →[,∞) by α(x,y) =
if x ⪯y or x ⪰y,
otherwise. α(x,y) =
if x ⪯y or x ⪰y,
otherwise. Clearly, T satisfies (), that is, Clearly, T satisfies (), that is, α(x,y)ψ
sd(Tx,Ty)
≤β
ψ
M(x,y)
ψ
M(x,y)
, α(x,y)ψ
sd(Tx,Ty)
≤β
ψ
M(x,y)
ψ
M(x,y)
, for all x,y ∈X. From condition (i), we have α(x,Tx) ≥. Moreover, for all x,y ∈X, from
the monotone property of T, we have α(x,y) ≥
⇒
x ⪰y or x ⪯y
⇒
Tx ⪰Ty or Tx ⪯Ty
⇒
α(Tx,Ty) ≥. Hence, the self-mapping T is α-admissible. Similarly, we can prove that T is triangular α-
admissible and so triangular α-orbital admissible. Now, if T is continuous, the existence
of a fixed point follows from Corollary .. Suppose now that (X,⪯,d) is regular. Let {xn}
be a sequence in X such that α(xn,xn+) ≥for all n and xn →x ∈X as n →∞. From the
regularity hypothesis and as X does not contain an infinite totally unordered subset, there
exists a subsequence {xnk} of {xn} such that xnk ⪯x or x ⪯xnk for all k. This implies from
the definition of α that α(xnk,x) ≥for all k. In this case, the existence of a fixed point
follows again from Corollary .. □ Hence, the self-mapping T is α-admissible. We have the following result. Similarly, we can prove that T is triangular α-
admissible and so triangular α-orbital admissible. Now, if T is continuous, the existence
of a fixed point follows from Corollary .. Suppose now that (X,⪯,d) is regular. Let {xn}
be a sequence in X such that α(xn,xn+) ≥for all n and xn →x ∈X as n →∞. From the
regularity hypothesis and as X does not contain an infinite totally unordered subset, there
exists a subsequence {xnk} of {xn} such that xnk ⪯x or x ⪯xnk for all k. This implies from
the definition of α that α(xnk,x) ≥for all k. In this case, the existence of a fixed point
follows again from Corollary .. □ □ In an analogous way, we derive the following results from Theorem .and Theo-
rem ., respectively. Corollary .Let (X,⪯) be a partially ordered set (which does not contain an infinite
totally unordered subset) and d be a b-metric-like on X with constant s ≥such that (X,d)
is complete. Let T : X →X be a nondecreasing mapping with respect to ⪯. Suppose that
there exist ψ ∈, β ∈Fs such that Corollary .Let (X,⪯) be a partially ordered set (which does not contain an infinite
totally unordered subset) and d be a b-metric-like on X with constant s ≥such that (X,d)
is complete. Let T : X →X be a nondecreasing mapping with respect to ⪯. Suppose that
there exist ψ ∈, β ∈Fs such that ψ
sd(Tx,Ty)
≤β
ψ
K(x,y)
ψ
K(x,y)
,
() ψ
sd(Tx,Ty)
≤β
ψ
K(x,y)
ψ
K(x,y)
,
() ψ
sd(Tx,Ty)
≤β
ψ
d(x,y)
ψ
d(x,y)
, () for all x,y ∈X with x ⪰y or y ⪰x. Suppose also that the following conditions hold: for all x,y ∈X with x ⪰y or y ⪰x. Suppose also that the following conditions hold: (i) there exists x∈X such that x⪯Tx; (i) there exists x∈X such that x⪯Tx; (ii) T is continuous or (ii)′ (X,⪯,d) is regular and d is continuous. (ii) T is continuous or (ii)′ (X,⪯,d) is regular and d is continuous. Then T has a fixed point u ∈X with d(u,u) = . Example .Let X = [,∞) define d : X × X →[,∞) by d(x,y) = |x – y|. Then (X,d)
is complete b-metric (so b-metric-like) space with constant s = . Define T : X →X and
α(x,y) : X × X →[,∞) as follows: Tx =
x
if x ∈[,],
x
x+
if x ∈(,∞), Tx =
x
if x ∈[,],
x
x+
if x ∈(,∞), and α(x,y) =
if x,y ∈[,],
otherwise. α(x,y) =
if x,y ∈[,],
otherwise. It is clear that T is triangular α-orbital admissible and we have α(,T) ≥. Moreover, X
is α-regular and d is continuous. It is clear that T is triangular α-orbital admissible and we have α(,T) ≥. Moreover, X
is α-regular and d is continuous. It is clear that T is triangular α-orbital admissible and we have α(,T) ≥. Moreover, X
is α-regular and d is continuous. Let ψ(t) = t, β(t) =
then clearly ψ ∈ and β ∈F. Moreover, T satisfies () for the
following reason: if x,y ∈[,], then α(x,y)ψ
d(Tx,Ty)
=
x
– y
= (x – y)
= β
ψ
d(x,y)
ψ(d(x,y). Otherwise, α(x,y)ψ
d(Tx,Ty)
= ≤β
ψ
d(x,y)
ψ(d(x,y). Therefore, by Theorem ., T has a fixed point x = . Example .Let X = {,,} define d : X × X →[,∞) by d(x,y) = (max{x,y})
. Then
(X,d) is complete b-metric-like space with constant s =
–=
such that d is continu-
ous. Define T : X →X by T = {(,),(,),(,)}. Let ψ(t) = t, β(t) =
e–t or β(t) =
+t
, then clearly ψ ∈ and β ∈F
. ψ
sd(Tx,Ty)
≤β
ψ
K(x,y)
ψ
K(x,y)
, () for all x,y ∈X with x ⪰y or y ⪰x where M(x,y) is defined as in (). Suppose also that the
following conditions hold: for all x,y ∈X with x ⪰y or y ⪰x where M(x,y) is defined as in (). Suppose also that the
following conditions hold: (i) there exists x∈X such that x⪯Tx; (i) there exists x∈X such that x⪯Tx; (ii) T is continuous or (ii)′ (X,⪯,d) is regular and d is continuous. (ii) T is continuous or (ii)′ (X,⪯,d) is regular and d is continuous. Then T has a fixed point u ∈X with d(u,u) = . ( )
( ) (
)
g
Then T has a fixed point u ∈X with d(u,u) = . Corollary .Let (X,⪯) be a partially ordered set (which does not contain an infinite
totally unordered subset) and d be a b-metric-like on X with constant s ≥such that (X,d)
is complete. Let T : X →X be a nondecreasing mapping with respect to ⪯. Suppose that
there exist ψ ∈, β ∈Fs such that ψ
sd(Tx,Ty)
≤β
ψ
Q(x,y)
ψ
Q(x,y)
,
() () for all x,y ∈X with x ⪰y or y ⪰x where M(x,y) is defined as in (). Suppose also that the
following conditions hold: Karapınar et al. Journal of Inequalities and Applications ( 2015) 2015:303 Page 21 of 22 (i) there exists x∈X such that x⪯Tx; (i) there exists x∈X such that x⪯Tx; (ii) T is continuous or (ii)′ (X,⪯,d) is regular and d is continuous. Then T has a fixed point u ∈X with d(u,u) = . (ii) T is continuous or (ii)′ (X,⪯,d) is regular and d is continuous. Corollary .Let (X,⪯) be a partially ordered set (which does not contain an infinite
totally unordered subset) and d be b-metric-like on X with constant s ≥such that (X,d)
is complete. Let T : X →X be a nondecreasing mapping with respect to ⪯. Suppose that
there exist ψ ∈, β ∈Fs such that Corollary .Let (X,⪯) be a partially ordered set (which does not contain an infinite
totally unordered subset) and d be b-metric-like on X with constant s ≥such that (X,d)
is complete. Let T : X →X be a nondecreasing mapping with respect to ⪯. ψ
sd(Tx,Ty)
≤β
ψ
K(x,y)
ψ
K(x,y)
, Suppose that
there exist ψ ∈, β ∈Fs such that ψ
sd(Tx,Ty)
≤β
ψ
d(x,y)
ψ
d(x,y)
,
() ψ
sd(Tx,Ty)
≤β
ψ
d(x,y)
ψ
d(x,y)
,
() ψ
sd(Tx,Ty)
≤β
ψ
d(x,y)
ψ
d(x,y)
, Note that
K(,) = , K(,) = K(,) =
, and clearly T satisfies () with L = Therefore, by
Corollary ., T has a fixed point x = . Page 22 of 22 Karapınar et al. Journal of Inequalities and Applications ( 2015) 2015:303 Received: 16 July 2015 Accepted: 15 September 2015 Received: 16 July 2015 Accepted: 15 September 2015 4 Conclusion It is clear that we can list several more results by replacing the b-metric-like space, with
some other abstract space, such as a b-metric space, a metric space, a metric-like space,
a partial metric space, and so on. Competing interests g
The authors declare that they have no competing interests. References References
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7. Bota, M-F, Chifu, C, Karapınar, E: Fixed point theorems for generalized (α∗-ψ)-´Ciri´c-type contractive multivalued
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All
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Hemimetabolous genomes reveal molecular basis of termite eusociality
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Nature ecology & evolution
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cc-by
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Hemimetabolous genomes reveal molecular basis of termite
eusociality Mark C. Harrison1,12, Evelien Jongepier1,12, Hugh M. Robertson2,12, Nicolas Arning1,
Tristan Bitard-Feildel1, Hsu Chao3, Christopher P. Childers4, Huyen Dinh3, Harshavardhan
Doddapaneni3, Shannon Dugan3, Johannes Gowin5,6, Carolin Greiner5,6, Yi Han3, Haofu
Hu7, Daniel S. T. Hughes3, Ann-Kathrin Huylmans8, Carsten Kemena1, Lukas P. M. Kremer1,
Sandra L. Lee3, Alberto Lopez-Ezquerra1, Ludovic Mallet1, Jose M. Monroy-Kuhn5,
Annabell Moser5, Shwetha C. Murali3, Donna M. Muzny3, Saria Otani7, Maria-Dolors
Piulachs9, Monica Poelchau4, Jiaxin Qu3, Florentine Schaub5, Ayako Wada-Katsumata10,
Kim C. Worley3, Qiaolin Xie11, Guillem Ylla9, Michael Poulsen7, Richard A. Gibbs3, Coby
Schal10, Stephen Richards3, Xavier Belles9,*, Judith Korb5,6,*, and Erich Bornberg-Bauer1,*
1Institute for Evolution and Biodiversity, University of Münster, Münster, Germany. Author Manuscript This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation,
distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source,
provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this
article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is
not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the
permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://
creativecommons.org/licenses/by/4.0/.Reprints and permissions information is available at www.nature.com/reprints. Author Manuscript *Correspondence and requests for materials should be addressed to X.B. or J.K. or E.B.-B. xavier.belles@ibe.upf-csic.es;
judith.korb@biologie.uni-freiburg.de; ebb@uni-muenster.de. Author contributions E.B.-B. conceived, managed and coordinated the project; M.C.H., E.J. and H.M.R. are joint first authors. J.K. conceived and managed
the C. secundus sequencing project, and coordinated termite-related analyses; S.R. conceived and managed the B. germanica
sequencing project; S.R., S.D., S.L.L., H.C., H.V.D., H.D., Y.H., J.Q., S.C.M., D.S.T.H., K.C.W., D.M.M. and R.A.G. carried out B. germanica library construction, genome sequencing and assembly; C.S. and A.W-.K. provided biological material through full-sib
mating for B. germanica; X.B. and C.S. co-managed the B. germanica analysis; M.P. and C.P.C. implemented Web Apollo data traces;
S.O. and M.P. provided biological material for M. natalensis; C.G., J.G., J.M.M.-K., A.M., F.S., H.H. and J.K. coordinated and carried
out DNA and RNA sequencing for C. secundus; M-D.P., X.B. and G.Y. coordinated transcriptome sequencing of B. germanica; L.M. performed automated gene prediction on C. secundus; E.J. HHS Public Access Author Manuscript Published in final edited form as: Published in final edited form as:
Nat Ecol Evol. 2018 March ; 2(3): 557–566. doi:10.1038/s41559-017-0459-1. Nat Ecol Evol. 2018 March ; 2(3): 557–566. doi:10.1038/s41559-017-0459-1. Hemimetabolous genomes reveal molecular basis of termite
eusociality Author Manuscript 3Human Genome Sequencing Center, Department of Human and Molecular Genetics, Baylor
College of Medicine, Houston, TX, USA. 4USDA-ARS, National Agricultural Library, Beltsville, MD, USA. 4USDA-ARS, National Agricultural Library, Beltsville, MD, USA. 5Evolutionary Biology & Ecology, University of Freiburg, Freiburg, Germany. 6Behavioral Biology, University of Osnabrück, Osnabrück, Germany. 7Ecology and Evolution, University of Copenhagen, Copenhagen, Denmark. 8Institute of Science and Technology Austria, Klosterneuburg, Austria. 9Institut de Biologia Evolutiva, CSIC-University Pompeu Fabra, Barcelona, Spain. 9Institut de Biologia Evolutiva, CSIC-University Pompeu Fabra, Barcelona, Spain. 10Department of Entomology and Plant Pathology, North Carolina State University, Raleigh, NC,
USA. 10Department of Entomology and Plant Pathology, North Carolina State University, Raleigh, NC,
USA. Author Manuscript 11China National GeneBank, Beijing Genomics Institute (BGI)-Shenzhen, Shenzhen, Chin 11China National GeneBank, Beijing Genomics Institute (BGI)-Shenzhen, Shenzhen, China. 12These authors contributed equally: Mark C. Harrison, Evelien Jongepier and Hugh M. Robertson. 12These authors contributed equally: Mark C. Harrison, Evelien Jongepier and Hugh M. Robertson. Hemimetabolous genomes reveal molecular basis of termite
eusociality and N.A. improved assembly and annotation for B. germanica & C. secundus, and compared and analysed genome sizes and quality. E.J., N.A. and L.P.M.K. analysed TEs; M.C.H. analysed CpG
patterns and signatures of selection; T.B-F., E.J., C.K., L.P.M.K. and A.L-E. performed orthology and phylogenetic analyses;
L.P.M.K., E.J., H.M.R. and M.C.H. analysed gene family evolution; A.L-E., E.J. and M.C.H. analysed transcriptomes and performed
differential expression analyses; T.B.-F. and A.L-E. carried out orthoMCL clustering; H.M.R. corrected gene models for
chemoreceptors; C.K. and E.J. corrected gene models for desaturases and elongases; A-K.H. and M.C.H. corrected gene models for
cytochrome P450s; E.B.-B. and M.C.H drafted and wrote the manuscript; X.B., M.-D.P. and J.K. contributed to sections of the
manuscript; E.J., L.P.M.K., A.L-E., C.K. and M.C.H. wrote and organized the Supplementary Information; L.P.M.K., N.A., A.L-E.,
M.C.H. and E.B.-B. prepared figures for the manuscript. All authors read, corrected and commented on the manuscript. C
i
i Author Manuscript Competing interests The authors declare no competing financial interests. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Life Science Reporting Summary. Further information on experimental design is available in the Life Sciences Reporting Summary. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Life Science Reporting Summary. Further information on experimental design is available in the Life Sciences Reporting Life Science Reporting Summary. Further information on experimental design is available in the Life Sciences Reporting Summary. Code availability. All custom-made scripts used in these analyses are available at the following repository: https://github.com/
ebbgroup/Genomic-comparisons-in-Blattodea. Code availability. All custom-made scripts used in these analyses are available at the following repository: https://github.com/
ebbgroup/Genomic-comparisons-in-Blattodea. Code availability. All custom-made scripts used in these analyses are available at the following repository: https://github.com/
ebbgroup/Genomic-comparisons-in-Blattodea. Data availability. The genome assembly of Blattella germanica is archived on NCBI under the accession PRJNA203136. The genome
assembly of Cryptotermes secundus is available on NCBI under the accession PRJNA381866. The additionally annotated genes for Z. nevadensis and M. natalensis are available from the Dryad Digital Repository: https://doi.org/10.5061/dryad.51d4r. Transcriptomic
reads generated in this study are available in SRA (B. germanica: PRJNA382128; C secundus: PRJNA382129; M. natalensis:
PRJNA382034). Supplementary information is available for this paper at https://doi.org/10.1038/s41559-017-0459-1. Page 2 Harrison et al. 2Department of Entomology, University of Illinois at Urbana-Champaign, Urbana, IL, USA. Nat Ecol Evol. Author manuscript; available in PMC 2019 April 25. Abstract Around 150 million years ago, eusocial termites evolved from within the cockroaches, 50 million
years before eusocial Hymenoptera, such as bees and ants, appeared. Here, we report the 2-Gb
genome of the German cockroach, Blattella germanica, and the 1.3-Gb genome of the drywood
termite Cryptotermes secundus. We show evolutionary signatures of termite eusociality by
comparing the genomes and transcriptomes of three termites and the cockroach against the
background of 16 other eusocial and non-eusocial insects. Dramatic adaptive changes in genes
underlying the production and perception of pheromones confirm the importance of chemical
communication in the termites. These are accompanied by major changes in gene regulation and
the molecular evolution of caste determination. Many of these results parallel molecular
mechanisms of eusocial evolution in Hymenoptera. However, the specific solutions are remarkably
different, thus revealing a striking case of convergence in one of the major evolutionary transitions
in biological complexity. Author Manuscript Eusociality, the reproductive division of labour with overlapping generations and
cooperative brood care, is one of the major evolutionary transitions in biology1. Although
rare, eusociality has been observed in a diverse range of organisms, including shrimps, mole
rats and several insect lineages2–4. A particularly striking case of convergent evolution
occurred within the holometabolous Hymenoptera and in the hemimetabolous termites
(Isoptera), which are separated by over 350 Myr of evolution5. Termites evolved within the
cockroaches around 150 Myr ago, towards the end of the Jurassic period6,7, about 50 Myr
before the first bees and ants appeared5. Therefore, identifying the molecular mechanisms
common to both origins of eusociality is crucial to understanding the fundamental signatures
of these rare evolutionary transitions. While the availability of genomes from many eusocial
and non-eusocial hymenopteran species8 has allowed extensive research into the origins of Author Manuscript Harrison et al. Page 3 eusociality within ants and bees9–11, a paucity of genomic data from cockroaches and
termites has precluded large-scale investigations into the evolution of eusociality in this
hemimetabolous clade. Author Manuscript Author Manuscript The conditions under which eusociality arose differ greatly between the two groups. Termites and cockroaches are hemimetabolous and so show a direct development, while
holometabolous hymenopterans complete the adult body plan during metamorphosis. In
termites, workers are immatures and only reproductive castes are adults12, while in
Hymenoptera, adult workers and queens represent the primary division of labour. Abstract Moreover,
termites are diploid and their colonies consist of both male and female workers, and usually
a queen and king dominate reproduction. This is in contrast to the haplodiploid system found
in Hymenoptera, in which all workers and dominant reproductives are female. It is therefore
intriguing that strong similarities have evolved convergently within the termites and the
hymenopterans, such as differentiated castes and a nest life with reproductive division of
labour. The termites can be subdivided into wood-dwelling and foraging termites. The
former belong to the lower termites and produce simple, small colonies with totipotent
workers that can become reproductives. Foraging termites (some lower and all higher
termites) form large, complex societies, in which worker castes can be irreversible12. For
this reason, higher, but not lower, termites can be classed as superorganismal13. Similarly,
within Hymenoptera, varying levels of eusociality exist. Here we provide insights into the molecular signatures of eusociality within the termites. We
analysed the genomes of two lower and one higher termite species and compared them to the
genome of the German cockroach, Blattella germanica, as a closely related non-eusocial
outgroup. Furthermore, differences in expression between nymphs and adults of the
cockroach were compared to differences in expression between workers and reproductives of
the three termites, to gain insights into how expression patterns changed along with the
evolution of castes. Using 15 additional insect genomes to infer background gene family
turnover rates, we analysed the evolution of gene families along the transition from non-
social cockroaches to eusociality in the termites. In this study, we concentrated particularly
on two hallmarks of insect eusociality, as previously described for Hymenoptera, with the
expectation that similar patterns occurred along with the emergence of termites. These are
the evolution of a sophisticated chemical communication, which is essential for the
functioning of a eusocial insect colony3,14,15, and major changes in gene regulation along
with the evolution of castes9,10. We also tested whether transposable elements spurred the
evolution of gene families that were essential for the transition to eusociality. Author Manuscript Nat Ecol Evol. Author manuscript; available in PMC 2019 April 25. Evolution of genomes, proteomes and transcriptomes Author Manuscript We sequenced and assembled the genome of the German cockroach, B. germanica
(Ectobiidae), and of the lower, drywood termite Cryptotermes secundus (Kalotermitidae; for
assembly statistics, see Supplementary Table 1). The cockroach genome (2.0 Gb) is
considerably larger than all three termite genomes. The genome size of C. secundus (1.30
Gb) is comparable to the higher, fungus-growing termite Macrotermes natalensis (1.31 Gb,
Termitidae)16, but more than twice as large as the lower, dampwood termite Zootermopsis
nevadensis (562 Mb, Termopsidae)17. The smaller genomes of termites compared to the Nat Ecol Evol. Author manuscript; available in PMC 2019 April 25. Page 4 Harrison et al. cockroach are in line with previous size estimations based on C-values18. The proteome of
B. germanica (29,216 proteins) is also much larger than in the termites, where we find the
proteome size in C. secundus (18,162) to be similar to those of the other two termites (M. natalensis: 16,140; Z. nevadensis: 15,459; Fig. 1). In fact, the B. germanica proteome was
the largest among all 21 arthropod species analysed here (Fig. 1). Strong evi-dential support
for over 80% of these proteins in B. germanica (see Supplementary Information) and large
expansions in many manually annotated gene families offer high confidence in the accuracy
of this proteome size. We also compared gene expression between the four species. When
comparing worker expression with queen expression in the termites and nymph expression
(fifth and sixth instars) with adult female expression in B. germanica, we found shifts in
specificity of expression for termites compared to the cockroach in several gene families
(Fig. 2). It has previously been reported for the primitively eusocial paper wasp Polistes
canadensis that the majority of caste-biased genes, especially those upregulated in workers,
are novel genes19. The authors suggested that this may be a feature of early eusociality. We
did not find the same pattern for the termites. Species-specific genes (those without an
orthologue) were not enriched for differentially expressed genes in any of the termites, with
slight peaks among Blattodea- and Isoptera-specific genes (Supplementary Fig. 1). Author Manuscript Author Manuscript Nat Ecol Evol. Author manuscript; available in PMC 2019 April 25. Expansion and positive selection of ionotropic receptors Insects perceive chemical cues from toxins, pathogens, food and pheromones with three
major families of chemoreceptors, the odorant (ORs), gustatory and ionotropic (IRs)
receptors22. ORs, in particular, have been linked to colony communication in eusocial
Hymenoptera, where they abound14,15,23. Interestingly, as previously detected for Z. nevadensis17, the OR repertoire is sub-stantially smaller in B. germanica and all three
termites compared to hymenopterans. IRs, on the other hand, which are less frequent in
hymenopterans, are strongly expanded in the cockroach and termite genomes (Fig. 3 and
Supplementary Fig. 3). Intronless IRs, which are known to be particularly divergent24, show
the greatest cockroach- and Blattodea-specific expansions (Fig. 3a, Blattodea-, Cockroach-
and Group D-IRs). By far the most IRs among all investigated species were found in B. germanica (455 complete gene models), underlining that the capacity for detecting many
differ- ent kinds of chemosensory cues is crucial for this generalist that thrives in
challenging, human environments. In line with a special- ization in diet and habitat, the total
number of IRs is lower within the termites (Z. nevadensis: 141; C. secundus: 135; M. natalensis: 75). Nevertheless, IRs are more numerous in termites than in all other analysed
species (except Nasonia vitripennis: 111). This is strik- ingly similar to the pattern for ORs
in Hymenoptera, which are also highly numerous in non-eusocial outgroups as well as in
eusocial sister species14,23,25. We scanned each IR group for signs of species-specific positive selection. Within the
Blattodea-specific intronless IRs, we found two codon positions under significant selection
for the higher termite M. natalensis (codeml site models 7 and 8; P = 5.4 × 10−5). Within a
subgroup of five sequences, this was more strongly pronounced with seven codon positions
under significant positive selection for M. natalensis (P < 1.7 × 10−10). The positively
evolving codons are situated within the two ligand-binding lobes of the receptors (Fig. 3c),
showing that a diversification of ligand specificity has occurred along with the transition to
higher eusociality and a change from wood-feeding to fungus-farming in M. natalensis. Only
two IRs were differentially expressed between nymphs and adult females in B. germanica. Underlining a change in expression along with the evolution of castes, we found 35 IRs to be
differentially expressed between workers and queens in Z. nevadensis, 11 in C. secundus and
10 in M. natalensis (Fig. 2 and Supplementary Table 10). Gene family expansions assisted by TEs in termites The transitions to eusociality in ants10 and bees9 have been linked to major changes in gene
family sizes. Similarly, we detected significant gene family changes on the branch leading to
the termites (seven expansions and ten contractions; Supplementary Fig. 2 and
Supplementary Table 2). The numbers of species-specific, significant expansions and
contractions of gene families varied within termites (Z. nevadensis: 15/5; C. secundus: 27/3;
M. natalensis: 24/20; Supplementary Fig. 2 and Supplementary Tables 3–5). Interestingly, in
B. germanica we measured 93 significant gene family expansions but no contractions
(Supplementary Table 6), which contributed to the large proteome. Author Manuscript The termite and cockroach genomes contain a higher level of repetitive DNA compared to
the hymenopterans we analysed (Fig. 1). C. secundus and B. germanica genomes both
contain 55% repetitive content (Supplementary Table 7), which is higher than in both
Z.nevadensis(28%)andthehighertermiteM.natalensis(46%;Fig. 1)20. As also found in Z. nevadensis and M. natalensis20, LINEs and especially the subfamily BovB were the most
abundant transposable elements (TEs) in the B. germanica and C. secundus genomes,
indicating that a proliferation of LINEs may have occurred in the ancestors of Blattodea
(cockroaches and termites). Author Manuscript We hypothesized that these high levels of TEs may be driving the high turnover in gene
family sizes within the termites and B. germanica21. Expanded gene families indeed had
more repetitive content within 10-kb flanking regions in all three termites (P < 1.3 × 10−8;
Wald t-test; Supplementary Tables 8 and 9), in particular in the higher termite M. natalensis. In contrast, gene family expansions were not correlated with TE content in flanking regions
for B. germanica. These results suggest that a major expansion of LINEs at the root of the
Blattodea clade contributed to the evolution of gene families within termites, probably via
unequal crossing-over21; however, the expansions in B. germanica were not facilitated by Harrison et al. Page 5 Page 5 TEs. It can therefore be speculated that the large expansion of LINEs within Blattodea
allowed the evolution of gene families that ultimately facilitated the transition to eusociality. Author Manuscript Nat Ecol Evol. Author manuscript; available in PMC 2019 April 25. Expansion and positive selection of ionotropic receptors The possible role of IRs in
pheromonal communication has been highlighted both in the cockroach Periplaneta
americana26 and in Drosophila melanogaster27, where several IRs show sex-biased
expression. Author Manuscript One group of ORs (orange clade in Fig. 3b) is evolving under significant positive selection
at codon positions within the second transmembrane domain in M. natalensis (codeml site
model; P = 1.1 × 10−11) and C. secundus (P = 5.6 × 10−16; Fig. 3d). Such a variation in the
transmembrane domain can be related to ligand-binding specificity, as has been shown for a
polymorphism in the third transmembrane domain for an OR in D. melanogaster28,29,
adding further support for an adaptive evolution of chemoreceptors, in line with the greater Harrison et al. Page 6 Page 6 need for a sophisticated colony communication in the termites. Similar to IRs, a higher
proportion of ORs were differentially expressed between workers and queens in the three
termites than between nymphs and adults in the cockroach (Fig. 2 and Supplementary Table
11), highlighting a change in expression and function along with the transition to eusociality. The evolution of chemoreceptors along with the emergence of the termites can also be
related to adaptation processes and changes in diet compared to the cockroach. Experimental
verification will help pinpoint which receptors are particularly important for communication. Author Manuscript CHC-producing enzymes have evolved caste-specificity Despite their different ancestry, both termites and eusocial hymenopterans are characterized
by the production of caste- specific cuticular hydrocarbons (CHCs)30–32, which are often
crucial for regulating reproductive division of labour and chemical communication. Accordingly, we find changes in the termites in three groups of proteins involved in the
synthesis of CHCs: desaturases (introduction of double bonds33), elongases (extension of C-
chain length34) and CYP4G1 (last step of CHC biosynthesis35). Author Manuscript Desaturases are thought to be important for division of labour and social communication in
ants36. As previously described for ants36, Desat B genes are the most abundant desaturase
family in the termites and the cockroach (Supplementary Table 12), especially in M. natalensis where we found ten gene copies (significant expansion; P = 0.0003;
Supplementary Table 5 and Supplementary Figure 4). As in ants, especially the first
desaturases (Desat A–Desat E) vary greatly in their expression between castes and species in
the three termites (Fig. 2 and Supplementary Table 13)36. In contrast to ants, where these
genes are under strong purifying selection36, for the highly eusocial termite M. natalensis,
we found significant positive selection within the Desat B genes (codeml site models 7 and
8; P = 1.1 × 10−16), indicating a diversification in function, possibly related to their greater
diversification of worker castes (major and minor workers, major and minor soldiers). Although desaturases are often discussed in the context of CHC production and chemical
communication, their biochemical roles are quite diverse36, and the positive selection we
observe for M. natalensis may, at least in part, be related to their rather different ecology of
foraging and fungus-farming rather than nest-mate recognition. Future experimental
verification of the function of these genes will help better understand these observed
genomic and transcriptomic patterns. Underlining an increased importance of CHC communication in termites, the expression
patterns of elongases (extension of C-chain length) differ considerably in the termites
compared to the cockroach (Fig. 2 and Supplementary Table 14). In contrast to B. germanica, in which elongases are both nymph- (five genes) and adult-biased (four genes),
only one or two elongase genes in each termite are queen-biased in their expression, while
many are worker-biased. As with the desaturases, a group of M. natalensis elongases also
reveal significant signals of positive selection (codeml branch- site test; P = 4 × 10−4),
further indicating a greater diversification of CHC production in this higher termite. Nat Ecol Evol. Author manuscript; available in PMC 2019 April 25. CHC-producing enzymes have evolved caste-specificity Author Manuscript The last step of CHC biosynthesis, the production of hydrocarbons from long-chain fatty
aldehydes, is catalysed by a P450 gene, CYP4G1, in D. melanogaster35. We found one copy Nat Ecol Evol. Author manuscript; available in PMC 2019 April 25. Harrison et al. Page 7 of CYP4G1 in B. germanica, Z. nevadensis and C. secundus, but three copies in M. natalensis, reinforcing the greater importance of CHC synthesis in this higher termite. Corroborating the known importance of maternal CHCs in B. germanica37, CYP4G1 is
overexpressed in female adults compared to nymphs (Fig. 2 and Supplementary Table 15). In each of the termites, however, CYP4G1 is more highly expressed in workers (or kings in
C. secundus) compared to queens (Fig. 2 and Supplementary Table 15), adding support that,
com- pared to cockroach nymphs, a change in the dynamics and turnover of CHCs in termite
workers has taken place. of CYP4G1 in B. germanica, Z. nevadensis and C. secundus, but three copies in M. natalensis, reinforcing the greater importance of CHC synthesis in this higher termite. Corroborating the known importance of maternal CHCs in B. germanica37, CYP4G1 is
overexpressed in female adults compared to nymphs (Fig. 2 and Supplementary Table 15). In each of the termites, however, CYP4G1 is more highly expressed in workers (or kings in
C. secundus) compared to queens (Fig. 2 and Supplementary Table 15), adding support that,
com- pared to cockroach nymphs, a change in the dynamics and turnover of CHCs in termite
workers has taken place. Author Manuscript Changes in gene regulation in termites The development of distinct castes underlying division of labour is achieved via differential
gene expression. Major changes in gene regulation have been reported as being central to the
transition to eusociality in bees9 and ants10. Accordingly, we found major changes in
putative DNA methylation patterns (levels per 1-to-1 orthologue) among the termites
compared to four other hemimetabolous insect species (Fig. 4a). This is revealed by CpG
depletion patterns (CpGo/e, observed versus expected number of CpGs), a reliable predictor
of DNA methylation38,39, correlating more strongly between the termites than among any of
the other analysed hemimetabolous insects (Fig. 4). In other words, within orthologous
genes, predicted DNA methylation levels differ greatly between termites and other
hemimetabolous species but remain conserved among termite species. Author Manuscript The predicted levels of DNA methylation correlated negatively with the caste-specificity of
expression for each of the termites. This is confirmed by a positive correlation between
CpGo/e (negative association with level of DNA methylation) and absolute log2(fold change)
of expression between queens and workers (Pearson’s r = 0.32 to 0.36; P < 2.2 × 10−16). The
caste-specific expression of putatively unmethylated genes in termites is reflected in the
enrichment of GO terms related to sensory perception, regulation of transcription, signalling
and development, whereas methylated genes are mainly related to general metabolic
processes (Fig. 4b and Supplementary Table 16). These results show strong parallels to
findings for eusocial Hymenoptera40–43. This is in stark contrast to the non-eusocial
cockroach, B. germanica, where there was only a very weak relationship between CpGo/e
and differential expression between nymphs and adult females (r = 0.14), nor were any large
differences apparent in enriched GO terms between putatively methylated and non-
methylated genes (Fig. 4b). Our results argue in favour of a diminished role of DNA methylation in caste-specific
expression within eusocial insects, as recently shown38,44. In fact, DNA methylation appears
to be important for the regulation of housekeeping genes because predicted methylated
genes are related to general biological processes (further supported by lower CpGo/e within
1-to-1 orthologues than in non-conserved genes)45, while caste-specific genes are ‘released’
from this type of gene regulation. However, a recent study linked caste-specific DNA
methylation to alternative splicing in Z. nevadensis46. Author Manuscript Major biological transitions are often accompanied by expansions of transcription factor
(TF) families, such as genes containing zinc-finger (ZF) domains47. We also observed large Nat Ecol Evol. Nat Ecol Evol. Author manuscript; available in PMC 2019 April 25. Changes in gene regulation in termites Author manuscript; available in PMC 2019 April 25. Page 8 Harrison et al. differences in ZF families within the termites compared to B. germanica. Many ZF families
were reduced or absent in termites, while different, unrelated ZF gene families were
significantly expanded (Supplementary Tables 2–6). Queen-biased genes were significantly
over-represented among ZF genes for each of the termites (P < 2 × 10−10; χ2 test;
Supplementary Table 17), indicating an important role of ZF genes in the regulation of genes
related to caste-specific tasks and colony organization in the termites. This is in contrast to
the significant under-representation of differentially expressed ZF genes within germanica (P
= 4.8 × 10−5; χ2-test). Interestingly, two other important TF families (bHLH and bZIP)47,
which were not expanded in the termites, showed no caste-specific expression pattern (P >
0.05), except bZIP genes, in which queen-biased genes were marginally over-represented for
M. natalensis (P = 0.049). These major upheavals in ZF gene families and their caste-
specific expression show that major changes in TFs accompanied the evolution of termites,
strikingly similar to the evolution of ants10. Author Manuscript Author Manuscript Nat Ecol Evol. Author manuscript; available in PMC 2019 April 25. Evolution of genes related to moulting and metamorphosis Hemimetabolous eusociality is characterized by differentiated castes, which represent
different developmental stages. This is in contrast to eusocial Hymenoptera, in which
workers and reproductives are adults. While cockroaches develop directly through several
nymphal stages before becoming reproductive adults, termite development is more
phenotypically plastic, and workers are essentially immatures (Fig. 2). In wood-dwelling
termites, such as C. secundus and Z. nevadensis, worker castes are non-reproductive
immatures that are totipotent to develop into other castes, while in the higher termite M. natalensis, workers can be irreversibly defined instars. It is therefore clear that a major
change during the evolution of termites occurred within developmental pathways. Accordingly, we found changes in expression and gene family size of several genes related
both to moulting and metamorphosis. In the synthesis of the moulting hormone, 20-hydroxyecdysone, the six Halloween genes
(five cytochrome P450s and a Rieske-domain oxygenase) play a key role48,49. Only one
Halloween gene, Shade (Shd; CYP314A1), which mediates the final step of 20- (five cytochrome P450s and a Rieske-domain oxygenase) play a key role48,49. Only one
Halloween gene, Shade (Shd; CYP314A1), which mediates the final step of 20-
hydroxyecdysone synthesis, is differentially expressed between the final nymphal stages and
adult females in B. germanica (Fig. 2 and Supplementary Table 18), consistent with its role
in the nymphal or imaginal moult. In the three termites, the Halloween genes show varying
caste-specific expression (Fig. 2 and Supplementary Table 18), showing that ecdysone plays
a significant role in the regulation of caste differences. Ecdysteroid kinase genes (EcK),
which con- vert the insect moulting hormone into its inactive state, ecdysone 22-phosphate,
for storage50, are only overexpressed in female adults compared to nymphs in B. germanica
(16/51 genes, Fig. 2 and Supplementary Table 19). In termites, however, where the gene
copy number is reduced (18 to 20 per species), these important moulting genes appear to
have evolved worker-specific functions (Fig. 2 and Supplementary Table 19). Author Manuscript Whereas 20-hydroxyecdysone promotes moulting, juvenile hormone (JH) represses imaginal
development in pre-adult instars51. JH is important in caste differentiation in eusocial
insects, including termites12,52. Haemolymph JH-binding proteins (JHBPs), which transport
JH to its target tissues53, are reduced within the termites (21 to 33 genes) but significantly Page 9 Harrison et al. Page 9 expanded in B. germanica (51 copies; P = 0018; Supplementary Table 6). Evolution of genes related to moulting and metamorphosis Thirteen of the
JHBP genes are overexpressed in adult females and only 8 in nymphs in B. germanica (Fig. 2 and Supplementary Table 20). In both Z. nevadensis and M. natalensis, on the other hand,
JHBPs are significantly more worker-biased (P < 0.01, χ2 test; Supplementary Table 20 and
Fig. 2). In C. secundus, expression is more varied, with four worker-biased, seven king-
biased and two queen-biased genes (Fig. 2 and Supplementary Table 20). Author Manuscript These changes in copy number and caste-specific expression of genes involved in moulting
and metamorphosis within termites compared to the German cockroach demonstrate that
changes occurred in the control of the developmental pathway along with the evolution of
castes. However, this interpretation needs to be experimentally verified. Nat Ecol Evol. Author manuscript; available in PMC 2019 April 25. Genome sequencing and assembly. Genomic DNA from a single Blattella germanica male from an inbred line (strain: American
Cyanamid = Orlando Normal) was used to construct two paired-end (180-bp and 500-bp
inserts) and one of the two mate-pair libraries (2-kb inserts). An 8-kb mate-pair library was
constructed from a single female. The libraries were sequenced on an Illumina HiSeq2000
sequencing platform. The 413 Gb of raw sequence data were assembled with Allpaths LG54,
and then scaffolded and gap-filled using the in-house tools Atlas-Link v.1.0 (https://
www.hgsc.bcm.edu/software/atlas-link) and Atlas gap-fill v.2.2. For Cryptotermes secundus,
three paired-end libraries (250-bp, 500-bp and 800-bp inserts) and three mate-pair libraries
(2-kb, 5-kb and 10-kb inserts) were constructed from genomic DNA that was extracted from
the head and thorax of 1,000 individuals, originating from a single, inbred field colony. The
libraries were sequenced on an Illumina HiSeq2000 sequencing platform. The C. secundus
genome was assembled using SOAPdenovo (v.2.04)55 with optimized parameters, followed
by gapcloser (v1.10, released with SOAPdenovo) and kgf (v1.18, released with
SOAPdenovo). Author Manuscript Repeat annotation. A custom C. secundus and B. germanica repeat library was constructed using a combination
of homology-based and de novo approaches, including RepeatModeler/RepeatClassifier
(http://www.repeatmasker.org/RepeatModeler/), LTRharvest/LTRdigest59 and
TransposonPSI (http://transposonpsi.sourceforge.net/). The ab initio repeat library was
complemented with the RepBase (update 29 August 2016)60 and SINE repeat databases,
filtered for redundancy with CD-hit and classified with RepeatClassifier. RepeatMasker
(version open-4.0.6, http://www.repeatmasker.org) was used to mask the C secundus and B. germanica genome. Repeat content for the other studied species (Fig. 1) was obtained from
the literature61–67. Author Manuscript Methods Author Manuscript Conclusions Author Manuscript
A Author Manuscript These results, considered alongside many studies on eusociality in Hymenoptera9, 10, 14,36,
provide evidence that major changes in gene regulation and the evolution of sophisticated
chemical communication are fundamental to the transition to eusociality in insects. Strong
changes in DNA methylation patterns correlated with broad-scale modifications of
expression patterns. Many of these modified expression patterns remained consistent among
the three studied termite species and occurred within protein pathways essential for eusocial
life, such as CHC production, chemoperception, ecdysteroid synthesis and JH transport. The
stronger patterns we observe for M. natalensis, especially within genes linked to chemical
communication, such as the expansion of Desat B and CYP4G1 genes and significant
positive selection in desaturases, elongases and IRs, may be associated with this termite’s
higher level of eusociality and its status as a superoganism13. The analysis of further higher
and lower termites would shed light on the generality of these patterns and possibly assist in
the distinction between the influences of eco- logical and eusocial traits. Many of the mechanisms implicated in the evolution of eusociality in the termites occurred
convergently around 50 Myr later in the phylogenetically distant Hymenoptera. However,
several details are unique due to the distinct conditions within which eusociality arose. One
important difference is the higher TE content within cockroaches and termites, which
probably facilitated changes in gene family sizes, supporting the transition to eusociality. However, the most striking difference is the apparent importance of IRs for chemical
communication in the termites, compared to ORs in Hymenoptera. According to our results,
the non-eusocial ancestors of termites possessed a broad repertoire of IRs, which favoured
the evolution of important functions for colony communication in these chemoreceptors
within the termites, whereas in the solitary ancestors of eusocial hymenopterans ORs were
most abundant14, 25. The parallel expansions of different chemoreceptor families in these
two independent origins of eusociality indicate that convergent selection pressures existed
during the evolution of colony communication in both lineages. Author Manuscript Page 10 Harrison et al. Transcriptome sequencing and assembly. For annotation purposes, 22 whole- body RNA-sequencing (RNA-Seq) samples from
various developmental stages were obtained for B. germanica. For C. secundus, RNA-Seq
libraries were obtained for three workers, four queens and four kings, based on degutted,
whole-body extracts. In addition, we sequenced ten Macrotermes natalensis RNA-Seq
libraries from three queens, one king and six pools of workers. All libraries were constructed
using the Illumina (TruSeq) RNA-Seq kit. Author Manuscript For protein-coding gene annotation, B. germanica reads were assembled with de novo
Trinity (version r2014–04-13)56. The C. secundus reads were assembled using Cufflinks on
reads mapped with TopHat (version2.2.1)57,58, de novo Trinity56 and genome-guided Trinity
on reads mapped with TopHat. Nat Ecol Evol. Author manuscript; available in PMC 2019 April 25. Protein orthology. In addition to B. germanica, C. secundus, Z. nevadensis and M. natalensis, 16 other insect
proteomes were included in our analyses: Locusta migratoria, Rhodnius prolixus, Ephemera
danica, Drosophila melanogaster, Aedes aegypti, Tribolium castaneum, Nasonia vitripennis,
Polistes canadensis, Apis mellifera, Harpegnathos saltator, Linepithema humile,
Camponotus floridanus, Pogonomyrmex barbatus, Solenopsis invicta, Acromyrmex
echinatior and Atta cephalotes; as well as for the centipede Strigamia maritima as an
outgroup (for sources, see Supplementary Table 22). These proteomes were grouped into
orthologous clusters with OrthoMCL81, with a granularity of 1.5. Author Manuscript Differential gene expression. The C. secundus and M. natalensis RNA-Seq libraries were complemented with nine
published Z. nevadensis libraries, yielding two to six libraries from workers, queens and
kings for each termite. These were compared to six of the B. germanica libraries: two from
fifth instar nymphs, two from sixth instar nymphs and two from adult females. Reads were
mapped to the genome using HiSat278. Read counts per gene were obtained using htseq-
count and DESeq279 was used for differential expression analysis. Differential expression
analysis between kings (males), queens (females) and workers (majors and minors combined
for M. natalensis) was assessed for the termites. For B. germanica we evaluated the
differential expression between adults and the two last nymphal stages combined, with the
assumption that the final nymphal stages are homologous to termite workers and the adult
females are homologous to termite queens. Genes were considered significantly
differentially expressed if P < 0.05 and log2(fold change) >|1| in order to account for
allometric differences as recommended in a previous study80. Author Manuscript Protein-coding gene annotation. The B. germanica genome was annotated with Maker (version 2.31.8)68, using the species-
specific repeat library, B. germanica transcriptome data (22 whole-body RNA-Seq samples) Nat Ecol Evol. Author manuscript; available in PMC 2019 April 25. Page 11 Harrison et al. and the Swiss-Prot/ UniProt database (last accessed: 21 January 2016) plus the C. secundus
and Zootermopsis nevadensis protein sequences for evidence-based gene model predictions. AUGUSTUS (version 3.2)69, GeneMark-ES Suite (version 4.21)70 and SNAP71 were used
for ab initio predictions. C. secundus protein-coding genes were predicted using homology-
based, ab initio and expression-based methods, and integrated into a final gene set (see
Supplementary Information). Gene structures were predicted by GeneWise72. The ab initio
annotations were predicted with AUGUSTUS73 and SNAP71, retained if supported by both
methods and integrated with the homology-based predictions using GLEAN74. Transcriptome-based gene models were merged with PASA75 and tested for coding potential
with CPC76 and OrfPredictor77. PASA gene models were merged with the homology-based
and ab initio gene set, retaining the PASA models in case of overlap. Desaturases, elongases,
chemosensory receptors, cytochrome P450s and genes involved in the juvenile hormone
pathway were manually curated in Blattodea. and the Swiss-Prot/ UniProt database (last accessed: 21 January 2016) plus the C. secundus
and Zootermopsis nevadensis protein sequences for evidence-based gene model predictions. AUGUSTUS (version 3.2)69, GeneMark-ES Suite (version 4.21)70 and SNAP71 were used
for ab initio predictions. C. secundus protein-coding genes were predicted using homology-
based, ab initio and expression-based methods, and integrated into a final gene set (see
Supplementary Information). Gene structures were predicted by GeneWise72. The ab initio
annotations were predicted with AUGUSTUS73 and SNAP71, retained if supported by both
methods and integrated with the homology-based predictions using GLEAN74. Author Manuscript Transcriptome-based gene models were merged with PASA75 and tested for coding potential
with CPC76 and OrfPredictor77. PASA gene models were merged with the homology-based
and ab initio gene set, retaining the PASA models in case of overlap. Desaturases, elongases,
chemosensory receptors, cytochrome P450s and genes involved in the juvenile hormone
pathway were manually curated in Blattodea. Author Manuscript Nat Ecol Evol. Author manuscript; available in PMC 2019 April 25. Gene family expansions and contractions. Author Manuscript For the analyses of gene family expansions and contractions, the hierarchical clustering
algorithm MC-UPGMA87 was used, with a ProtoLevel cutoff of 80 (ref. 88). Protein families
were further divided into sub-families if they contained more than 100 proteins in a single
species, or more than an average of 35 proteins per species. Proteins were blasted against the
RepeatMasker TE database (E-value < 10−5) and clusters where > 50% of the proteins were
identified as transposable elements were discarded. Clade- and species-specific protein
family expansions and contractions, were identified with CAFE v3.089 using the same
protocol as in previous studies9,10 (see also Supplementary Information). IR and OR identification, phylogeny and structure. Ionotropic receptors (IRs) were identified using two custom hidden Markov models
(HMMs) obtained with hmmbuild and hmmpress of the HMMER suite82. The first HMM
comprises the IR’s ion channel and ligand-binding domain based on a MAFFT83 protein
alignment of 76 IRs from 15 species (Supplementary Table 23). The second HMM was built
to distinguish IRs from iGluRs, IR8a and IR25a, which have an additional amino-terminal Nat Ecol Evol. Author manuscript; available in PMC 2019 April 25. Harrison et al. Page 12 Page 12 domain24. For this we built an HMM from 48 protein sequences (Supplementary Table 23). The proteomes were scanned with pfam_scan and the two custom HMMs, where proteins
that matched the IR HMM, but not the amino- terminal domain HMM were annotated as
IRs. Odorant receptors (ORs) were identified on the basis of the Pfam domain PF02949 (7tm
OR). domain24. For this we built an HMM from 48 protein sequences (Supplementary Table 23). The proteomes were scanned with pfam_scan and the two custom HMMs, where proteins
that matched the IR HMM, but not the amino- terminal domain HMM were annotated as
IRs. Odorant receptors (ORs) were identified on the basis of the Pfam domain PF02949 (7tm
OR). Author Manuscript Multiple sequence alignments of IRs and ORs were obtained with hmmalign82, using the
Pfam OR HMM PF02949 and custom IR HMM to guide the alignment. Gene trees were
computed with FastTree84 (options: -pseudo -spr 4 –mlacc 2 -slownni) and visualized with
iTOL v385. Putative IR ligand-binding residues and structural regions were identified on the
basis of the alignments with melanogaster IRs and iGluRs of known structure86. TE-facilitated expansions. The repeat content in the 10-kb flanking regions of B. germanica, C. secundus, Z. nevadensis and M. natalensis genes was calculated using bedtools90. Coding DNA
sequences (CDSs) from neighbouring genes were removed and the repeat content was
analysed using generalized linear mixed models (glmmPQL implemented in the R91 package
MASS92) with binomial error distribution. Fixed predictors included gene family expansion,
species ID and their interaction. Cluster ID was fitted as a random factor to avoid pseudo-
replication. Significance was assessed on the basis of the Wald t-test (R package aod93) at α
< 0.05. Main and interaction effects for each of the genomic regions are listed in
Supplementary Table 8. Model parameters are listed in Supplementary Table 8. Author Manuscript Nat Ecol Evol. Author manuscript; available in PMC 2019 April 25. Supplementary Material Refer to Web version on PubMed Central for supplementary material. Tests for positive selection. To test for positive selection within gene families of interest, site model tests 7 and 8 were
performed (model = 0; NSsites = 7 8) on species-specific CDS alignments, or branch-site
test (model = 2; NSsites = 2; fix_ omega = 1 for null model and 0 for alternative model) on
multi-species alignments. Protein sequences were aligned using MAFFT83 with the E-INS-i
strategy, and CDS alignments were created using pal2nal.pl94. Phylogenetic trees were
created with FastTree84. Alignments were trimmed using Gblocks (settings: -b2 = 21; -b3 =
20; -b4 = 5; -b5 = a). Models were compared using likelihood-ratio test and where P < 0.05,
Bayes empirical Bayes results were consulted for codon positions under positive selection (P
< 0.05). Author Manuscript Nat Ecol Evol. Author manuscript; available in PMC 2019 April 25. Page 13 Page 13 Harrison et al. CpG depletion patterns and GO enrichment. Author Manuscript To estimate DNA methylation, we compared observed to expected CpG counts within CDS
sequences38,39. A low CpGo/e indicates a high level of DNA methylation, as the cytosines of
methylated CpGs often mutate to thymines. Expected CpG counts were calculated by
dividing the product of cytosine and guanine counts by the sequence length. The principal
component analysis in Fig. 4 was created using the R function prcomp on log- transformed
CpGo/e values for all 1-to-1 orthologues for the seven hemimetabolous species. These
orthologues were extracted from the OrthoMCL results. The three-dimensional (3D) plot
was created with the plot3d command from the R package rgl. CpG-depleted (first quartile) and -enriched (fourth quartile) genes were tested for
enrichment of Gene Ontology terms. Pfam protein domains were obtained for germanica, Z. nevadensis, C. secundus and M. natalensis protein sequences using PfamScan95. Corresponding GO terms were obtained with Pfam2GO. GO-term over-representation was
assessed using the TopGO96 package in R. Enrichment analysis was performed using the
weight algorithm selecting nodesize = 10 to remove terms with fewer than ten annotated GO
terms. After that, GO terms classified as significant (topGOFisher < 0.05) were visualized
using the R package tagcloud (https://cran.r-project.org/web/packages/tagcloud/). Author Manuscript Acknowledgements We thank O. Niehuis for allowing use of the unpublished E. danica genome, J. Gadau and C. Smith for comments
and advice on the manuscript, and J. Schmitz for assistance with analyses and proofreading the manuscript. J.K. thanks Charles Darwin University (Australia), especially S. Garnett and the Horticulture and Aquaculture team, for
providing logistic support to collect C. secundus. The Parks and Wildlife Commission, Northern Territory, the
Department of the Environment, Water, Heritage and the Arts gave permission to collect (Permit number 36401)
and export (Permit WT2010–6997) the termites. USDA is an equal opportunity provider and employer. M.C.H. and
E.J. are supported by DFG grant BO2544/11–1 to E.B.-B. J.K. is supported by University of Osnabrück and DFG
grant KO1895/16–1. X.B. and M.-D.P. are supported by Spanish Ministerio de Economía y Competitividad
(CGL2012–36251 and CGL2015–64727-P to X.B., and CGL2016–76011-R to M.-D.P.), including FEDER funds,
and by Catalan Government (2014 SGR 619). C.S. is supported by grants from the US Department of Housing and
Urban Development (NCHHU-0017–13), the National Science Foundation (IOS-1557864), the Alfred P. Sloan
Foundation (2013–5-35 MBE), the National Institute of Environmental Health Sciences (P30ES025128) to the
Center for Human Health and the Environment, and the Blanton J. Whitmire Endowment. M.P. is supported by a
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Press, Baltimore, 2007). Nat Ecol Evol. Author manuscript; available in PMC 2019 April 25. Page 18 Harrison et al. Fig. 1. Phylogenetic, genomic and proteomic comparisons of 20 insect species. From left to right: a phylogenetic tree of 20 insect species with Strigamia maritima
(centipede) as the outgroup (species of newly sequenced genomes presented in this study are
underlined); level of eusociality (one red insect: simple eusociality; two red insects:
advanced eusociality; black fly: non-eusocial); fractions of repetitive content (yellow) within
genomes of selected species (for sources, see Supplementary Information); proportions of
species-specific gene family expansions (green), contractions (red) and stable gene families
(black), the size of the pies represents the relative size of the gene family change (based on
total numbers); a bar chart showing protein orthology across taxonomic groups within each
genome. Ma, million years ago. Nat Ecol Evol. Author manuscript; available in PMC 2019 April 25. References Smar, Strigamia maritima; Edan, Ephemera danica; Rpro,
Rhodnius prolixus; Nvit, Nasonia vitripennis; Amel, Apis mellifera; Pcan, Polistes
canadensis; Hsal, Harpegnathos saltator; Lhum, Linepithema humile; Cflo, Camponotus
floridanus; Pbar, Pogonomyrmex barbatus; Sinv, Solenopsis invicta; Aech, Acromyrmex
echinatior; Acep, Atta cephalotes; Tcas, Tribolium castaneum; Aaeg, Aedes aegypti; Dmel,
Drosophila melanogaster; Lmig, Locusta migratoria; Bger, Blattella germanica; Znev,
Zootermopsis nevadensis; Csec, Cryptotermes secundus; Mnat, Macrotermes natalensis. Author Manuscript Author Manuscript Fig. 1. Phylogenetic, genomic and proteomic comparisons of 20 insect species. Fig. 1. Phylogenetic, genomic and proteomic comparisons of 20 insect species. From left to right: a phylogenetic tree of 20 insect species with Strigamia maritima
(centipede) as the outgroup (species of newly sequenced genomes presented in this study are
underlined); level of eusociality (one red insect: simple eusociality; two red insects:
advanced eusociality; black fly: non-eusocial); fractions of repetitive content (yellow) within
genomes of selected species (for sources, see Supplementary Information); proportions of
species-specific gene family expansions (green), contractions (red) and stable gene families
(black), the size of the pies represents the relative size of the gene family change (based on
total numbers); a bar chart showing protein orthology across taxonomic groups within each
genome. Ma, million years ago. Smar, Strigamia maritima; Edan, Ephemera danica; Rpro,
Rhodnius prolixus; Nvit, Nasonia vitripennis; Amel, Apis mellifera; Pcan, Polistes
canadensis; Hsal, Harpegnathos saltator; Lhum, Linepithema humile; Cflo, Camponotus
floridanus; Pbar, Pogonomyrmex barbatus; Sinv, Solenopsis invicta; Aech, Acromyrmex
echinatior; Acep, Atta cephalotes; Tcas, Tribolium castaneum; Aaeg, Aedes aegypti; Dmel,
Drosophila melanogaster; Lmig, Locusta migratoria; Bger, Blattella germanica; Znev,
Zootermopsis nevadensis; Csec, Cryptotermes secundus; Mnat, Macrotermes natalensis. g
p y g
p
g
(centipede) as the outgroup (species of newly sequenced genomes presented in this study are
underlined); level of eusociality (one red insect: simple eusociality; two red insects: Author Manuscript Author Manuscript Fig. 2. Comparison of developmental pathways between B. germanica, the lower termites Z. nevadensis and C. secundus, and the higher termite M. natalensis. Shown from left to right are: a simple phylogeny97 describing important novelties along the
evolutionary trajectory to termites (numbers in brackets are genome sizes); life cycles;
differential expression (log2(fold change) > 1 and P < 0.05; DESeq279; sample sizes are
shown in the last column) between workers and queens (between nymphs and adult females
in B. Nat Ecol Evol. Author manuscript; available in PMC 2019 April 25. References germanica) of the selected gene families (Desat, desaturases; Elong, elongases;
H’ween, Halloween genes) and total numbers within all genes; the numbers denote total
numbers of genes in each gene family. Harrison et al. Page 19 Harrison et al. Harrison et al. Page 19 Author Manuscript Author Manuscript Fig. 2. Comparison of developmental pathways between B. germanica, the lower termites Z. nevadensis and C. secundus, and the higher termite M. natalensis. Fig. 2. Comparison of developmental pathways between B. germanica, the lower termites Z. nevadensis and C. secundus, and the higher termite M. natalensis. Shown from left to right are: a simple phylogeny97 describing important novelties along the
evolutionary trajectory to termites (numbers in brackets are genome sizes); life cycles;
differential expression (log2(fold change) > 1 and P < 0.05; DESeq279; sample sizes are
shown in the last column) between workers and queens (between nymphs and adult females
in B. germanica) of the selected gene families (Desat, desaturases; Elong, elongases;
H’ween, Halloween genes) and total numbers within all genes; the numbers denote total
numbers of genes in each gene family. Author Manuscript Author Manuscript Page 20 Page 20 Harrison et al. g Fig. 3. Expansions, contractions and positive selection within iRs and ORs in termites. a,b, IR (a) and OR (b) gene trees of 13 insect species. In each tree, only well-supported
clades (support values > 85) that include B. germanica or termite genes are highlighted
within the gene trees. The lengths of the coloured bars represent the number of genes per
species within each of these clades. The red asterisk in a denotes the putative root of
intronless IRs. c, The upper schematic diagram depicts the 2D structure of an IR, containing
ligand-binding lobes (S1 and S2), transmembrane regions (TM1–3) and the pore domain (P). Below, the sequence of the domains along the peptide is represented, showing that the sites,
Nat Ecol Evol Author manuscript; available in PMC 2019 April 25 Author Manuscript Author Manuscript Author Manuscript Fig. 3. Expansions, contractions and positive selection within iRs and ORs in termites. Author Manuscript Fig. 3. Expansions, contractions and positive selection within iRs and ORs in termites. a,b, IR (a) and OR (b) gene trees of 13 insect species. In each tree, only well-supported
clades (support values > 85) that include B. germanica or termite genes are highlighted
within the gene trees. Nat Ecol Evol. Author manuscript; available in PMC 2019 April 25. Nat Ecol Evol. Author manuscript; available in PMC 2019 April 25. References The lengths of the coloured bars represent the number of genes per
species within each of these clades. The red asterisk in a denotes the putative root of
intronless IRs. c, The upper schematic diagram depicts the 2D structure of an IR, containing
ligand-binding lobes (S1 and S2), transmembrane regions (TM1–3) and the pore domain (P). Below, the sequence of the domains along the peptide is represented, showing that the sites, Fig. 3. Expansions, contractions and positive selection within iRs and ORs in termites. a,b, IR (a) and OR (b) gene trees of 13 insect species. In each tree, only well-supported
clades (support values > 85) that include B. germanica or termite genes are highlighted
within the gene trees. The lengths of the coloured bars represent the number of genes per
species within each of these clades. The red asterisk in a denotes the putative root of
intronless IRs. c, The upper schematic diagram depicts the 2D structure of an IR, containing
ligand-binding lobes (S1 and S2), transmembrane regions (TM1–3) and the pore domain (P). Below, the sequence of the domains along the peptide is represented, showing that the sites, Nat Ecol Evol. Author manuscript; available in PMC 2019 April 25. Harrison et al. Page 21 which are under significant positive selection (red bars; codeml site models 7 and 8) within
Blattodea IRs for M. natalensis (P < 1.7 × 10−10; likelihood-ratio test, 5 sequences, 413
aligned codons), are all situated within the ligand-binding lobes and on or around the
putative ligand-binding sites (asterisks)86. d, The same representation for ORs, which
include eight transmembrane regions. Positive selection was found for M. natalensis (P = 1.1
× 10−10; 5 sequences, 1,001 aligned codons) and C. secundus (P = 5.6 × 10−16; likelihood
ratio test, 26 sequences, 1,913 aligned codons) of the orange clade, each at two codon
positions within the second transmembrane region and at a third position within the carboxy-
terminal extracellular region for M. natalensis. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Page 22 Harrison et al. Fig. 4. CpGo/e of seven hemimetabolous insects. a, Principal component analysis (PCA) of predicted DNA methylation patterns among 2,664
1-to-1 orthologues, estimated via CpGo/e. Nat Ecol Evol. Author manuscript; available in PMC 2019 April 25. Nat Ecol Evol. Author manuscript; available in PMC 2019 April 25. References The spheres represent the positions of the species
within the 3D PCA, with the distance between the spheres representing the similarity of
CpGo/e between species at each orthologue; the curves are the distribution of CpGo/e, with
et al. Page 22 Author Manuscript Author Manuscript Author Manuscript Author Manuscript Fig. 4. CpGo/e of seven hemimetabolous insects. Author Manuscript g
p
o/e
a, Principal component analysis (PCA) of predicted DNA methylation patterns among 2,664
1-to-1 orthologues, estimated via CpGo/e. The spheres represent the positions of the species
within the 3D PCA, with the distance between the spheres representing the similarity of
CpGo/e between species at each orthologue; the curves are the distribution of CpGo/e, with
the dotted line showing CpGo/e = 1. b, Tag clouds of enriched (P < 0.05; Fisher test, weight
algorithm, topGO96) GO terms (biological processes) among the lower (left) and the higher
quartile (right) of CpGo/e within termites (top) and B. germanica (bottom). For termites, Nat Ecol Evol. Author manuscript; available in PMC 2019 April 25. genes were merged from all three species for analysing GO term enrichment. Number of
enriched genes and total number of genes in topGO enrichment tests (low CpGo/e/high
CpGo/e/gene universe): B. germanica (3,291/1,842/11,409); termites (6,754/4,600/25,910). High CpGo/e indicates a low level of DNA methylation and vice versa. Page 23 Harrison et al. Page 23 Page 23 genes were merged from all three species for analysing GO term enrichment. Number of
enriched genes and total number of genes in topGO enrichment tests (low CpGo/e/high
CpGo/e/gene universe): B. germanica (3,291/1,842/11,409); termites (6,754/4,600/25,910). High CpGo/e indicates a low level of DNA methylation and vice versa. genes were merged from all three species for analysing GO term enrichment. Number of
enriched genes and total number of genes in topGO enrichment tests (low CpGo/e/high
CpGo/e/gene universe): B. germanica (3,291/1,842/11,409); termites (6,754/4,600/25,910). High CpGo/e indicates a low level of DNA methylation and vice versa. Author Manuscript Author Manuscript
A Author Manuscript Author Manuscript
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PAM50 Breast Cancer Subtyping by RT-qPCR and Concordance with Standard Clinical Molecular Markers
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BMC medical genomics
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RESEARCH ARTICLE Open Access PAM50 Breast Cancer Subtyping by RT-qPCR
and Concordance with Standard Clinical
Molecular Markers Roy RL Bastien1, Álvaro Rodríguez-Lescure2, Mark TW Ebbert1, Aleix Prat3,4, Blanca Munárriz5, Leslie Rowe1,
Patricia Miller1, Manuel Ruiz-Borrego6, Daniel Anderson1, Bradley Lyons1, Isabel Álvarez7, Tracy Dowell1,
David Wall1, Miguel Ángel Seguí8, Lee Barley1, Kenneth M Boucher9, Emilio Alba10, Lisa Pappas9, Carole A Davis11,
Ignacio Aranda12, Christiane Fauron1, Inge J Stijleman11, José Palacios13, Antonio Antón14, Eva Carrasco15,
Rosalía Caballero15, Matthew J Ellis16, Torsten O Nielsen17, Charles M Perou3, Mark Astill1,
Philip S Bernard1,11,19*† and Miguel Martín18† Bastien et al. BMC Medical Genomics 2012, 5:44
http://www.biomedcentral.com/1755-8794/5/44 Bastien et al. BMC Medical Genomics 2012, 5:44
http://www.biomedcentral.com/1755-8794/5/44 * Correspondence: philip.bernard@hci.utah.edu
†Equal contributors
1The ARUP Institute for Clinical and Experimental Pathology, Salt Lake City,
UT, USA
11Department of Pathology, University of Utah Health Sciences Center/
Huntsman Cancer Institute, Salt Lake City, UT, USA
Full list of author information is available at the end of the article * Correspondence: philip.bernard@hci.utah.edu
†Equal contributors
1The ARUP Institute for Clinical and Experimental Pathology, Salt Lake City,
UT, USA
11Department of Pathology, University of Utah Health Sciences Center/
Huntsman Cancer Institute, Salt Lake City, UT, USA
Full list of author information is available at the end of the article
© 2012 Bastien et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited. * Correspondence: philip.bernard@hci.utah.edu
†Equal contributors
1The ARUP Institute for Clinical and Experimental Pathology, Salt Lake City,
UT, USA
11Department of Pathology, University of Utah Health Sciences Center/
Huntsman Cancer Institute, Salt Lake City, UT, USA
Full list of author information is available at the end of the article
l l
d C
l
d
h
O
l d
b
d
d
h
f h C Abstract Background: Many methodologies have been used in research to identify the “intrinsic” subtypes of breast cancer
commonly known as Luminal A, Luminal B, HER2-Enriched (HER2-E) and Basal-like. The PAM50 gene set is often
used for gene expression-based subtyping; however, surrogate subtyping using panels of immunohistochemical
(IHC) markers are still widely used clinically. Discrepancies between these methods may lead to different treatment
decisions. Methods: We used the PAM50 RT-qPCR assay to expression profile 814 tumors from the GEICAM/9906 phase III
clinical trial that enrolled women with locally advanced primary invasive breast cancer. All samples were scored at a
single site by IHC for estrogen receptor (ER), progesterone receptor (PR), and Her2/neu (HER2) protein expression. Equivocal HER2 cases were confirmed by chromogenic in situ hybridization (CISH). Single gene scores by IHC/CISH
were compared with RT-qPCR continuous gene expression values and “intrinsic” subtype assignment by the PAM50. High, medium, and low expression for ESR1, PGR, ERBB2, and proliferation were selected using quartile cut-points
from the continuous RT-qPCR data across the PAM50 subtype assignments. Results: ESR1, PGR, and ERBB2 gene expression had high agreement with established binary IHC cut-points (area
under the curve (AUC) ≥0.9). Estrogen receptor positivity by IHC was strongly associated with Luminal (A and B)
subtypes (92%), but only 75% of ER negative tumors were classified into the HER2-E and Basal-like subtypes. Luminal A tumors more frequently expressed PR than Luminal B (94% vs 74%) and Luminal A tumors were less
likely to have high proliferation (11% vs 77%). Seventy-seven percent (30/39) of ER-/HER2+ tumors by IHC were
classified as the HER2-E subtype. Triple negative tumors were mainly comprised of Basal-like (57%) and HER2-E
(30%) subtypes. Single gene scoring for ESR1, PGR, and ERBB2 was more prognostic than the corresponding IHC
markers as shown in a multivariate analysis. (Continued on next page) © 2012 Bastien et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited. Page 2 of 12 Bastien et al. BMC Medical Genomics 2012, 5:44
http://www.biomedcentral.com/1755-8794/5/44 (Continued from previous page)
Conclusions: The standard immunohistochemical panel for breast cancer (ER, PR, and HER2) does not adequately
identify the PAM50 gene expression subtypes. Measurement of PCR efficiency, limits of detection, and
limits of quantification Breast cancer cell lines (BT474, MCF7, MDA-MB-231,
MDA-MB-436, MDA-MB-453, MDA-MB-468, SKBR3
and T47D) were cultured, pelleted and processed into
FFPE tissue blocks. The RNA was extracted, pooled, re-
verse transcribed, and serially diluted at 2-fold increments
from 2.56μg to 0.039ng per assay, which corresponds to a
range of 7.11ng to 108.51fg of cDNA per reaction well. Each gene was measured in triplicate per RT-qPCR
run on the Roche LC480 and 2 runs were performed
for each of the 17 dilutions. A detailed description of
methods used to calculate PCR efficiency, limits of de-
tection and limits of quantification can be found in
Additional file 3. Abstract Although there is high agreement between biomarker scoring by
protein immunohistochemistry and gene expression, the gene expression determinations for ESR1 and ERBB2 status
was more prognostic. from reduction mammoplasties or grossly uninvolved
breast tissue and 155 primary invasive breast cancers. These samples were collected from 2005–2009 under
IRB approved protocols at the University of Utah and
the University of North Carolina at Chapel Hill. Clinical-
pathological information associated with the samples is
based on the College of American Pathology (CAP) and
American Joint Committee on Cancer (AJCC) standards
at the time of collection (Additional file 1). Subtype classifi-
cation and single and meta-gene (proliferation) scores
were predicted on an independent test set of 814 sam-
ples from the GEICAM/9906 clinical trial, a rando-
mized Phase 3 trial of fluorouracil, epirubicin, and
cyclophosphamide
alone
or
followed
by
paclitaxel
[22]. Patients that were hormone receptor positive
(ER and/or PR positive by IHC) were given adjuvant
tamoxifen. The
hormone
receptor
status
for
these
samples was evaluated at a single site (Department of
Pathology, Hospital General Universitario de Alicante)
using immunohistochemistry (IHC) for progesterone
receptor (PR) (clone PgR636, DAKO, Glostrup, Den-
mark) and estrogen receptor (ER) (clone 1D5, DAKO,
Glostrup, Denmark) (Additional file 2). The scores for
the proportion of dyed cells and intensity were summed to
obtain a total Allred Score [23]. Measurement of HER2 ex-
pression was performed by Herceptest™(DAKO, Glostrup,
Denmark) and samples with scores of 2+ by IHC were
confirmed by CISH, following the ASCO/CAP guide-
lines [24]. The clinical data for the training set and
GEICAM/9906 test set are summarized in Table 1. Background For over a decade, research studies have used gene expres-
sion to classify invasive breast cancers into biologically and
clinically distinct subtypes that have become known as
Luminal A, Luminal B, HER2-Enriched (HER2-E) and
Basal-like [1-3]. Subtype information has repeatedly shown
to be an independent predictor of survival in breast cancer
when used in multivariate analyses with standard clinical-
pathological variables [3-6]. In 2009, Parker et al. derived a
minimal gene set (PAM50) for classifying “intrinsic”
subtypes of breast cancer [3,7]. The PAM50 gene set has
high agreement in classification with larger “intrinsic”
gene sets previously used for subtyping [1,3,4,8], and is
now commonly employed [9-12]. There are several multi-gene expression tests clinically
available for determining risk of relapse in early stage
breast cancer, including the 21-gene recurrence score
[13] (Oncotype DxW, Genomic Health Inc, Redwood City,
CA, www.oncotypedx.com), the 14-gene distant metastasis
signature [14] (BreastOncPx™, US Labs, Irvine, CA,
www.uslabs.net), the 97-gene histologic grade predictor
[15] (MapQuant Dx™Genomic Grade, Ipsogen, Marseilles,
France and New Haven, CT, USA, www.ipsogen.com), and
the 70-gene prognosis signature [16] (MammaPrintW,
Agendia, Irvine, CA, www.agendia.com). The molecu-
lar signature of proliferation is perhaps the strongest
variable in all these tests for determining outcome in
ER + breast cancer. In addition to gene expression profiling by micro-
array or RT-qPCR [2-4,8,17,18], many studies have
used immunohistochemical panels to identify subtypes
[19-21]. For example, high grade ER+/HER2- tumors
and ER+/HER2+ tumors are often considered Luminal
B, while ER-/HER2+ are considered HER2-E subtype
and triple negative tumors are considered Basal-like. In
this study, we assess agreement between histopathology/
IHC status and PAM50 classification for subtype, ESR1,
PGR, ERBB2, and proliferation. Scaling single and Meta-Gene scores The PAM50 subtype assay can also provide quantitative
and qualitative gene expression scores for the standard
biomarkers
usually
measured
semi-quantitatively
by
IHC: ESR1/ER, PGR/PR and ERBB2/HER2. In addition,
the PAM50 contains many cell cycle regulated genes that
can be combined into a meta-gene for proliferation
(CENPF, ANLN, CDC20, CCNB1, CEP55, MYBL2, MKI67,
UBE2C, RRM2, and KIF2C). The meta-gene for prolif-
eration were selected because they had strong correl-
ation within the associated dendrogram of the training
set cluster. The quantitative scale of 1–10 for the single
genes and proliferation was derived by rescaling the
original log-expression ratios from the training set and
included a 10% buffer on either side of the original
values to allow for values that were higher or lower
than what was encountered in the training set. Any
new values that were less than 0 or greater than 10
were truncated at 0 and 10, respectively. Fixed cut-points (low vs. intermediate/high) for the
single genes (ESR1, PGR, and ERBB2) and proliferation
were directly applied from the training set to the GEI-
CAM/9906 test set. Receiver Operator Characteristic
(ROC) curves were generated by dichotomizing IHC data
and treating RT-qPCR data as a continuous variable. Selection of prototype samples for the RT-qPCR training
set random error into the call to determine the frequency
of switching subtype [26]. Training set samples were run across 3 batches of PCR
plates manufactured at ARUP Laboratories (ARUP Labora-
tories, Salt Lake City, UT, www.aruplab.com). The method
to identify prototype samples representing the subtypes has
been previously described [3]. Briefly, hierarchical clustering
(median centered by gene, Pearson correlation, centroid-
linkage) [25] was performed on the RT-qPCR data and Sig-
Clust was run at each node of the dendrogram beginning at
the root and stopping when the test was no longer signifi-
cant (p> 0.001). A “centroid” was generated for each sub-
type in the training set using the average expression for
each gene across all prototype samples of a given subtype. Single sample subtype prediction was performed by
calculating a Spearman rank correlation coefficient
between the gene expression values of an individual
sample compared to each of the centroid gene values
for Luminal A, Luminal B, HER2-Enriched, Basal-like,
and Normal. The subtype classification for the new
sample is assigned to the centroid with the highest
correlation. Training set, subtype stability, and classification
accuracy We identified 154 prototypic samples from the RT-
qPCR data by hierarchical clustering of the PAM50 clas-
sifier genes, and statistical selection from the dendro-
gram by SigClust [27]. The training set was comprised
of 53 Luminal A, 27 Luminal B, 32 HER2-enriched, 38
Basal-like and 4 Normal-like (Figure 1). The 10-fold
cross validation had 91.6% concordance (multi-rater
kappa score of 0.885) with the initial SigClust subtype
assignments (Additional file 4). 10-Fold cross validation to determine stability of selected
prototypes The 154 prototype samples identified by SigClust were
randomly split into 10 groups. Nine of the 10 groups
were used to calculate new centroids for each of the 5
possible subtype assignments. Each sample from the
remaining group was then assigned a subtype based
on closest proximity to the newly calculated cen-
troids using Spearman's Rho. The process of calculat-
ing centroids using 9 of the 10 groups and predicting
on the remaining group was repeated leaving out a
different group each time. Samples and clinical data There was ethical review and approval for all protocols
used in this study from the respective centers involved
and all subjects gave written informed consent to par-
ticipate. A training set was developed using 171 breast
samples, comprised of 16 “normal” breast tissue samples Page 3 of 12 Bastien et al. BMC Medical Genomics 2012, 5:44
http://www.biomedcentral.com/1755-8794/5/44 Bastien et al. BMC Medical Genomics 2012, 5:44
http://www.biomedcentral.com/1755-8794/5/44 Table 1 Patient characteristics Table 1 Patient characteristics
Variable
Training set data
n = 154 Total (%)
Variable
Test set data
n = 814 Total (%)
Age (years)
Median
55.5
Age (years)
Median
50.4
(range)
26 – >92
(range)
23.1 – 76.2
Menopausal status
Pre
49 (31.8)
Menopausal status
Pre
439 (53.9)
Post
101 (65.6)
Post
375 (46.1)
Unknown
4 (2.6)
Primary tumor size
T1
63 (40.9)
Primary tumor size
T1
338 (41.5)
T2
69 (44.8)
T2
430 (52.8)
T3
17 (11.0)
T3
46 (5.7)
Unknown
1 (0.6)
Reduction Mamoplasty
4 (2.6)
Nodal status
0
95 (61.7)
Nodal status
0
0
1 – 3
54 (35.1)
1 – 3
503 (61.8)
> 3
0 (0)
> 3
311 (38.2)
Unknown
1 (0.6)
Reduction Mamoplasty
4 (2.6)
Histopathologic grade*
G1
23 (14.9)
Histopathologic grade*
G1
107 (13.1)
G2
45 (29.2)
G2
335 (41.2)
G3
80 (51.9)
G3
313 (38.5)
GX
2 (1.3)
GX
59 (7.2)
Reduction Mamoplasty
4 (2.6)
Estrogen receptor^
Positive
100 (64.9)
Estrogen receptor^
Positive
644 (79.1)
Negative
49 (31.8)
Negative
170 (20.9)
Unknown
1 (0.6)
Reduction Mamoplasty
4 (2.6)
Progesterone receptor^^
Positive
82 (53.2)
Progesterone receptor^^
Positive
567 (69.7)
Negative
67 (43.5)
Negative
247 (30.3)
Unknown
1 (0.6)
Reduction Mamoplasty
4 (2.6)
HER2 status
Positive
37 (24.0)
HER2 status
Positive
116 (14.3)
Negative
111 (72.1)
Negative
698 (85.7)
Unknown
2 (1.3)
Reduction Mamoplasty
4 (2.6)
Ki67 IHC
Unknown
154 (100)
Ki67 IHC
Positive
236 (29.6)
Negative
561 (70.4)
PAM50 Intrinsic Subtype
Luminal A
53 (34.4)
PAM50 Intrinsic Subtype
Luminal A
277 (34.0)
Luminal B
27 (17.5)
Luminal B
261 (32.1)
HER2-enriched
32 (20.8)
HER2-enriched
174 (21.4)
Basal-like
38 (24.7)
Basal-like
70 (8.6)
Normal-like
4 (2.6)
Normal-like
32 (3.9)
*Grade based on Nottingham-Bloom-Richardson scoring. ER
i i
i
d
l
i i
l i Test set data
n = 814 Total (%) Page 4 of 12 Bastien et al. BMC Medical Genomics 2012, 5:44
http://www.biomedcentral.com/1755-8794/5/44 Measurements of assay reproducibility Reproducibility of the PAM50 assay was determined
using 3 cell lines (MCF7, ME16C and SKBR3) and a
pool of Luminal A prototype samples that were each run
12 times (3 runs across 4 batches of PAM50 plates) over
30 days. Variation in each gene measurement was
assessed using the difference between the mean calibrator
crossing point (CP) and each sample replicate CP (ΔCP). The square root of the mean CV2 for ΔCP was used to es-
timate the variation for each gene within plate, within
batch, and across batches. Higher gene CVs may be due to
lower concentration of a single gene within a sample. We
used the technical variability in measuring each gene to
further assess the stability of the categorical subtype call in
the GEICAM/9906 test set samples. Since the biology be-
tween subtypes is a continuum and some samples may
have close proximity to more than 1 prototypic sub-
type, we used a Monte-Carlo simulation to introduce Interference from normal breast tissue contamination Figure 1 Clinical PAM50 RT-qPCR breast cancer training set. Hierarchical clustering of RT-qPCR data for the PAM50 classifier genes
normalized to the 5 control genes using 171 FFPE procured breast samples. Statistical selection using SigClust identified the 5 significant groups
previously identified and designated as Luminal A (dark blue), Luminal B (light blue), HER2-E (pink), Basal-like (red), and Normal (green). The 16
non-neoplastic samples (grey), from reduction mammoplasty and grossly uninvolved breast tissues, all Clustered together and away from the
invasive cancers. SigClust identified 4 reduction mammoplasty samples (green) that were used to train the Normal subtype. within the GEICAM/9906 test set. Additional single and
meta-gene cut-points for the training and test sets can
be found in Additional file 6. Comparisons between the
gene expression and IHC data for GEICAM/9906 gave
good overall agreement with a high area under the curve
(AUC) for ESR1/ER (AUC = 0.90), PGR/PR (AUC = 0.90),
and ERBB2/HER2 (AUC = 0.95) (Figure 3). Rather than
re-optimize the cut-points on the test set, the fixed cut- to a classification of Normal, with the exception of
Luminal B, which switched to Luminal A. The switch
from Luminal B to Luminal A required 50% contribu-
tion from the normal breast tissue signature. Interfer-
ence data from the introduction of normal breast
tissue RNA into each of the subtypes is provided in
Additional file 5. During the dilution series for HER2-E
with “normal” there was switching in the ESR1 score
between intermediate and low suggesting that both
samples had similar ESR1 expression near the cut-off
for those scores. Table 2 Cut-points for quantitative gene scores Table 2 Cut-points for quantitative gene scores
Score ranges
Genes/Meta-genes
Low
Intermediate
High
ESR1 (ER)†
0 - 5.2
>5.2 - 7.6
>7.6 - 10
PGR (PR){
0 - 5.1
>5.1 - 7.4
>7.4 - 10
ERBB2 (HER2)}
0 - 5.6
>5.6 - 7.5
>7.5 - 10
Proliferation*
0 - 3.9
>3.9 - 5.3
>5.3 - 10
†high ESR1 = above median expression for Luminal A; low ESR1 = below
median expression for HER2-enriched. Interference from normal breast tissue contamination Interference from normal breast tissue contamination
A major source of subtype misclassification comes from
having normal tissue within the tumor sample [28]. We assessed the effect of having contaminating normal
tissue within the tumor sample by diluting out RNA
from tumor subtypes with pooled RNA from “normal”
reduction mammoplasties (0%, 25%, 50% and 75%). Primary tumors were pooled to represent Luminal A
and HER2-E samples while cell lines were used to
represent Luminal B (MCF7) and Basal-like (ME16C). The changes in subtype classification occurred in a
systematic fashion with all subtypes switching directly Page 5 of 12 Bastien et al. BMC Medical Genomics 2012, 5:44
http://www.biomedcentral.com/1755-8794/5/44 FGFR4
ERBB2
GRB7
BLVRA
BAG1
BCL2
CXXC5
ESR1
GPR160
FOXA1
MLPH
NAT1
SLC39A6
MAPT
PGR
MDM2
TMEM45B
MMP11
ACTR3B
CDC6
CCNE1
EXO1
CDCA1
KNTC2
BIRC5
CENPF
ANLN
CDC20
CCNB1
CEP55
MYBL2
MKI67
UBE2C
RRM2
KIF2C
MELK
TYMS
PTTG1
ORC6L
UBE2T
CDH3
EGFR
KRT17
KRT14
KRT5
FOXC1
MIA
SFRP1
PHGDH
MYC
Figure 1 Clinical PAM50 RT-qPCR breast cancer training set. Hierarchical clustering of RT-qPCR data for the PAM50 classifier genes
normalized to the 5 control genes using 171 FFPE procured breast samples. Statistical selection using SigClust identified the 5 significant groups
previously identified and designated as Luminal A (dark blue), Luminal B (light blue), HER2-E (pink), Basal-like (red), and Normal (green). The 16
non-neoplastic samples (grey), from reduction mammoplasty and grossly uninvolved breast tissues, all Clustered together and away from the
invasive cancers. SigClust identified 4 reduction mammoplasty samples (green) that were used to train the Normal subtype. FGFR4
ERBB2
GRB7
BLVRA
BAG1
BCL2
CXXC5
ESR1
GPR160
FOXA1
MLPH
NAT1
SLC39A6
MAPT
PGR
MDM2
TMEM45B
MMP11
ACTR3B
CDC6
CCNE1
EXO1
CDCA1
KNTC2
BIRC5
CENPF
ANLN
CDC20
CCNB1
CEP55
MYBL2
MKI67
UBE2C
RRM2
KIF2C
MELK
TYMS
PTTG1
ORC6L
UBE2T
CDH3
EGFR
KRT17
KRT14
KRT5
FOXC1
MIA
SFRP1
PHGDH
MYC
Figure 1 Clinical PAM50 RT-qPCR breast cancer training set. Hierarchical clustering of RT-qPCR data for the PAM50 classifier genes
normalized to the 5 control genes using 171 FFPE procured breast samples. Statistical selection using SigClust identified the 5 significant groups
previously identified and designated as Luminal A (dark blue), Luminal B (light blue), HER2-E (pink), Basal-like (red), and Normal (green). The 16
non-neoplastic samples (grey), from reduction mammoplasty and grossly uninvolved breast tissues, all Clustered together and away from the
invasive cancers. SigClust identified 4 reduction mammoplasty samples (green) that were used to train the Normal subtype. Subtype, immunohistochemistry, and RT-qPCR gene
scores The RT-qPCR values for ESR1, PGR, ERBB2, and prolif-
eration were evaluated across prototypic samples in the
training set. High, intermediate, and low cut points were
made based on the continuous distribution of expression
across the tumor subtypes. The cut-points for each of
the scores and how they were determined is presented
in Table 2. Figure 2 shows the expression and cut-points
for ESR1 in the training set and how these compare p
}high ERBB2 = above median expression for HER2-enriched; low ERBB2 = below
lowest quartile expression for HER2-enriched. *high Proliferation = above highest quartile expression for Luminal A; low
Proliferation = below lowest quartile expression for Luminal A. *high Proliferation = above highest quartile expression for Luminal A; low
Proliferation = below lowest quartile expression for Luminal A. Page 6 of 12 Bastien et al. BMC Medical Genomics 2012, 5:44
http://www.biomedcentral.com/1755-8794/5/44 Figure 2 ESR1 score cut-offs using training set and the GEICAM/9906 testing set. The ESR1 score is provided as a qualitative call of high,
intermediate, or low. The cut-offs were based on the continuous expression of ESR1 across prototype samples in the training set. Each circle on the
box plot represents an individual sample that is color coded according to IHC status. The cut-points between high, intermediate, and low classes were
individually derived from the training set samples (A). Data from ESR1 gene expression over the GEICAM 9906 samples (B) are plotted on the same
scale as the training set. Samples are colored according to ER IHC positivity (red) or negativity (blue) determined at a central facility. Figure 2 ESR1 score cut-offs using training set and the GEICAM/9906 testing set. The ESR1 score is provided as a qualitative call of high,
intermediate, or low. The cut-offs were based on the continuous expression of ESR1 across prototype samples in the training set. Each circle on the
box plot represents an individual sample that is color coded according to IHC status. The cut-points between high, intermediate, and low classes were
individually derived from the training set samples (A). Data from ESR1 gene expression over the GEICAM 9906 samples (B) are plotted on the same
scale as the training set. Samples are colored according to ER IHC positivity (red) or negativity (blue) determined at a central facility. Subtype, immunohistochemistry, and RT-qPCR gene
scores Although the HER2-E subtype is often thought of as
being ER-, only 36% (63/174) were ER- by IHC and 44%
(76/174) were low ESR1 score. Seventeen percent of
HER2-E samples were called triple-negative. Of the
clinically HER2+ group by IHC/CISH, approximately
two-thirds (69/113 = 61%) were HER2-E and one-third
were Luminal B (37/113 = 33%) subtype (Figure 4). Using
the qPCR cut-off for ERBB2 expression, we found that
98% (609/624) of samples that were low ERBB2 were also
HER2- by IHC/CISH, while 53% (109/190) of tumors with points based on the training set were used and it showed
high sensitivity/specificity, although a slightly higher
false positive rate for ERBB2 than would have been
selected by eye. Ninety-two
percent
(497/538)
of
Luminal
(A/B)
tumors were ER + by IHC and 99% (530/538) had an
intermediate-high ESR1 score (Tables 3 and 4). Luminal
A tumors more frequently expressed PR/PGR than
Luminal B tumors using either IHC (94% vs 74%) or
qPCR (95% vs 61%). False positive rate
True positive rate
0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
AUC: 0.90
Sensitivity: 0.84
Specificity: 0.85
B)
False positive rate
True positive rate
0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
AUC: 0.90
Sensitivity: 0.96
Specificity: 0.74
A)
False positive rate
True positive rate
0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
AUC: 0.95
Sensitivity: 0.94
Specificity: 0.85
C)
ER/ESR1
PR/PGR
HER2/ERBB2
Figure 3 Receiver Operator Characteristic (ROC) curves for ESR1, PGR, and ERBB2 for the GEICAM/9906 test set. ROC curves for the
GEICAM/9906 test set were generated using the clinical IHC status (positive vs. negative) for ER, PR, and HER2/neu as compared to the continuous
RT-qPCR data for ESR1, PGR, and ERBB2. The cut-points for sensitivity/specificity are based on the training set. False positive rate
True positive rate
0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
AUC: 0.95
Sensitivity: 0.94
Specificity: 0.85
C)
HER2/ERBB2 False positive rate
True positive rate
0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
AUC: 0.90
Sensitivity: 0.96
Specificity: 0.74
A)
ER/ESR1 False positive rate
True positive rate
0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0.4
0.6
0.8
1.0
AUC: 0.90
Sensitivity: 0.84
Specificity: 0.85
B)
PR/PGR False positive rate Figure 3 Receiver Operator Characteristic (ROC) curves for ESR1, PGR, and ERBB2 for the GEICAM/9906 test set. Subtype, immunohistochemistry, and RT-qPCR gene
scores ROC curves for the
GEICAM/9906 test set were generated using the clinical IHC status (positive vs. negative) for ER, PR, and HER2/neu as compared to the continuous
RT-qPCR data for ESR1, PGR, and ERBB2. The cut-points for sensitivity/specificity are based on the training set. Page 7 of 12 Page 7 of 12 Bastien et al. BMC Medical Genomics 2012, 5:44
http://www.biomedcentral.com/1755-8794/5/44 Table 3 Histological scoring across PAM50 subtypes
Grade
ER
PR
HER2
LumA
G1-68 (25%)
Neg-19 (7%)
Neg-16 (6%)
Neg-273 (99%)
n = 277
G2-142 (51%)
Pos-258 (93%)
Pos-261 (94%)
Pos-4 (1%)
G3-39 (14%)
GX-28 (10%)
LumB
G1-25 (10%)
Neg-22 (8%)
Neg-68 (26%)
Neg-224 (86%)
n = 261
G2-111 (43%)
Pos-239 (92%)
Pos-193 (74%)
Pos-37 (14%)
G3-111 (43%)
GX-14 (5%)
HER2-E
G1-6 (3%)
Neg-63 (36%)
Neg-93 (53%)
Neg-105 (60%)
n = 174
G2-65 (37%)
Pos-111 (64%)
Pos-81 (47%)
Pos-69 (40%)
G3-96 (55%)
GX-7 (4%)
Basal
G1-0 (0%)
Neg-63 (90%)
Neg-62 (89%)
Neg-67 (96%)
n = 70
G2-4 (6%)
Pos-7 (10%)
Pos-8 (11%)
Pos-3 (4%)
G3-61 (87%)
GX-5 (7%) Table 3 Histological scoring across PAM50 subtypes
Grade
ER
PR
HER2
LumA
G1-68 (25%)
Neg-19 (7%)
Neg-16 (6%)
Neg-273 (99%)
n = 277
G2-142 (51%)
Pos-258 (93%)
Pos-261 (94%)
Pos-4 (1%)
G3-39 (14%)
GX-28 (10%)
LumB
G1-25 (10%)
Neg-22 (8%)
Neg-68 (26%)
Neg-224 (86%)
n = 261
G2-111 (43%)
Pos-239 (92%)
Pos-193 (74%)
Pos-37 (14%)
G3-111 (43%)
GX-14 (5%)
HER2-E
G1-6 (3%)
Neg-63 (36%)
Neg-93 (53%)
Neg-105 (60%)
n = 174
G2-65 (37%)
Pos-111 (64%)
Pos-81 (47%)
Pos-69 (40%)
G3-96 (55%)
GX-7 (4%)
Basal
G1-0 (0%)
Neg-63 (90%)
Neg-62 (89%)
Neg-67 (96%)
n = 70
G2-4 (6%)
Pos-7 (10%)
Pos-8 (11%)
Pos-3 (4%)
G3-61 (87%)
GX-5 (7%) were low in all 3 genes by qPCR (Table 5). Conversely,
only 56% (57/101) and 67% (60/90) of triple negatives
defined by IHC/CISH or qPCR were Basal-like, respect-
ively. There was no difference (p > 0.05) in ESR1, PGR or
ERBB2 expression by qPCR in Basal-like tumors, regard-
less of being triple-negative or non-triple negative by
IHC/CISH (Figure 5). Additional file 7 shows a comparison of unsupervised
hierarchical clustering with supervised subtype assign-
ment and single marker scores for GEICAM/9906. In
general, the supervised classification agreed with the
sample associated dendrogram clusters. The side branches
of the dendrogram clusters are less correlated to other
samples and reflect the continuum in the biology, espe-
cially between Luminal A, Luminal B and HER2-E sub-
types. Prognostic significance of gene expression versus
standard methods for ER and HER2 status Prognostic significance of gene expression versus
standard methods for ER and HER2 status
Although there was high agreement between IHC/CISH
and RT-qPCR measurements for ER/ESR1 and HER2/
ERBB2, we wanted to assess whether the two different
methods provided equivalent prognostic information. When tested in a multivariate Cox model for overall sur-
vival, only the RT-qPCR assignments were selected in
the final Cox model in the GEICAM/9906 test set
(Table 6). When all patients with locally advanced breast
cancer were stratified, regardless of chemotherapy regimen
(FEC vs FEC-T), both classifications for assessing ER/ESR1
and HER2/ERBB2 status were significantly associated with
outcome (Figure 6). Since endocrine therapy was based on
ER status determined by IHC, those ER + samples that
were ESR1- (29/154 = 19%) would have received adjuvant
tamoxifen and conversely those patients with ER- tumors
that were ESR1+ (45/660 =7%) would not have received
therapy. When separating outcome based on agreement
and disagreement between the methods, we find that
women with ER+/ESR1+ tumors have similar outcomes
to women with ER-/ESR1+ tumors, and women with
ER-/ESR1- tumors have similar outcomes to women
with ER+/ESR1- tumors. This shows that the RT-qPCR
assignment is more prognostic and accurate than IHC
for ER. intermediate-high ERBB2 expression were HER2+. How-
ever, analyses just within the HER2-E subtype showed 71%
(66/93) of tumors with high ERBB2 gene expression were
HER2+ by IHC/CISH. Ninety percent (63/70) of Basal-like tumors were ER-
by IHC and 96% (67/70) were low ESR1 score. Further-
more, 81% (57/70) of Basal-like tumors were triple nega-
tive (ER-/PR-/HER2-) by IHC/CISH and 86% (60/70) Table 4 Single gene scores and proliferation across
PAM50 subtypes
Proliferation
ESR1^
PGR^
ERBB2^
LumA
Low-104 (38%)
Neg-0 (0%)
Neg-15 (5%)
Neg-254
(92%)
n = 277
Intermediate-116
(42%)
Pos-277
(100%)
Pos-262
(95%)
Pos-23 (8%)
High-57 (11%)
LumB
Low-4 (2%)
Neg-8 (3%)
Neg-101
(39%)
Neg-194
(74%)
n = 261
Intermediate-55
(21%)
Pos-253
(97%)
Pos-160
(61%)
Pos-67
(26%)
High-202 (77%)
HER2-
E
Low-15 (9%)
Neg-76
(44%)
Neg-112
(64%)
Neg-81
(47%)
n = 174
Intermediate-60
(34%)
Pos-98 (56%)
Pos-62
(36%)
Pos-93
(53%)
High-99 (57%)
Basal
Low-0 (0%)
Neg-67
(96%)
Neg-67
(96%)
Neg-66
(94%)
n = 70
Intermediate-3 (4%)
Pos-3 (4%)
Pos-3 (4%)
Pos-4 (6%)
High-67 (96%)
^ RT-qPCR status based on intermediate-high (positive) versus low (negative). Table 4 Single gene scores and proliferation across
PAM50 subtypes Subtype, immunohistochemistry, and RT-qPCR gene
scores The HER2-E and Basal-like subtypes cluster
away from the Luminal tumors and have similar gene
expression profiles overall; however, standard IHC/
CISH biomarkers poorly define these subtypes. Discussion Many studies have tried to identify the gene expression-
based “intrinsic” subtypes using a variety of methods for
the sake of simplicity, cost, and available technologies. Methods that can be used from formalin-fixed, paraffin-
embedded tissues are optimal since this is how samples
are procured and archived in most pathology departments. Bastien et al. BMC Medical Genomics 2012, 5:44
http://www.biomedcentral.com/1755-8794/5/44 Bastien et al. BMC Medical Genomics 2012, 5:44
http://www.biomedcentral.com/1755-8794/5/44 Page 8 of 12 Figure 4 Association between HER2 status and “intrinsic” subtype. Figure (A) shows the subtype distribution within HER2+ samples by IHC/
CISH. Figure (B) shows the ER/HER2 status for samples only within the HER2-E subtype. Figure 4 Association between HER2 status and “intrinsic” subtype. Figure (A) shows the subtype distribution within HER2+ samples by IHC/
CISH. Figure (B) shows the ER/HER2 status for samples only within the HER2-E subtype. the PAM50 RT-qPCR subtypes were prognostic [30]. In
another randomized study (NCIC.CTG MA.5) that
assessed PAM50 subtype sensitivity to anthracycline-
based chemotherapy, it was shown that the HER2-E
subtype received the most benefit, while women with
Basal-like tumors had no benefit from this aggressive
treatment [31]. This study and the MA.5 trial found that
only about two-thirds of clinically Her2+ tumors are
classified as HER2-E and about the same percent of
triple negatives are classified as Basal-like. Thus, only a
subset of the IHC defined groups overlap with PAM50
subtype classification, which may have ramifications for
clinical trial findings and predicting therapy benefit. The two preferred technologies for gene expression profil-
ing from FFPE tissues are RT-qPCR [17,18] and Nano-
string nCounter [29]. The nCounter system uses color-
coded probes that bind directly to the RNA transcript
without reverse transcription and PCR amplification. While these methods have high agreement for gene
quantification, other methodologies may lead to differ-
ent conclusions and treatment decisions. Discussion For instance,
in the NCIC.CTG MA.12 clinical trial that randomized
pre-menopausal women with primary breast cancer to
tamoxifen versus placebo it was found that a panel 6
IHC antibodies for subtyping was not prognostic but Table 5 Surrogate subtyping by 3-marker scoring
Luminal A
Luminal B
HER2-E
Basal-
like
ER+/PR+/HER2- (n = 471)
244 (52%)
170 (36%)
53 (11%)
4 (1%)
ESR1+/PGR+/ERBB2-
(n = 397)
239 (60%)
127 (32%)
31 (8%)
0 (0%)
ER+/PR-/HER2- (n = 81)
12 (15%)
47 (58%)
19 (23%)
3 (4%)
ESR1+/PGR-/ERBB2-
(n = 101)
15 (15%)
65 (64%)
18 (18%)
3 (3%)
ER-/PR-/HER2+ (n = 39)
0 (0%)
7 (18%)
30 (77%)
2 (5%)
ESR1-/PGR-/ERBB2+
(n = 51)
0 (0%)
6 (12%)
41 (80%)
4 (8%)
ER-/PR-/HER2- (n = 101)
4 (4%)
10 (10%)
30 (30%)
57
(56%)
ESR1-/PGR-/ERBB2- (n = 90)
0 (0%)
2 (2%)
28 (31%)
60
(67%)
ER+/PR+/HER2+ (n = 45)
2 (4%)
18 (40%)
25 (56%)
0 (0%)
ESR1+/PGR+/ERBB2+
(n = 80)
23 (29%)
33 (41%)
24 (30%
0 (0%)
ER+/PR-/HER2+ (n = 18)
0 (0%)
4 (22%)
14 (78%)
0 (0%)
ESR1+/PGR-/ERBB2+
(n = 53)
0 (0%)
28 (53%)
25 (47%)
0 (0%)
ER-/PR+/HER2- (n = 25)
14 (56%)
4 (16%)
3 (12%)
4 (16%)
ESR1-/PGR+/ERBB2- (n = 7)
0 (0%)
0 (0%)
4 (57%)
3 (43%)
ER-/PR+/HER2+ (n = 2)
1 (50%)
1 (50%)
0 (0%)
0 (0%)
ESR1-/PGR+/ERBB2+
(n = 3)
0 (0%)
0 (0%)
3 (100%)
0 (0%) Table 5 Surrogate subtyping by 3-marker scoring Table 5 Surrogate subtyping by 3-marker scoring Receiver Operator Characteristic (ROC) curves are
commonly used in medicine to optimize the sensitivity/
specificity of an assay depending on the purpose of the
test (i.e. screening, monitoring, prognosis, etc.) [32]. In
clinical pathology, ROC curves are often used to validate
a new methodology against an existing “gold” standard. A major limitation to this approach is that cut-offs are
then determined by comparison to an often less than
perfect reference. We used an approach for selecting
single (ESR1, PGR, ERBB2) and meta-gene (prolifera-
tion) cut-offs that was based on the distribution of
expression of these markers across the different sub-
types. This method showed to be reproducible in an
independent test set. The ROC curves showed high agreement between RT-
qPCR and the standard IHC biomarkers. Discussion In addition, the patients
included in the test set were locally advanced and
received chemotherapy that can cause chemotherapy
induced amenorrhea and a reduction in ovarian func-
tion [36], which again may benefit the luminal sub-
type most. In order to determine if there was a prognostic dif-
ference between the RT-qPCR and IHC we included
both methods in a Cox proportional hazards model
and showed that gene expression remained significant
in the multivariate analysis and replaced IHC. Fur-
thermore, the outcome plots for women with tumors
scored positive for ER by IHC but negative for ESR1
had outcomes similar to women that were ER-/ESR1-. Conversely, women with ER- tumors by IHC but
positive for ESR1 had similar outcomes to women
with ER+/ESR1+ disease. Thus, despite the fact that
patients were treated in favor of the IHC diagnosis
(i.e. ER + disease was treated with adjuvant tamoxifen)
the course of disease was in agreement with the gene
expression determination. The better prognosis seen
in the ESR1+ but ER- subtype is curious since these
patients
would
not
have
been
given
adjuvant The Normal subtype was developed from reduction
mammoplasty “normal” breast tissue and serves as a
quality control measure since these cases would be con-
sidered to have an insufficient amount of tumor tissue
to make a tumor subtype call. Interference studies
showed that the introduction of “normal” breast tissue
RNA caused a systematic shift in subtype assignment
with subtypes switching to Normal, except Luminal B
which changed to Luminal A. None of the assignment switches occurred until the
introduction of 50% “normal” breast tissue RNA. The
greatest risk of misclassification would come from Lu-
minal B subtypes masquerading as Luminal A tumors
because of “normal” tissue contamination [28]; however,
these tumors maintain a high proliferation score sug-
gesting they are still a high risk Luminal tumor. Table 6 Univariate and multivariate analyses of prognostic factors in GEICAM/9906
MVA analysis for OS
Univariate analysis
Multivariate analysis (backward/forward stepwise selection)
Signatures
HR
Lower 95%
Upper 95%
p-value
HR
Lower 95%
Upper 95%
p-value
ARM FEC-P vs. FEC
0.708
0.528
0.948
0.021
0.734
0.543
0.993
0.045
Grade 3 vs. 1-2
1.745
1.297
2.346
<0.001
1.335
0.962
1.853
0.084
Nodes >3 vs. 1-3
2.103
1.574
2.808
<0.001
1.882
1.391
2.546
0.000
Tumor size >2 cm vs. Discussion ESR1 had high
sensitivity although the cut-off for ER + status was 10%
positive staining nuclei, whereas the new recommendation
for determining ER status is 1% [33]. These borderline
cases for ER positivity may be better characterized by the
overall subtype biology. For ERBB2, there was high
specificity, which is optimal since confirmatory CISH
or FISH would only be performed when it was un-
certain if the gene was truly amplified [34]. It has
been suggested that the use of single gene RT-qPCR
measurement for ERBB2 is insufficient for determin-
ing HER2 positive samples that may benefit from
trastuzumab/HerceptinW therapy [35]. Dabbs et al. Bastien et al. BMC Medical Genomics 2012, 5:44
http://www.biomedcentral.com/1755-8794/5/44 Page 9 of 12 Page 9 of 12 A)
B)
C)
0
2
4
6
8
10
p=0.24
nonTNBC
TNBC
0
2
4
6
8
10
p=0.572
nonTNBC
TNBC
0
2
4
6
8
10
p=0.168
nonTNBC
TNBC
Figure 5 Relative transcript abundance for ESR1, PGR, and ERBB2 in the Basal-like subtype. There was no difference (p > 0.05) in (A) ESR1,
(B) PGR, or (C) ERBB2 expression by qPCR in Basal-like tumors, regardless of being called triple-negative or non-triple negative by IHC/CISH. A)
0
2
4
6
8
10
p=0.24
nonTNBC
TNBC C)
0
2
4
6
8
10
p=0.168
nonTNBC
TNBC
There was no difference (p > 0.05) in (A) ESR1,
ti
t i l
ti
b IHC/CISH B)
0
2
4
6
8
10
p=0.572
nonTNBC
TNBC A) C) B) Figure 5 Relative transcript abundance for ESR1, PGR, and ERBB2 in the Basal-like subtype. There was no difference (p > 0.05) in (A) ESR1,
(B) PGR, or (C) ERBB2 expression by qPCR in Basal-like tumors, regardless of being called triple-negative or non-triple negative by IHC/CISH. found that the negative predictive value for determin-
ing HER2/ERBB2 status was high between the Her-
cepTest
and
the
GHI
Oncotype
Dx
qPCR
assay
(99%); but the concordance for positive HER2/ERBB2
samples was only 28%. In contrast, we showed that
the
concordance
between
HER2
(IHC/CISH)
and
ERBB2
(RT-qPCR)
is
greater
than
90%
when
restricted to the HER2-E subtype. endocrine blockade therapy. However, gene expression
for ESR1 may be identifying the “true” luminal origin
of these tumors which have a better prognosis, re-
gardless of therapy [30]. Additional files Data from single and
meta-gene expression score over the GEICAM 9906 samples are plotted
on the 1–10 scale. The cut-points between high, intermediate, and low
classes were individually derived from the training set. Samples are color-
coded according to immunohistochemistry positivity (red) or negativity
(blue), except in the case of the training set proliferation score where
samples are colored by high, intermediate or low proliferation class. Luminal score samples are colored as being ER+/PR+, ER + or
PR + (positive, red), and ER-/PR- (negative, blue). Additional file 7: Hierarchical clustering for GEICAM 9906. A
comparison of unsupervised hierarchical clustering with supervised
subtype assignment and single marker scores for GEICAM 9906. Additional file 1: Clinical-pathological information associated with
training set subtypes. Clinical-pathological information associated with
the 171 samples included in the training set. Additional file 1: Clinical-pathological information associated with
training set subtypes. Clinical-pathological information associated with
the 171 samples included in the training set. Additional file 2: Clinical-pathological information and PAM50 data
associated with GEICAM/9906 test set. Clinical-pathological
information and PAM50 RT-qPCR results associated with the 814 samples
included in the GEICAM9906 test set. Additional file 2: Clinical-pathological information and PAM50 data
associated with GEICAM/9906 test set. Clinical-pathological information and PAM50 RT-qPCR results associated with the 814 samples
included in the GEICAM9906 test set. Additional file 3: Additional materials and methods. Methods for
plate manufacturing, PCR, calculation of log-expression ratios, PCR-
efficiency, limits of detection, and limits of quantification are described
(Additional files 8 and 9). Additional file 4: 10-fold cross validation of training set. Each gene
was measured in triplicate per RT-qPCR run on the Roche LC480 and 2
runs were performed for each of the 17 dilutions. The prototype samples
identified by SigClust were split into 10 groups and nine of the 10
groups were used to calculate new centroids for each of the 5 possible
subtype assignments. Additional file 5: Interference in subtype call and single/meta-gene
scores from normal contamination. Interference by normal cell
contamination of subtype call and single and meta-gene classes is shown. The changes in subtype classification occurred in a systematic fashion with
all subtypes switching to a classification of Normal/Insufficient, with the
exception of Luminal B, which switched to Luminal A. Additional file 6: Single and meta-gene cutoffs. Data from single and
meta-gene expression score over the GEICAM 9906 samples are plotted
on the 1–10 scale. Discussion ≤2cm
2.089
1.510
2.890
<0.001
1.724
1.224
2.427
0.002
Age >50 vs ≤50
1.189
0.890
1.589
0.242
1.012
0.999
1.026
0.078
ER status by IHC + vs. -
0.619
0.449
0.855
0.004
-
-
-
-
ER status by GE + vs. -
0.536
0.387
0.741
<0.001
0.630
0.438
0.906
0.013
Clinical HER2 status + vs. -
1.389
0.9532
2.024
0.0872
-
-
-
- Table 6 Univariate and multivariate analyses of prognostic factors in GEICAM/9906 Bastien et al. BMC Medical Genomics 2012, 5:44
http://www.biomedcentral.com/1755-8794/5/44 Page 10 of 12 0
2
4
6
8
10
0.0
0.2
0.4
0.6
0.8
1.0
0
2
4
6
8
10
0.0
0.2
0.4
0.6
0.8
1.0
years
years
GEICAM 9906 Overall Survival
ESR1-/ERBB2-
ESR1-/ERBB2+
ESR1+/ERBB2+
ESR1+/ERBB2-
RT-qPCR
IHC
Overall Survival Proportion
Overall Survival Proportion
Log Rank p=0.00247
Log Rank p=0.00149
0
2
4
6
8
10
0.0
0.2
0.4
0.6
0.8
1.0
years
RT-qPCR versus IHC
Overall Survival Proportion
Log Rank p=0.00173
ESR1-/ER-
ESR1-/ER+
ESR1+/ER-
ESR1+/ER+
A)
B)
C)
ER-/Her2-
ER-/Her2+
ER+/Her2+
ER+/Her2-
Figure 6 Kaplan-Meier plots of overall survival in GEICAM 9906 data set. When stratifying all patients with locally advanced breast cancer,
regardless of chemotherapy regimen (FEC vs FEC-T), both RT-qPCR (A) and IHC/CISH (B) molecular classifications for assessing ESR1/ER and ERBB2/
Her2 status were significant. However, the separation of the survival curves suggests that ER-status as assessed by qPCR has prognostic superiority
to IHC (C). Discussion 0
2
4
6
8
10
0.0
0.2
0.4
0.6
0.8
1.0
years
GEICAM 9906 Overall Survival
IHC
Overall Survival Proportion
Log Rank p=0.00247
B)
ER-/Her2-
ER-/Her2+
ER+/Her2+
ER+/Her2- 0
2
4
6
8
10
0.0
0.2
0.4
0.6
0.8
1.0
years
ESR1-/ERBB2-
ESR1-/ERBB2+
ESR1+/ERBB2+
ESR1+/ERBB2-
RT-qPCR
Overall Survival Proportion
Log Rank p=0.00149
A) l
0
2
4
6
8
10
0.0
0.2
0.4
0.6
0.8
1.0
years
RT-qPCR versus IHC
Overall Survival Proportion
Log Rank p=0.00173
ESR1-/ER-
ESR1-/ER+
ESR1+/ER-
ESR1+/ER+
C) 0
2
4
6
8
10
0.0
0.2
0.4
0.6
0.8
1.0
years
GEICAM 9906 Overall Survival
IHC
Overall Survival Proportion
Log Rank p=0.00247
0
2
4
6
8
10
0.0
0.2
0.4
0.6
0.8
1.0
years
RT-qPCR versus IHC
Overall Survival Proportion
Log Rank p=0.00173
ESR1-/ER-
ESR1-/ER+
ESR1+/ER-
ESR1+/ER+
B)
C)
ER-/Her2-
ER-/Her2+
ER+/Her2+
ER+/Her2-
l
d
h
f
ll
h l
ll
d
d b GEICAM 9906 Overall Survival GEICAM 9906 Overall Survival Figure 6 Kaplan-Meier plots of overall survival in GEICAM 9906 data set. When stratifying all patients with locally advanced breast cancer,
regardless of chemotherapy regimen (FEC vs FEC-T), both RT-qPCR (A) and IHC/CISH (B) molecular classifications for assessing ESR1/ER and ERBB2/
Her2 status were significant. However, the separation of the survival curves suggests that ER-status as assessed by qPCR has prognostic superiority
to IHC (C). Additional files The cut-points between high, intermediate, and low
classes were individually derived from the training set. Samples are color-
coded according to immunohistochemistry positivity (red) or negativity
(blue), except in the case of the training set proliferation score where
samples are colored by high, intermediate or low proliferation class. Luminal score samples are colored as being ER+/PR+, ER + or
PR + (positive, red), and ER-/PR- (negative, blue). Additional files A fifth tumor type that has often been referred to
as “Normal-like” has been suggested to be an artifact
of having too few tumor cells and a large background
of normal breast cells in the sample. Our mixing
experiments here support this hypothesis and show
that
when
increasing
amounts
of
“normal”
tissue
RNA
is
added
to
a
tumor
it
switches
into
the
Normal-like
group. It
is,
however,
suspected
that
some tumors now called Normal-like may be put into
the recently described Claudin-low classification [37]. The Claudin-low subtype is mostly triple-negative,
shares biomarkers in common with normal breast
epithelial cells and Basal-like tumors, and may be
caused by deficiency in either BRCA1or p53, or both;
however there is no clinical indication for Claudin-
low, and most are typically classified as Basal-like. There are now many more groups of tumors being
identified with transcriptome and copy number vari-
ance analyses [38,39]. The overlap between these new
groups, existing subtypes, and standard biomarkers
already in practice should allow for more personalized
treatments and better outcomes in the future. Additional file 1: Clinical-pathological information associated with
training set subtypes. Clinical-pathological information associated with
the 171 samples included in the training set. Additional file 2: Clinical-pathological information and PAM50 data
associated with GEICAM/9906 test set. Clinical-pathological
information and PAM50 RT-qPCR results associated with the 814 samples
included in the GEICAM9906 test set. Additional file 3: Additional materials and methods. Methods for
plate manufacturing, PCR, calculation of log-expression ratios, PCR-
efficiency, limits of detection, and limits of quantification are described
(Additional files 8 and 9). Additional file 4: 10-fold cross validation of training set. Each gene
was measured in triplicate per RT-qPCR run on the Roche LC480 and 2
runs were performed for each of the 17 dilutions. The prototype samples
identified by SigClust were split into 10 groups and nine of the 10
groups were used to calculate new centroids for each of the 5 possible
subtype assignments. Additional file 5: Interference in subtype call and single/meta-gene
scores from normal contamination. Interference by normal cell
contamination of subtype call and single and meta-gene classes is shown. The changes in subtype classification occurred in a systematic fashion with
all subtypes switching to a classification of Normal/Insufficient, with the
exception of Luminal B, which switched to Luminal A. Additional file 6: Single and meta-gene cutoffs. Authors’ contributions RRLB participated in design of the study, generating data, and drafting the
manuscript. ARL participated in recruiting patients, collecting samples and
clinical data, and reviewing the manuscript. MTWE participated in design of
the study, bioinformatics and statistical analysis, and drafting the manuscript. AP participated in bioinformatics and statistical analysis. BM participated in
recruiting patients, collecting samples and clinical data, and reviewing the
manuscript. LR participated in design of the study. PM participated in design
of the study. MRB participated in recruiting patients, collecting samples and
clinical data, and reviewing the manuscript. DA participated in design of the
study. BL participated in manufacturing of the PCR plates. IA performed IHQ/
CISH on GEICAM/9906 samples and participated in reviewing the manuscript. TD participated in manufacturing of the PCR plates. DW participated in
manufacturing of the PCR plates. MAS participated in recruiting patients,
collecting samples and clinical data, and reviewing the manuscript. LB
participated in manufacturing of the PCR plates. KMB participated in
statistical analysis. EA participated in recruiting patients, collecting samples
and clinical data, and reviewing the manuscript. LP participated in statistical
analysis. CAD participated in sample preparation and organization. IA
participated in recruiting patients, collecting samples and clinical data, and
reviewing the manuscript. CF participated in robotics design. IJS participated
in data generation and reviewing the manuscript. JP participated in
recruiting patients, collecting samples and clinical data, and reviewing the
manuscript. AA participated in recruiting patients, collecting samples and
clinical data, and reviewing the manuscript. EC participated in design of the
study and reviewing the manuscript. RC participated in design of the study,
managing the collection of samples and central laboratory activity, and
reviewing the manuscript. MJE participated in reviewing the manuscript. TON participated in reviewing the manuscript. CMP participated in collecting
samples and clinical data, and reviewing the manuscript. MA participated in
the conceiving and design of the study. PSB participated in conceiving and
design of the study, and drafting the manuscript. MM participated in design
of the study, recruiting patients, collecting samples and clinical data, and
reviewing the manuscript. All authors read and approved the final
manuscript. Conclusions Within plate, within plate batch and across plate batch coefficient of
variation for the 50 classifier and 5 housekeeper genes of the PAM50
were calculated using cell lines and a tumor samples. References 7. Tibshirani R, Hastie T, Narasimhan B, Chu G: Diagnosis of multiple cancer
types by shrunken centroids of gene expression. Proc Natl Acad Sci U S A
2002, 99(10):6567–6572. 8. Sorlie T, Tibshirani R, Parker J, Hastie T, Marron JS, Nobel A, Deng S, Johnsen
H, Pesich R, Geisler S, et al: Repeated observation of breast tumor
subtypes in independent gene expression data sets. Proc Natl Acad Sci U
S A 2003, 100(14):8418–8423. 9. Ellis MJ, Suman VJ, Hoog J, Lin L, Snider J, Prat A, Parker JS, Luo J,
DeSchryver K, Allred DC, et al: Randomized phase II neoadjuvant
comparison between letrozole, anastrozole, and exemestane for
postmenopausal women with estrogen receptor-rich stage 2 to 3 breast
cancer: clinical and biomarker outcomes and predictive value of the
baseline PAM50-based intrinsic subtype--ACOSOG Z103. J Clin Oncol
2011, 29(17):2342–2349. Received: 6 April 2012 Accepted: 31 August 2012
Published: 4 October 2012 Received: 6 April 2012 Accepted: 31 August 2012
Published: 4 October 2012 Acknowledgements
h
k This work was supported by the Huntsman Cancer Institute (HCI)/
Foundation, the ARUP Institute for Clinical and Experimental Pathology, and
NCI grants U01 CA114722-01 and P30 CA42014-19. A. Prat is supported by
the Translational Oncology fellowship of the Sociedad Española de
Oncologia Médica (SEOM). We thank the TRAC facility and Research
Informatics at HCI, and the UNC Tissue Procurement Facility for contributing
samples. We also appreciate the guidance of Dr. Joel S. Parker. 10. Esserman LJ, Berry DA, Cheang MC, Yau C, Perou CM, Carey L, DeMichele A,
Gray JW, Conway-Dorsey K, Lenburg ME, et al: Chemotherapy response
and recurrence-free survival in neoadjuvant breast cancer depends on
biomarker profiles: results from the I-SPY 1 TRIAL (CALGB 150007/
150012; ACRIN 6657). Breast Cancer Res Treat 2012, 132(3):1049–1062. Conclusions Compiling small biomarker panels for the purpose of
“intrinsic” subtyping is of limited value in identifying
PAM50 based subtypes. Gene expression scoring for
ESR1 and ERBB2 has good agreement with the corre-
sponding protein biomarkers (ER and HER2) and may
have more prognostic power. Additional file 7: Hierarchical clustering for GEICAM 9906. A
comparison of unsupervised hierarchical clustering with supervised
subtype assignment and single marker scores for GEICAM 9906. Page 11 of 12 Bastien et al. BMC Medical Genomics 2012, 5:44
http://www.biomedcentral.com/1755-8794/5/44 of Medical Oncology, Hospital Universitario Virgen del Rocío, Sevilla, Spain. 7Department of Medical Oncology, Hospital de Donostia, San Sebastián,
Spain. 8Department of Medical Oncology, Corporatiò Sanitaria Parc Taulí,
Sabadell, Spain. 9Department of Oncological Sciences, Huntsman Cancer
Institute, Salt Lake City, UT, USA. 10Department of Medical Oncology, Hospital
Universitario Virgen de la Victoria, Málaga, Spain. 11Department of Pathology,
University of Utah Health Sciences Center/Huntsman Cancer Institute, Salt
Lake City, UT, USA. 12Department of Pathology, Hospital General Universitario
de Alicante, Alicante, Spain. 13Department of Pathology, Hospital Virgen del
Rocio, Sevilla, Spain. 14Department of Medical Oncology, Hospital
Universitario Miguel Servet, Zaragoza, Spain. 15Spanish Breast Cancer
Research Group, GEICAM, Madrid, Spain. 16Department of Oncology,
Washington University, St. Louis, MO, USA. 17Department of Anatomical
Pathology, University of British Columbia, Vancouver, Canada. 18Department
of Medical Oncology, Hospital General Universitario Gregorio Marañón,
Universidad Complutense, Madrid, Spain. 19Huntsman Cancer Institute, 2000
Circle of Hope, Salt Lake City, UT 84112, USA. Additional file 8: PCR Efficiency, limits of detection, and limits of
quantification. Supplemental table listing the efficiency of PCR, limits of
detection, and limits of quantification for the 50 classifier and 5
housekeeper genes of the PAM50. Data are from 34 runs across 17
dilutions from a mixture of 8 breast cancer cell lines. Additional file 9: Reproducibility of PAM50 gene measurements. Within plate, within plate batch and across plate batch coefficient of
variation for the 50 classifier and 5 housekeeper genes of the PAM50
were calculated using cell lines and a tumor samples. Additional file 8: PCR Efficiency, limits of detection, and limits of
quantification. Supplemental table listing the efficiency of PCR, limits of
detection, and limits of quantification for the 50 classifier and 5
housekeeper genes of the PAM50. Data are from 34 runs across 17
dilutions from a mixture of 8 breast cancer cell lines. Additional file 9: Reproducibility of PAM50 gene measurements. Competing interests ARUP Laboratories Inc. has a financial interest in the commercial offering of
the subject matter. PSB receives research funding from the ARUP Institute for
Clinical and Experimental Pathology, although he is not an employee of
ARUP. PSB, CMP, and MJE have equity interest in Bioclassifier LLC, which has
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Soybean Oligosaccharides Supplementation Attenuates the Fecal Odor Compounds by Modulate the Cecal Microbiota of Broilers
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Soybean Oligosaccharides Supplementation
Attenuates the Fecal Odor Compounds by Modulate
the Cecal Microbiota of Broilers
Haiying Liu
Shenyang Agricultural University
https://orcid.org/0000-0002-2372-8526
Xin Li
Shenyang Agricultural University
Xin Zhu
Shenyang Agricultural University
Weiguo Dong
Shenyang Agricultural University
Guiqin Yang
(
yguiqin@syau.edu.cn
)
Shenyang Agricultural University
Research
Keywords: 16S rRNA, soybean oligosaccharides, chlortetracycline, odor compounds, cecal microbiota,
broilers
Posted Date: June 17th, 2020
DOI: https://doi.org/10.21203/rs.3.rs-35493/v1
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License.
Read Full License Soybean Oligosaccharides Supplementation
Attenuates the Fecal Odor Compounds by Modulate
the Cecal Microbiota of Broilers
Haiying Liu
Shenyang Agricultural University
https://orcid.org/0000-0002-2372-8526
Xin Li
Shenyang Agricultural University
Xin Zhu
Shenyang Agricultural University
Weiguo Dong
Shenyang Agricultural University
Guiqin Yang
(
yguiqin@syau.edu.cn
)
Shenyang Agricultural University
Research
Keywords: 16S rRNA, soybean oligosaccharides, chlortetracycline, odor compounds, cecal microbiota,
broilers
Posted Date: June 17th, 2020
DOI: https://doi.org/10.21203/rs.3.rs-35493/v1
License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Research Posted Date: June 17th, 2020 License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. R
d F ll Li License:
This work is licensed under a Creative Commons Attribution 4.0 International
License. Read Full License Page 1/21 Page 1/21 Page 1/21 Abstract Background: Abatement of odor emissions in poultry production is very important for the quality and
safety in poultry industries and benefit to the environment. Background: Abatement of odor emissions in poultry production is very important for the quality and
safety in poultry industries and benefit to the environment. Methods: This study was conducted to evaluate the effects of the dietary supplementation of different
levels soybean oligosaccharides (SBO) in comparison with chlortetracycline (CHL) on major odor-causing
compound in excreta and cecal microbiota of broiler chickens. One-day-old broiler chickens were
assigned to 6 treatments with 6 replicate pens (10 birds/pen) for the 42-day experiment, including, the
negative control (NC) fed a basal diet, the positive control (PC) fed a basal diet with CHL, and the basal
diet with SBO at 0.5, 2.0, 3.5, and 5.0 g/kg, respectively. Fresh excreta was sampled for analysis odor
compounds by high performance liquid chromatography. Cecum content was collected to analyze the
cecal microbiota by 16S rRNA sequencing. Results: The excreta indole concentration of broilers fed 2.0, 3.5 and 5.0 g/kg SBO and CHL diets were
significantly decreased (P < 0.01) compared to NC. Excreta skatole concentration (P < 0.001) and pH (P <
0.05) were decreased by SBO and CHL. Formate concentration of birds fed 3.5 and 5.0 g/kg SBO diets
were higher than that of birds fed other diets (P < 0.001). The acetate concentration (P = 0.003) were
increased in birds fed 3.5 g/kg SBO diet. Deep sequencing 16S rRNA revealed that the composition of the
cecal microbial digesta slightly or significantly changed by the supplementation of SBO or CHL. SBO
decreased the abundance of Bacteroides, Bilophila, and Escherichia, which were related to indole and
skatole concentration of excreta. While CHL had strong tendency to enrich Ruminococcus and reduce
Rikenella. Conclusion: These results indicated that supplementation of dietary SBO was beneficial in attenuating
the concentration of odor causing compounds and impact the composition cecal microbiota of broilers. Background However, the change and
relationship between the compounds of major odor compounds and the intestinal microbiota are not
elucidated by adding dietary SBO. It also remains unknown whether SBO and antibiotic modulate the
intestinal microbiota in distinct manners. We hypothesized that the mode of action of different levels of dietary SOB or antibiotic effect on odor-
causing compounds content in excreta and occurs through the composition of cecum microbiota of
broilers. Therefore, it is necessary to better understand how cecal bacterial communities react to these
feed additives and suitable level of addition. In these study, we used high-throughput sequencing of the
V4 region of the bacterial 16S rRNA gene to assess the alter of structure cecal bacterial community and
the change of major odor compounds of excreta in broiler chickens after fed either a commercial diet free
of antibiotics (NC), the same basal diet supplemented with a sub-therapeutic level of chlortetracycline
(PC), or the basal diet supplemented with 0.5 g/kg to 5.0 g/kg of SBO (0.5SBO, 2.0SBO, 3.5SBO, or
5.0SBO). The new light on their produce mechanism and may allow SBO as targeted manipulation of the
intestinal microbiota to reduce odor compounds and improve animal health and productivity in the future. Background Broiler chickens are an indispensable source of animal protein for human beings, more than 65 billion
chickens are produced annually in the word [1]. Meanwhile, broilers production also brings a huge amount
of odor substances. Abatement of odor emissions in poultry production is very important for the quality
and safety in poultry industries and benefit to the environment [2, 3]. The odor compounds, mainly
including skatole (3-methylindole), indoles and volatile organic compounds, are produced by the
microbial degradation of the nutrient substrates in the large intestine[4, 5]. These substances could cause
the decline in performance, meat quality and welfare of broilers. Billions of microbes inhabit in the large
intestine and act as mediators of these reactions of broilers. The component of diet is thought to be the
key factor in influencing the composition and metabolic activity of the intestinal microbiota, and further
effects in the production of odor compounds [6–8]. Therefore, altering the ingredient of the diet may be
possible to reduce odor emission from the source of odor [9, 10]. Page 2/21 Soybean oligosaccharides (SBO) are one group of non-digestible carbohydrates, which are extracted from
soybean seeds, primarily consist of stachyose, raffinose, and sucrose [11, 12]. While the exact mode of
action remains elusive, it is usually considered that dietary SBO could be selectively fermented by some
types of intestinal bacteria in the large intestine, resulting in decrease the contents of the major odor-
causing compounds and better growth performance of animals [3, 13]. Several studies have shown a link
between the cecal microbiota and odor-causing compounds of animals by dietary SBO. For example,
diets with SBO significantly changed the population of Bifidobacteria, Lactobacillus and E. coli, and
decreased the content fecal NH3 in broilers [14]. SBO supplementation modifies the intestinal ecosystem
in weaned piglets and has potentially beneficial effects on the gut [15]. Similar study in mice revealed that
administration of SBO improved the numbers of benefical intestinal microbes [16]. In vitro studies have
revealed that SBO can improve the gut microbiota balance in colon and modulate its metabolism in pigs
[7], changed in the intestinal microbiota and reduced the production of odor compounds in broilers [17,
18]. Antibiotics work primarily by reshaping the intestinal microbiota [19], which is known as a unique
ecosystem to play a crucial role in host health and metabolism [20–22]. DNA isolation The microbial DNA was isolated from cecal content using a QIAamp Fecal DNA Isolation Kits (Qiagen Co.,
Ltd., Beijing, China) according to the manufacturer’s instructions. The quantity and quality of extracted
DNA was checked by agarose gel electrophoresis (1.5%) and Nanodrop spectrophotometer (Nyxor
Biotech, Paris, France) analysis. Sampling procedures, determination of odor compounds
and VFAs On d 40, 41 and 42, fresh excreta were sampled from each replicate group using plastic trays below each
cages within 2 min after excretion. The dropped fresh excreta were collected from the tray and transferred
into plastic tubes. The total amount of collected excreta samples from the same replicate were pooled
stored in a freezer at − 20℃ for skatole and indole analyses. The pH of excreta was measured using the
pH meter (PB-10, Sartorius, Goettingen, Germany). Concentrations of indole, skatole, VFAs (formic,
acetate, propionate, and butyrate) and lactate in the excreta were measured using HPLC equipment
(model Agilent 1100, Agilent Technologies, Santa Clara, CA) as described Yang et al. [3, 8]. On d 42, one bird per replicate of each treatment was randomly selected, euthanized by cervical
dislocation. The contents of ceca were collected under sterile conditions by dissection and pooled two
contents by treatment. Samples were immediately frozen in liquid nitrogen and were stored at -80 ℃ for
further microbiota analysis through 16S rRNA sequencing. Experimental design, birds and diets A total of 360 day-of-hatch commercial mixed sex Archer Abor+-hybird broiler chicks were obtained from a
Commercial Hatchery (Shenyang, China) and randomly assigned to one of six dietary treatments with 6
replicates (n = 10) per treatment in a completely randomized block design. All birds were fed a non-
medicated common commercial-type broiler starter (d 0–21) and grower (d 22 to 42) corn-soybean meal
basal diets and offered water ad libitum. Detailed information about the basal diets is summarized in
Additional file 1. The six treatment groups were as follows: negative control (NC), basal diet was no
additives; positive control (PC), the same basal diet supplemented with 40 mg/kg chlortetracycline (CHL), Page 3/21 Page 3/21 Page 3/21 and SBO at 0.5 g/kg (0.5SBO), 2.0 g/kg (2.0SBO), 3.5 g/kg, (3.5SBO), and 5.0 g/kg (5.0SBO), respectively. The basal diets were commercially manufactured feed which formulated to meet NRC (1994)
requirements without antibiotics. The SBO was extracted from soybean meal, and the main ingredient
was stachyose, with purity of 80.92% [23]. The chickens were raised in cages pens in an environmentally controlled broiler house. The ambient
temperature on 1 d was 35 ± 2 ℃, and was decreased 3 ℃ per week until it reached 26 ℃, thereafter, the
temperature was kept constant until 42 d. The light was provided 24 h constant for the entire period of the
experiment. PCR amplification and sequencing The following primers were applied for the amplification of the variable V4 region of the bacterial 16S
rRNA gene: 515F 5ˊ-barcode-GTGCCAGCMGCCGCGGTAA-3ˊand 806R: 5ˊ-GGACTACHVGGGTWTCTAAT-
3ˊ, where the barcode is an 8-base sequence unique to each sample. PCRs were carried out in triplicate
50 µl reactions containing 0.5 µmol L− 1 forward and reverse primers, 5 µl tenfold reaction buffer, Page 4/21 Page 4/21 250 nmol L− 1 dNTP, 0.2 µl FastPfu Polymerase and 20 ng template DNA. Thermal cycling consisted of
3 min initial denaturation at 95 ℃, followed by 30 cycles at 95 ℃ for 30 s, 50 ℃ for 30 s, and 72 ℃ for
60 s, and a final extension at 72 ℃ for 7 min. About 400–450 bp fragment of PCR products was obtained
and purified with GenJET Gel Extraction Kit (Thermo Scientific Co., USA). 250 nmol L− 1 dNTP, 0.2 µl FastPfu Polymerase and 20 ng template DNA. Thermal cycling consisted of
3 min initial denaturation at 95 ℃, followed by 30 cycles at 95 ℃ for 30 s, 50 ℃ for 30 s, and 72 ℃ for
60 s, and a final extension at 72 ℃ for 7 min. About 400–450 bp fragment of PCR products was obtained
and purified with GenJET Gel Extraction Kit (Thermo Scientific Co., USA). 250 nmol L− 1 dNTP, 0.2 µl FastPfu Polymerase and 20 ng template DNA. Thermal cycling consisted of
3 min initial denaturation at 95 ℃, followed by 30 cycles at 95 ℃ for 30 s, 50 ℃ for 30 s, and 72 ℃ for
60 s, and a final extension at 72 ℃ for 7 min. About 400–450 bp fragment of PCR products was obtained
and purified with GenJET Gel Extraction Kit (Thermo Scientific Co., USA). Sequencing libraries were generated using NEB Next® Ultra™ DNA Library Prep Kit (New England Biolabs
Co., US) following manufacturer’s recommendations and index codes were added. The library quality was
assessed on the Qubit@2.0 Fluorometer (Thermo Scientific Co., US) and Agilen Bioanalyzer 2100 system. At last, samples were sequenced on an IIlumina MiSeq 2500 instrument using a 250 bp paired-end reads
protocol at the Mymbio Tech Company of Beijing. Paired-end sequencing with dual index reads was
performed with automated cluster generation. Bioinformatic analysis Paired-end reads from the original fragments were merged using FLASH version 1.2.7 [24]. 16S rRNA
reads were decoded based on sample specific barcodes and processed to remove low quality reads data. The quality average score of reads was above 20, joining together the sequences and removed the
sequences shorter less than 100 bp reads using Mothur software to filer data. Chimeras and error
sequences of optimized data were removed using QIME (version 1.8.0) [25] software package by
clustering data into operational taxonomic units (OTUs) for species classification under 0.97 similarity
[26, 27]. Rarefaction analysis and good’s coverage were determined to quantify the coverage and
sampling effort, and statistic the abundance information of each OUT in samples. Collector’s curves for
richness observations (ACE and Chao1) and Shannon diversity index were calculated. Beta diversity
analysis was performed using UniFrac to compare the results of principal coordinate analysis (PCoA)
using the community ecology package R-forge and generate PCoA figures. Besides, using the Number
Cruncher Statistical System software (NCSS 20017; Kaysville, UT, USA), by the unweighted pair-group
(UPGMA) method and Manhattan distance with no scaling, double hierarchical analysis was conducted
[28]. Statistical analysis Data were analyzed by one-way analysis of variance protocol using SPSS 19.0 (Statistical Package for
Social Science; SPSS Inc., Chicago). Replicates were considered the experimental units for data of odor
compounds and VFAs. Differences between the means were compared using LSD multiple range tests,
and statistical significance was set at P < 0.05 level. For microbial data, means separation was via four
nonorthogonal contrasts of (i) negative control treatment vs. others, (ii) negative control treatment vs. SBO treatments, (iii) positive control treatment vs. others, (iv) positive control treatment vs. SBO
treatments. Pearson correlation coefficients (r) were used to evaluate the relationship between odor
compounds and genera. Page 5/21 Page 5/21 Results
Effect of in-feed SBO on odor compounds and VFAs in
excreta Results
Effect of in-feed SBO on odor compounds and VFAs in
excreta Effect of in-feed SBO on odor compounds and VFAs in
excreta
Table 1
Effect of soybean oligosaccharides on indole (ng/g), skatole (ng/g) concentration, and pH in excreta of
broilers during excreta collection. Item
Dietary Treatmentsd
Statistics
NC
PC
0.5SBO
2.0SBO
3.5SBO
5.0SBO
SEMe
P-value
Indole
111.07a
60.58bc
87.85ab
40.49c
21.71c
37.92c
8.040
0.003
Skatole
34.23a
23.90 cd
28.18b
22.42d
18.68e
26.23bc
0.964
< 0.001
pH
6.53a
6.49ab
6.29abc
6.21abc
6.12c
6.30abc
0.043
0.023
a, b, cMeans in a column within treatment grouping without a common superscript letter differ (P <
0.05). dNC, negative control; PC, positive control, NC with 40 mg/kg chlortetracycline; 0.5SBO, 2.0SBO,
3.5SBO, and 5.0SOB: NC with 0.5, 2.0, 3.5, and 5.0 g/kg soybean oligosaccharides respectively. eSEM, standard error of the mean. a, b, cMeans in a column within treatment grouping without a common superscript letter differ (P <
0.05). dNC, negative control; PC, positive control, NC with 40 mg/kg chlortetracycline; 0.5SBO, 2.0SBO,
3.5SBO, and 5.0SOB: NC with 0.5, 2.0, 3.5, and 5.0 g/kg soybean oligosaccharides respectively. eSEM, standard error of the mean. The effect of dietary supplementation of different levels SBO on the concentration of skatole and indole,
and pH in excreta of broilers on final days are presented in Table 1. The treatments supplementation of
SBO decreased indole and skatole of excreta in broiler chickens compared to the NC treatment (P < 0.05). The indole (P = 0.003) concentration was significant decreased when supplementation of SBO at 2.0, 3.5
and 5.0 g/kg compared to NC, with the average 70% lower than the NC; while there is no significant
differences with PC. The skatole concentrations (P < 0.001) were significantly lower when
supplementation of SBO or CHL than NC, and it was lowest when SBO supplementation at 3.5 g/kg, with
less 45.4% and 12.8% than NC and PC, respectively. VFAs and lactate data are shown for excreta in broiler chickens fed dietary supplements on the final days
in Table 2. The birds fed 3.5 and 5.0 g/kg SBO has higher formate than that of the other groups (P <
0.001). While feeding birds with diets containing 3.5 g/kg SBO increased acetate level in excreta
compared to other treatments (P = 0.003). The propionate, butyrate and lactate level of excreta were not
affected by the dietary treatments (P > 0.05). Results
Effect of in-feed SBO on odor compounds and VFAs in
excreta Page 6/21 Table 2
Effect of soybean oligosaccharides on VFAs and lactate concentrations in excreta of broilers (mg/g)
Item
Dietary Treatmentsc
Statistics
NC
PC
0.5SBO
2.0SBO
3.5SBO
5.0SBO
SEMd
P-value
Formate
0.187b
0.176b
0.277b
0.300b
0.561a
0.542a
0.033
< 0.001
Acetate
0.237b
0.223b
0.244b
0.258b
0.293a
0.256b
0.006
0.003
Propionate
0.256
0.224
0.227
0.244
0.237
0.259
0.022
0.999
Butyrate
0.229
0.140
0.136
0.258
0.212
0.186
0.017
0.327
Lactate
4.108
4.016
4.565
4.378
4.249
4.058
0.188
0.964
a, bMeans in a column within treatment grouping without a common superscript letter differ (P < 0.05). cNC, negative control; PC, positive control, NC with 40 mg/kg chlortetracycline; 0.5SBO, 2.0SBO,
3.5SBO, and 5.0SBO: NC with 0.5, 2.0, 3.5, and 5.0 g/kg soybean oligosaccharides respectively. dSEM, standard error of the mean. Table 2
Effect of soybean oligosaccharides on VFAs and lactate concentrations in excreta of broilers (mg/g)
Item
Dietary Treatmentsc
Statistics
NC
PC
0.5SBO
2.0SBO
3.5SBO
5.0SBO
SEMd
P-value Table 2 cNC, negative control; PC, positive control, NC with 40 mg/kg chlortetracycline; 0.5SBO, 2.0SBO,
3.5SBO, and 5.0SBO: NC with 0.5, 2.0, 3.5, and 5.0 g/kg soybean oligosaccharides respectively. dSEM, standard error of the mean. Effect of in-feed SBO on the cecal microtiota diversity Broiler chicks were fed a non-antibiotics corn-soybean basal diet supplemented without or with different
levels of SBO and antibiotic for six weeks before collection of cecal content samples for each treatment. Following bacterial DNA isolation and sequencing of the V4 region of the 16S rRNA gene. A total of 4 479
533 raw sequences reads were obtained, and 3 420 000 high-quality sequences were used for further
analysis, with an average of 190 000 sequences per sample. There was detected 9 821 difference
phylotypes among all samples for OUTs at distance (species level). At OUT level, rarefaction curves was
performed and indicated that there were sufficient sequences for sampling to detect the diversity of
majority bacteria. The estimators of community richness (ACE and Chao), diversity (Simpson and
Shannon index) and good’s coverage are shown in Table 3. There was no significant differences in the
OUT, ACE, Chao1, Simpson, Shannon, and good’s coverage indices in the dietary treatments, but there was
a trend significant for OUT, ACE, and the Chao1 (P < 0.10). OUT, ACE, and the Chao1 were more for NC
compared with others (P = 0.090, 0.064, and 0.099), and ACE, and the Chao1were more for NC compared
with SBO treatments (P = 0.070 and 0.100). Page 7/21 Table 3
The bacterial diversity analysis of samples in different treatment of broilers cecum contents
Item
Dietary Treatmentsa
Statistics
NC
PC
0.5SBO
2.0SBO
3.5SBO
5.0SBO
SEMb
Contrastc
(P-value)
OTU
2266
1761
1841
1862
1989
1742
84.2
1(0.090)
ACE
2856
2287
2271
2282
2485
2169
102
1(0.064),
2(0.070)
Chao1
2749
2276
2281
2262
2456
2183
95.8
1(0.099),
2(0.100)
Simpson
0.975
0.945
0.913
0.919
0.907
0.958
0.012
Shannon
6.87
6.17
6.29
6.28
6.32
6.71
0.180
good’s
coverage
0.997
0.997
0.997
0.998
0.997
0.998
0.0001
aNC, negative control; PC, positive control, NC with 40 mg/kg chlortetracycline; 0.5SBO, 2.0SBO,
3.5SBO, and 5.0SBO: NC with 0.5, 2.0, 3.5, and 5.0 g/kg soybean oligosaccharides respectively. bSEM, standard error of the mean. c1 = NC vs. others; 2 = NC vs. SBO treatments (0.05 < P < 0.1). Table 3 aNC, negative control; PC, positive control, NC with 40 mg/kg chlortetracycline; 0.5SBO, 2.0SBO,
3.5SBO, and 5.0SBO: NC with 0.5, 2.0, 3.5, and 5.0 g/kg soybean oligosaccharides respectively. bSEM
t
d
d
f th c1 = NC vs. others; 2 = NC vs. SBO treatments (0.05 < P < 0.1). Effect of in-feed SBO on the cecal microtiota diversity Using the Bray-Curtis and Jaccard indices determined β-diversity to reveal the differences in cacal
microbiota composition among individual treatments. While there was no obvious segregation of the
microbiota that birds fed different diets based on the Bray-Curtis index (Fig. 1A), NC and PC groups were
relatively separated from all other groups using the Jaccard index (Fig. 1B). Effect of in-feed SBO on the bacterial genus of cecal
microbiota At the genus level, the sequences from samples corresponded to 177 genera. The double hierachal cluster
analysis on the top 50 most abundant taxa (Fig. 3) indicated that the proportion of unknown flora was
the highest (56.73–75.98%) in groups, and other dominant genus included those of the taxa Bacteroides
(1.47–12.33%), Ruminococcus (2.74–4.61%), Phascolacrctobacterium (0.18–5.99%), Rikenella (0.65–
6.96%), Oscillospira (1.96–3.89%), Faecalibacterium (1.36–5.05%), AF12 (0.52–2.40%), Helicobacter
(0.34–2.49%), while the relative abundance of other taxa was below 2%. The unknown bacteria was less for the mean of NC diet compared with others (P = 0.047) (Fig. 4;
Additional file 3). Compared with NC, supplementation of PC and SBO (P = 0.006) or supplementation of
SBO (P = 0.009) showed lower percentage of Bcteroides (Additional file 3), and lowest was observed at
3.5 mg/kg SBO (Fig. 4). The abundance of Ruminococcus was greater for PC than for the birds fed SBO
and NC or SBO (P = 0.028 and P = 0.024, respectively), the mean of fed PC was significant greater than
the birds fed SBO at 0.5, 3.5, and 5.0 mg/kg. The percentage of Rikenella was less for PC than for other
treatments (P = 0.048). The Escherichia was greater for NC vs. others (P = 0.026) or SBO treatments (P =
0.030). Bilophila was greater for NC vs. others (P = 0.063) or SBO treatments (P = 0.071), which were close
to significant level. There were higher abundance of Faecalibacterium for supplementation of SBO levels
of 2.0, 3.5 and 5.0 mg/kg, and higher Lactobacillus for 0.5, 2.0 mg/kg SBO levels compared to fed NC or
PC, although no significant difference among the experimental treatments. Effect of in-feed SBO on the bacterial phylum of cecal
i
bi t Page 8/21
On the classification basis of sequences from the cecal samples, 7 different phyla were identified at the
phylum level, which distributed across all the treatment groups (Fig. 2). The others, the remaining phyla
with sequence frequencies of < 1%, were considered low abundance. The pyrosequencing results showed
that Bacterodetes (43.16–54.74%) and Firmicutes (39.62–51.70%) were the dominant phyla for cecal
content, accounting for more than 90% (90.57–94.86%) of the total bacterial abundance in broiler
chickens fed dietary supplements on d 42 (Additional file 2). The percentage of Proteobacteria showed
higher for NC than for PC and supplementation SBO diets (P = 0.049) (Additional file 2). The abundance
of Tenericutes was slightly less for the mean of NC or PC compared with SBO diets (P = 0.101 and P =
0.100, respectively). Feeding birds with diet PC decreased Actinobacteria compared to containing SBO
diets and NC (P = 0.023) or only SBO diets (P = 0.026). There were no significant differences between the Page 8/21 experimental treatments in the abundance of Bacteroidetes (P = 0.916), Firmicutes (P = 0.908), Unknown
(P = 0.629), Cyanobacteria (P = 0.208) or others (P = 0.676). The correlation coefficient between the skatole levels of
excreta and bacteria at genus in cecum of broilers As shown by Table 4, the relationships among odor compounds and cecal digesta microbiota were
observed in SBO and control diets of broilers. Indole was highly correlated with Bilophila (P < 0.1), while
skatole was highly related to Bacteroides (P < 0.1), Bilophila (P < 0.05), and Escherichia (P < 0.1). There
were high r (positive) for acetate versus Faecalibacterium (P < 0.05). Propionate was significantly
correlated with Unknown (negative), Bacteroides, and Rikenella (P < 0.05), and lactate was highly
correlated with Lactobacillus (P < 0.05). Page 9/21 Table 4
The correlation coefficient between the odor compound levels of excreta and bacteria at genus in cecum
of broilers
Itema
Unk
Bac
Rum
Rik
Fae
Bil
Lac
Esc
Indole
-0.059
0.493
0.007
-0.104
-0.641
0.751*
-0.017
0.628
Skatole
-0.376
0.744*
-0.049
0.295
-0.536
0.861**
-0.270
0.800*
Formate
-0.384
-0.192
-0.530
0.564
0.699
-0.382
-0.220
-0.281
Acetate
-0.257
-0.234
-0.595
0.224
0.908**
-0.336
0.097
-0.288
Propionate
-0.908**
0.858**
-0.182
0.880**
0.397
0.590
-0.550
0.709
Butyrate
-0.498
0.541
-0.062
0.253
0.743
0.301
0.004
0.382
Lactate
0.507
-0.319
-0.646
-0.358
0.020
-0.303
0.902**
-0.433
pH
-0.131
0.470
0.582
-0.035
-0.707
0.654
-0.453
0.628
aUnk = Unknown; Bac = Bacteroides; Rum = Ruminococcus; Rik = Rikenella; Fae = Faecalibacterium; Bil
= Bilophila; Lac = Lactobacillus; Esc = Escherichia. *P < 0.1; **P < 0.05. Discussion However, this technique lack the
depth and precision to reveal specific changes in bacterial composition. Subsequently, the method next-
generation sequencing of the bacterial 16S rRNA gene could offer more information on specific changes
in certain bacterial populations. However, there is scarce reports about using high-throughput sequencing
study the changes on microbial communities of cecum by supplementation SBO in broilers. The
approach of high-throughput sequencing provided a much accurate and in-depth estimation of cecal
microbial diversity in broilers. Therefore, the results of present study provided the information of the
ecology of cecal bacterial communities. In line with the previously reports on cecal microbiota of broilers
[1, 30], it was dominated by Bacteroidetes and Firmicutes at the phylum level, regardless of diet. The
metabolism favours the fermentation of cellulose and stach etc in the cecum, results in higher microbial
diversity and dominance of the saccarolytic and anaerobic order of Bacteroides and Clostridiales [1]. The
most abundant genera detected in this study were unclassified, Bacteroides, Ruminococcus,
Phascolacrctobacterium, Rikenella, Oscillospira, Faecalibacterium, and the unclassifed bacteria was the
most. The results were quite similar with those obtained by Wen et al. and Zhu [1, 30]. Bjerrum et al. [30]
reported that bacterial strains closely related to Faecalibacterium prausnitzii were dominant in the ceca. Phascolacrctobacterium, Rikenella, Oscillospira, Faecalibacterium, and the unclassifed bacteria was the
most. The results were quite similar with those obtained by Wen et al. and Zhu [1, 30]. Bjerrum et al. [30]
reported that bacterial strains closely related to Faecalibacterium prausnitzii were dominant in the ceca. Phascolacrctobacterium, Rikenella, Oscillospira, Faecalibacterium, and the unclassifed bacteria was the
most. The results were quite similar with those obtained by Wen et al. and Zhu [1, 30]. Bjerrum et al. [30]
reported that bacterial strains closely related to Faecalibacterium prausnitzii were dominant in the ceca. Based on the phylogenetic diversity of bacterial communities and number of OTUs, SBO and CHL did not
change the chicken cecal bacterial community membership, but the treatments significantly altered
relative abundance of certain taxa in the cecum. This observation is in accordance with the study of Zhu
et al. [30] who reported that SBO addition (0.6%) altered the chicken cecal microbiota. When added
frucotooligosaccharide in broiler diets, the total number of anaerobes and Lactobacilli in the ceca
increased while the number of E. Coli decreased [32]. Similar results were observed by Baurhoo et al by
mannanoligosaccharide [33]. Discussion Odor emission from chicken excreta is produced mainly by the microbial degradation the substrates in
the cecum of broilers, which is a serious environmental problem in broilers industry. Strategies to mitigate
emissions are needed. Among the strategies to reduce odor and gas emissions in the broiler industry,
dietary supplementation of feed additives including SBO are becoming an accepted strategy because of
their beneficial effects against intestinal pathogenic microbes and reduces the production of odor
compounds. The aim of this study was to evaluate the effects of different levels of dietary SBO and CHL
on odor-causing compounds in excreta and the composition of cecum microbiota of broilers. At the end of the experiment, birds fed SBO has indole reduced by 21–80% and impairment of skatole by
33–64% compared with negative treatment. The value of skatole was decreased 22% between 3.5SBO
treatment and antibiotic treatment. Also, 3.5 g/kg SBO supplementation decreased the pH, increased
acetate in excreta compared with both control treatments, while 3.5 g/kg and 5.0 g/kg SBO had higher
formate than that both control treatments. The produced short chain fat acids from fermented
indigestible residues by the abundant microorganisms present in the ceca. Oligosaccharides are able to
enter into the ceca in generous amounts and being fermented there by microflora [24]. Our former study
has shown similar results [3], when supplementing 1.25 g/kg SBO in broiler decreased indole, skatole and
the pH in broiler excreta. Additionally, it has been observed that SBO could decreased the concentration of
indole and skatole, and pH value by intestinal microbiota of broilers in vitro [18]. Page 10/21 Page 10/21 Overall, in this study, the results demonstrated that the dietary supplementation with SBO might be a
useful strategy to attenuate the production of odor in broilers. SBO supplementation decreased the
concentration of indole, skatole and pH, and enhance the concentration of part of organic acid of excreta
in broilers. The results of correlation analysis further indicated that the odor compounds were associated
with intestinal microbial community. Broilers fed SBO and CHL had some alterations in the alpha and
beta diversity, though non-significant, which indices of the cecal microbiota and these modifications
promoted relevant changes of the microbial community structure. In the early animal trials and in vitro studies, we used the molecular technique of denaturing gradient gel
electrophoresis revealed a shift in the microbiota composition [3, 8, 18]. Discussion In present study, Bacteroides, Bilophila and Escherichia were significantly
decreased in the cecum by SBO (Fig. 4). While Ruminococcus (Fig. 4), Lachnospiraceae and
Coriobacteriaceae (Additional file 4) were enriched, Rikenella (Fig. 4) appeared to be diminished by
chlortetracycline. These results are consistent with earlier observations that in-feed antibiotic
preferentially enriched butyrate-producing bacteria [19], SBO inhibited the growth of pathogenic microbes,
and reduced the production of odor compounds [3, 14, 30]. Previous studies have found that
supplementation of broiler diets with a mixture of chlortetracycline and other antibiotic increased both
Ruminococcaceae and Lachnospiraceae [34]. It is known that both Lachnospiraceae and
Ruminococcaceae produce butyrate [19, 35, 36]. Page 11/21 Page 11/21 The most dramatic effect of SBO supplementation is differential reduction of the members of four most
dominant bacterial families (Lachnospiraceae, Bacteroidaceae, Desulfovibrionaceae,and
Enterobacteriaceae) in the chicken cecum (Additional file 4). SBO increased the population of a group of
lactic acid bacteria in vitro, genera Lactobacillus, Pediococcus, Weissella, and Leuconostoc in the cecal
contents of young broiler chickens [17]. In broilers, supplementation of 1.25 g/kg SBO showed a higher
cecal bacterial diversity, along with lower excreta indole and skatole production, it also increased the
excreta total VFA concentration and decreased the pH value, when compared with that of the broilers fed
the control diet [3]. Dietary supplementation with SBO increased the diversity of intestinal microflora and
elevated the numbers of some presumably beneficial intestinal bacteria, (eg, Bifidobacterium sp,
Faecalibacterium prausnitzii, Fusobacterium prausnitzii, and Roseburia), also increased the concentration
of short-chain fatty acid in the intestinal lumen, and reduced the numbers of bacteria with pathogenic
potential (eg, Escherichia coli, Clostridium, and Streptococcus) and the concentration of several protein-
derived catabolites (eg, isobutyrate, isovalerate, and ammonia) in piglets[15]. In contrast, Ma et al. [16]
described that intragastric administration of 4.0 g/kg body weight SBO significantly enhanced the
proliferation of Bifidobacteria and lactic bacteria, and increased numbers of Enterococci and decreased
numbers of Clostridium perfringens in the fecal contents of mouse. Two lactic acid bacterial genera including Lactobacillus and Enterococcus were differentially regulated by
SBO among the abundant OTUs in the cecum, however, no statistical differences were observed on the
relative abundance of these bacterial genera between treatments, which may be related with the adding
levels of SBO (Additional file 3). Lactic acid bacteria provide a myriad of beneficial effects to the host,
and are widely used as probiotics in animal production [37, 38]. Discussion It is interesting to note that Lactobacillus
was upregulated by 0.5, 2.0, and 3.5 g/kg SBO, while the Enterococcus was obviously reduced in response
to 0.5, 2.0, and 3.5 g/kg SBO. An upregulation in the Lactobacillus abundance is consistent with earlier
observations that SBO administration was associated with population of the Lactobacillus species [17,
39]. The lactate concentration in excreta was significantly positive correlation with Lactobacillus in the
cecum (Table 4). Both Lactobacillus spp. and acetate production in the cecum of chickens by xylo-
oligosaccharides treated diets have been shown to be increased, may promote intestinal health [40].The
abundance of Enterococcus was reduced by lower levels of SBO (Additional file 3). Enterobacter
aerogenes has been showed to be responsible for the degradation of L-tryptophan to skatole in cecum of
broilers [18]. A lone Proteobacteria (Escherichia) was also reduced by all levels of SBO and CHL, and
significantly positive correlation with the levels of skatole in excreta, which is in agreement of earlier
reports of a downregulation of Escherichia in response to in-feed SBO[16, 18]. Escherichia is a typical
harmful bacterium in the intestinal tract that is involved in the degradation of tryptophan to indole [41]. These results collectively suggest that SBO has a strong tendency improve the intestinal microbial
balance [3] by favouring a quick proliferation of beneficial strains and inhibiting the growth of pathogenic
microbes [7] and then contributes to reduce the production of odor compounds [18, 42]. Our results show that a higher positive correlation for acetate versus Faecalibacterium, which is a
bacterial stain known to ferment mono-oligosaccharides and oligosaccharides into butyrate and lactate
[31]. The intestinal metabolite skatole is generated by decarboxylation of indoleacetic acid by Bacteroides Page 12/21 Page 12/21 spp. and Clostridium spp. [43]. Indole formation is converted from tryptophan via the action of the
enzyme tryptophanase, which is expressed in many Gram-positive bacterial species including Escherichia
coli, Clostridium spp. and Bacteroides spp. [44–46]. Our results show that a positive correlation for indole
versus Bilophila, skatole versus Bacteroides, Bilophila and Escherichia, which is in agreement of earlier
report of Uncultured Lachnospiraceae bacterium and Bacteroides sp. are associated with the production
of major odor-causing compounds in the excreta of broilers [3]. Indole and skatole are mainly produced by the microbial degradation of several substrates of cecum of
broilers. Conclusions In summary, our data indicates that dietary SBO supplementation modulated the relative abundance of
some specific bacteria without changing the whole microbial structure. Decreased the relative abundance
of Bacteroides, Rikenella, and Escherichia bacteria in cecum and elevated fecal acetate concentration in
chickens fed SBO might be an intestinal skatole mitigating attribute and may contribute to ameliorate the
producing of odor-causing compound during chicken production. Discussion The present study demonstrated that supplementation of broilers dietary SBO significantly
reduced the production of odor compounds in excreta, and this effect was associated with cecal
microbiota composition, in comparison to its un-supplemented control counterpart, and these partly
similar to those of CHL individuals. Indole and skatole are mainly produced by the microbial degradation of several substrates of cecum of
broilers. The present study demonstrated that supplementation of broilers dietary SBO significantly
reduced the production of odor compounds in excreta, and this effect was associated with cecal
microbiota composition, in comparison to its un-supplemented control counterpart, and these partly
similar to those of CHL individuals. Funding The National Natural Science Foundation of China (No. 31772618 and 31372328) supported this study. The National Natural Science Foundation of China (No. 31772618 and 31372328) supported this study. Acknowledgment The authors are grateful to the staff of the Department of Animal Science and Technology of the
Shenyang Agricultural University for their valuable assistance in conducting the experiments. Abbreviations SBO: Soybean oligosaccharides; CHL: Chlortetracycline; NC: Negative control; PC: Positive control; NRC:
National research council; VFA: Volatile fatty acid; HPLC: High performance liquid chromatography; OTUs:
Operational taxonomic units; PCoA: Principal coordinate analysis; SEM: Standard error of the mean Availability of data and materials Page 13/21 All data generated or analyzed during this study are available from the corresponding author by request. The datasets supporting the conclusions of this article are included in the article. All data generated or analyzed during this study are available from the corresponding author by request. The datasets supporting the conclusions of this article are included in the article. Competing interests All authors approved the submission of this manuscript and declare no conflicts of interest. The
manuscript has not been previously published and is not under consideration for publication elsewhere. The agencies that funded this research had no role in the study design, analysis, or writing of this article. Consent for publication Not applicable. Ethics approval and consent to participate All animal trials were conducted in accordance with the Institutional Animal Care and Use Committee of
Shenyang Agricultural University. Author details College of Animal Science and Veterinary Medicine, Shenyang Agricultural University, Shenyang, China. Conflicts of Interest The authors declare no conflict of interest. Authors’ Contributions Guiqin Yang conceived and designed the experiments; Haiying Liu, Xin Li and Weiguo Dong performed the
experiments. Haiying Liu, Xin Li, Xin Zhu analyzed and interpreted the data. Haiying Liu and Li Xin, and
Guiqin Yang drafted and revised the manuscript. All authors contributed to data interpretation and
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20:709–715. Figure 1 The β-diversity of cecal microbiota of 42-day-old broilers. Principal components (PCs) 1 and 2 accounted
for 10.9 and 9.55% (A), for 45.26 and 16.64% of the variance (B), respectively. NC, negative control; PC,
positive control, NC with 40 mg/kg chlortetracycline; 0.5SBO, 2.0SBO, 3.5SBO, and 5.0SBO: NC with 0.5,
2.0, 3.5, and 5.0 g/kg soybean oligosaccharides respectively. Figure 2
The microbiome compositions in cecum at phylum level for 16S rRNA sequences in cecal digesta of Figures Page 17/21 Figure 1
The β-diversity of cecal microbiota of 42-day-old broilers. Principal components (PCs) 1 and 2 accounted
for 10.9 and 9.55% (A), for 45.26 and 16.64% of the variance (B), respectively. NC, negative control; PC,
positive control, NC with 40 mg/kg chlortetracycline; 0.5SBO, 2.0SBO, 3.5SBO, and 5.0SBO: NC with 0.5,
2.0, 3.5, and 5.0 g/kg soybean oligosaccharides respectively. Figure 4 Relative abundance of the predominant genera in different level SBO groups. Values are presented as the
mean ± SEM (n = 3 per group), with the treatments not sharing a common letter considered significantly
different (p < 0.05). NC, negative control; PC, positive control, NC with 40 mg/kg chlortetracycline; 0.5SBO,
2.0SBO, 3.5SBO, and 5.0SBO: NC with 0.5, 2.0, 3.5, and 5.0 g/kg soybean oligosaccharides respectively. Figure 2 The microbiome compositions in cecum at phylum level for 16S rRNA sequences in cecal digesta of
broilers fed with different levels soybean oligosaccharides and control diets. NC, negative control; PC,
positive control, NC with 40 mg/kg chlortetracycline; 0.5SBO, 2.0SBO, 3.5SBO, and 5.0SBO: NC with 0.5,
2.0, 3.5, and 5.0 g/kg soybean oligosaccharides respectively. Page 18/21 Page 18/21 Figure 3
Heat map of relative abundance of the predominant genera in different level SBO groups. Both rows and
columns were clustered using the Euclidean distance and average linkage. NC, negative control; PC,
positive control, NC with 40 mg/kg chlortetracycline; 0.5SBO, 2.0SBO, 3.5SBO, and 5.0SBO: NC with 0.5,
2.0, 3.5, and 5.0 g/kg soybean oligosaccharides respectively. Figure 3 Heat map of relative abundance of the predominant genera in different level SBO groups. Both rows and
columns were clustered using the Euclidean distance and average linkage. NC, negative control; PC,
positive control, NC with 40 mg/kg chlortetracycline; 0.5SBO, 2.0SBO, 3.5SBO, and 5.0SBO: NC with 0.5,
2.0, 3.5, and 5.0 g/kg soybean oligosaccharides respectively. Heat map of relative abundance of the predominant genera in different level SBO groups. Both rows and
columns were clustered using the Euclidean distance and average linkage. NC, negative control; PC,
positive control, NC with 40 mg/kg chlortetracycline; 0.5SBO, 2.0SBO, 3.5SBO, and 5.0SBO: NC with 0.5,
2.0, 3.5, and 5.0 g/kg soybean oligosaccharides respectively. Page 19/21 Page 19/21 Page 19/21 Figure 4
Relative abundance of the predominant genera in different level SBO groups. Values Figure 4
Relative abundance of the predominant genera in different level SBO groups. Values are presented as the
mean ± SEM (n = 3 per group), with the treatments not sharing a common letter considered significantly
different (p < 0.05). NC, negative control; PC, positive control, NC with 40 mg/kg chlortetracycline; 0.5SBO,
2.0SBO, 3.5SBO, and 5.0SBO: NC with 0.5, 2.0, 3.5, and 5.0 g/kg soybean oligosaccharides respectively. supplement6.docx Supplementary Files Page 20/21 This is a list of supplementary files associated with this preprint. Click to download. supplement6.docx Page 21/21
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Cardiovascular CT in Cyanotic Congenital Heart Disease
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Current Cardiovascular Imaging Reports (2019) 12: 30
https://doi.org/10.1007/s12410-019-9507-3
CARDIAC COMPUTED TOMOGRAPHY (B CHOW AND G SMALL, SECTION EDITORS)
Cardiovascular CT in Cyanotic Congenital Heart Disease
Anjali Chelliah 1,2 & Amee M. Shah 3 & Kanwal M. Farooqi 3 & Andrew J. Einstein 4 & B. Kelly Han 5
Published online: 7 June 2019
# The Author(s) 2019
Abstract
Purpose of Review To discuss the role of cardiovascular computed tomography (CT) in diagnosing and managing a spectrum of
cyanotic congenital heart defects before and after repair and to review practical imaging considerations in patients with specific
cyanotic lesions.
Recent Findings As CT technology has rapidly advanced and radiation doses have dramatically decreased, cardiovascular CT
has provided a lower-risk, high-quality alternative to diagnostic cardiac catheterization or cardiac magnetic resonance imaging
(MRI) and has been increasingly utilized in nearly every type of cyanotic congenital heart defect.
Summary Cardiovascular CT can provide rapid, high-resolution images to aid in preoperative planning and postoperative
surveillance of cyanotic heart disease. To optimize each study, protocols should be carefully tailored to specific defects and to
each patient’s unique clinical and hemodynamic considerations.
Keywords Cardiac computed tomography . Pediatrics . Congenital heart disease . Cyanotic congenital heart disease . Single
ventricle disease
Introduction
Congenital heart disease (CHD)—structural abnormalities of
the heart and cardiac vessels that occur in fetal development—
is the most common type of birth defect, affecting 8 to 9 of
every 1000 children [1]. Approximately 25% of these children
have cardiac lesions characterized as cyanotic [2, 3]. In cyanotic forms of CHD, a net right-to-left cardiac shunt allows
desaturated systemic venous blood to bypass the lungs and
enter the systemic arterial circulation. This shunt leads to
decreased arterial saturations and an increased deoxygenated
hemoglobin level that manifests as central cyanosis when it
exceeds 5 g/dl. A broad spectrum of complex defects, including tricuspid or pulmonary atresia and total anomalous pulmonary venous return (TAPVR), can cause right-to-left
shunts. In defects such as tetralogy of Fallot (TOF) or pulmonary atresia with intact ventricular septum (PA/IVS), right
ventricular outflow tract obstruction leads to decreased pulmonary blood flow and intracardiac shunting. Mixing of pulmonary and systemic circulations, as seen in truncus
This article is part of the Topical Collection on Cardiac Computed
Tomography
* Anjali Chelliah
ac2967@cumc.columbia.edu
Amee M. Shah
ams79@cumc.columbia.edu
1
Division of Pediatric Cardiology, Goryeb Children’s Hospital,
Morristown Medical Center, Morristown, NJ, USA
2
Division of Pediatric Cardiology, Columbia University Irving
Medical Center, New York, NY, USA
3
Division of Pediatric Cardiology, Columbia University Irving
Medical Center and Morgan-Stanley Children’s Hospital of New
York-Presbyterian, New York, NY, USA
4
Division of Cardiology, Department of Medicine, and Department of
Radiology, Columbia University Irving Medical Center and New
York-Presbyterian Hospital, New York, NY, USA
5
The Children’s Heart Clinic at The Children’s Hospitals and Clinics
of Minnesota and The Minneapolis Heart Institute and Foundation,
Minneapolis, MN, USA
Kanwal M. Farooqi
kf2549@cumc.columbia.edu
Andrew J. Einstein
ae2214@cumc.columbia.edu
B. Kelly Han
khan@chc-pa.org
30 Page 2 of 14
arteriosus and d-transposition of the great arteries (d-TGA),
can also cause a net right-to-left shunt. Defects with left-sided
cardiac obstruction, like hypoplastic left heart syndrome, typically cause low cardiac output and shock but can sometimes
present with desaturation from right-to-left shunting across a
patent ductus arteriosus to provide systemic blood flow [4].
Cyanosis can also present in older unrepaired patients with
longstanding systemic to pulmonary shunting, such as an
unrestrictive ventricular septal defect, in whom pulmonary
arterial hypertension has developed and reversed the shunt
to right-to-left, known as Eisenmenger syndrome.
While a growing percentage of CHD patients are diagnosed
prenatally by obstetric ultrasound or echocardiogram, in the
USA, about two-thirds are discovered postnatally [5].
Postnatally diagnosed cyanotic CHD often presents acutely
in the newborn period or may be identified by routine pulse
oximetry screening in the newborn nursery [6–8]. It typically
requires surgical repair, palliation, or catheter-based intervention in the neonatal period or early infancy to alleviate
desaturation. Therefore, even when a defect is prenatally diagnosed, the precise anatomic details that affect management
must be determined quickly after birth. Congenital cardiologists often have a limited time window in the first hours to
days of life to image the heart and decide upon the timing of
intervention, procedure type, and surgical approach. These
decisions depend in part on the infinitely many anatomic variations found in complex CHD, affecting visceroatrial situs,
ventriculoarterial connections, great vessel anatomy and size,
coronary artery arrangement, the presence, location, and size
of septal defects, and systemic and pulmonary venous
anatomy.
As management techniques are refined and CHD survival
improves, the patient population has grown rapidly to approximately 2.4 million in the USA, including 300,000 with complex defects that include repaired or palliated cyanotic CHD
[9]. The growing need for lifelong surveillance after repair has
increased the demand for congenital cardiac imaging, which
serves as a cornerstone of complex CHD management to assess residual lesions, monitor function, identify complications,
and plan subsequent interventions.
Transthoracic echocardiography is the primary tool used to
assess cardiac anatomy and function. It is readily available,
relatively inexpensive, and poses little risk, even to unstable
patients. However, despite typically good acoustic windows in
infants and children, it can be difficult to determine details of
small, often sub-centimeter structures and extracardiac vessels
and reproducibly assess ventricular size and function.
Echocardiographic windows are more limited in adult CHD
patients, especially those with extensive sternal scarring after
multiple interventions. Diagnostic cardiac catheterizationbased angiography, historically a mainstay of congenital cardiac imaging, provides superior spatial and temporal resolution [10]. However, invasive angiography is increasingly used
Curr Cardiovasc Imaging Rep (2019) 12: 30
primarily for hemodynamic evaluation rather than anatomic
definition due to its risks. Catheterization is invasive, requires
central vascular access, requires sedation or anesthesia in
young patients, and exposes patients to radiation. Thus, the
use of invasive catheterization to define complex congenital
heart defect anatomy has declined with advances in noninvasive imaging [11–13].
In the last few decades, cardiovascular MRI has emerged as
a high-quality, “one-stop shop” modality for imaging CHD. It
permits three-dimensional (3D) visualization of cardiac structures and precise measurement of ventricular size and function
without radiation. It is considered the gold standard for
assessing the right ventricle (RV) in repaired tetralogy of
Fallot [14]. In patients with cyanotic CHD, MRI flow sequences can quantify valve regurgitation and assess the ratio
of pulmonary to systemic blood flow, differential pulmonary
blood flow, and collateral flow [15]. Myocardial parametric
mapping by MRI can detect micro-level fibrosis and edema
[16]. Gadolinium contrast-enhanced images can also create
magnetic resonance (MR) angiograms, identify focal myocardial fibrosis, and assess perfusion during rest and stress [17,
18]. However, these studies are very time-consuming. Even
with advances in real-time and free-breathing imaging, congenital cardiac MRIs can last up to an hour or more and require patients to remain motionless and comply with repeated
breath holds [15, 19]. Children and some older patients with
developmental delays therefore often require general anesthesia, raising concerns about potentially deleterious effects on
developing brains in children whose cyanotic lesions are already associated with neurocognitive delays [20–22].
Anesthesia induction also carries a higher risk of cardiac arrest
and mortality from arrest in children with CHD, particularly in
cyanotic single ventricle patients who are unrepaired or partially palliated [23, 24]. The use of MRI is also limited in
patients with implanted devices that may be ferromagnetic
or cause significant imaging artifact; this applies to many
repaired cyanotic CHD patients with coils, stents, pacemakers,
and defibrillators. Cardiac MRI poses additional technical
challenges in neonates and infants, whose fast heart rates
and small structures limit spatial resolution, signal-to-noise
ratio, and the accuracy of volume and flow measurements
[15]. Finally, recent data emerging on the risks of gadolinium
deposition in the central nervous system and other organs have
prompted an FDA advisory on gadolinium contrast, making
MRI a less benign imaging option [25, 26].
Benefits of Cardiovascular CT in Cyanotic CHD
As the risk-benefit ratio of cardiac MRI has shifted, cardiac
CT has played a rapidly expanding role in managing complex
CHD and planning surgical and catheter-based interventions
[12, 27]. Technical advances have driven much of this
Curr Cardiovasc Imaging Rep (2019) 12: 30
increase [28]. Older scanners were limited by long scan times
and low temporal resolution, but arguably, the most significant
barrier to their use was high radiation doses.
Radiation exposure is of concern in all children but is particularly a matter of concern for those with cyanotic CHD
[29]. They may undergo numerous chest x-rays, catheterizations, nuclear stress tests, and lung perfusion scans. A recent
study from a large pediatric cardiac center documented cumulative lifetime radiation effective doses of up 77 mSv among
their CHD patients [30]. Another group reported that single
ventricle patients received on average 10 mSv per year during
the first 3–4 years of life [31]. Older CT scanners, using a
retrospectively electrocardiogram (ECG)-gated scan mode,
have been reported to expose CHD patients to effective doses
of up to 28 mSv per cardiac scan [32]. Since then, innovations
such as prospective ECG gating, ECG-controlled tube current
modulation, high-pitch helical scanning, lower tube potentials, wider detector coverage, and iterative reconstruction
techniques have dramatically lowered radiation exposure
[33, 34]. Today, sub-milliSievert scans are achievable in many
children [35]. However, effective doses still vary widely, and
recently published pediatric cardiac-specific dose-length
product conversion or “k-“factors suggest that k-factors, and
therefore effective cardiac radiation doses, in children are considerably higher than previously believed [32, 36]. In an effort
to minimize radiation doses and standardize imaging parameters across pediatric centers, the Image Gently alliance’s
“Have-a-Heart” campaign recently published radiation management guidelines for pediatric cardiovascular CT [32].
Several large congenital cardiac centers have also recently
established an international congenital cardiovascular CT registry to establish best practice guidelines for radiation exposure and anesthesia use in CHD patients [37].
Newer CT scanner platforms also allow rapid image acquisition that decreases the need for sedation or anesthesia. With
dual-source scan technology and wide detector coverage,
studies can be obtained in a single heartbeat without requiring
a breath hold for most indications. General anesthesia and
paralysis are not necessary in most patients [35, 36]. While
cardiac MRI in critically ill and often unstable patients poses
significant risks because of study length, need for sedation, the
need to change MRI-incompatible ventilators and lines, and
difficulties with closely monitoring patients inside the scanner,
cardiac CT mitigates these risks. CT can be done in patients on
ventilators, or even on extracorporeal membrane oxygenation
(ECMO) or ventricular assist device support, as long as the
patient can be transported safely to the scanner [38, 39]. The
same study can also evaluate for pulmonary artery embolism
or thrombosis, which in particular has been reported in patients with Eisenmenger physiology, and image the lungs
and airways, which are often abnormal in CHD patients [40,
41]. These factors make cardiac CT an attractive option for
imaging cyanotic CHD patients.
Page 3 of 14 30
In recent years, cardiac CT has also helped to advance the
use of patient-specific 3D printing and augmented or virtual
reality modeling for use in planning complex intracardiac repairs and other CHD interventions [42]. Digital 3D modeling
and printing aid users in “mentally rotating” imaging data,
which allows them to better understand complex spatial relationships within the heart and extracardiac structures [43].
Any volumetric Digital Imaging and Communications in
Medicine (DICOM) dataset, including contrast-enhanced
and non-contrast MR angiograms and 3D echo data, can be
used to generate models, but CT is the most common source of
3D cardiac models and is considered the easiest to use [42,
44]. It confers submillimeter isotropic spatial resolution, and
its bright contrast facilitates the process of segmenting or identifying anatomic structures to be printed. Figure 1 demonstrates a cardiac CT angiogram performed in a 1-day-old cyanotic neonate with prenatally diagnosed complex double outlet RV with an inlet ventricular septal defect, d-malposed great
arteries with the aorta rightward of the pulmonary artery, and
an aortic coarctation. The CT, performed with an effective
radiation dose of about 0.3 mSV, was used delineate the relationships of the ventricles, VSD, and outflows and to create a
detailed, hollowed-out scale model of the patient’s heart.
Although the infant was initially anticipated to undergo a
staged repair over the first months of life to connect each
ventricle to its appropriate outflow, the CT and 3D model
permitted the surgeon to plan a complex intracardiac baffle
repair that was successfully performed in a single operation
at 2 weeks of age [45].
The Role of Cardiovascular CT in Managing
Specific Cyanotic Heart Defects
Cardiovascular CT has been utilized to diagnose and manage
a broad spectrum of cyanotic cardiac defects by delineating
anatomic details, assessing cardiac relationships to
extracardiac structures, and identifying post-intervention complications [12, 27]. In the following section, we will review its
uses as they pertain to specific cyanotic defects.
Tetralogy of Fallot and Other Right Ventricular
Outflow Tract Obstructive Lesions
TOF is the most common form of cyanotic CHD, accounting
for 5% of congenital heart defects [46]. In TOF, anterior deviation of the conal portion of the ventricular septum obstructs
blood flow through the RV outflow tract and directs
desaturated blood though a ventricular septal defect into the
systemic circulation. RVoutflow tract obstruction falls along a
spectrum ranging from minimal subpulmonary stenosis to
pulmonary atresia or, in its most severe form, absent main
and branch pulmonary arteries with multiple aortopulmonary
30 Page 4 of 14
Curr Cardiovasc Imaging Rep (2019) 12: 30
Fig. 1 a Segmentation of a
cardiac CT in a 1-day-old neonate
with complex double outlet right
ventricle, d-malposed great
vessels, and an aortic coarctation,
in preparation for rapid
prototyping. b 3D-printed and
digital cardiac models (lateral
walls removed to show
intracardiac anatomy)
collaterals (MAPCAs) arising from the aortic arch to feed the
lungs. Preoperatively, most TOF anatomy can be determined
by echocardiogram. However, an estimated 5% to 12% of
these patients have coronary artery abnormalities, such as an
accessory left anterior descending coronary arising from the
right coronary and crossing the pulmonary outflow tract,
which can preclude a full neonatal repair [47–49]. Echo may
be unable to definitively assess coronary artery anatomy, but
CT has been shown to identify coronary anomalies in young
children with unrepaired TOF with 97% to 100% sensitivity
and specificity [50, 51].
In TOF with MAPCAs, the tortuous network of very small
collateral vessels is rarely seen well on echocardiogram or
even on cardiac MRI. Traditional management strategy involved obtaining a diagnostic cardiac catheterization under
general anesthesia within a few days of birth. However, the
excellent spatial resolution of cardiovascular CT makes it an
optimal modality to visualize even distal aortopulmonary collateral anatomy [52–54]. In many institutions, CT has become
the first-line imaging modality to delineate aortopulmonary
collateral anatomy upon birth or initial patient presentation.
CT can determine the presence and anatomy of native pulmonary arteries, courses of the MAPCAs, the lung segments they
supply, and the relationship of each collateral to the airways
and esophagus (Fig. 2). Catheterization may therefore be delayed until the patient is older, decreasing risk of vessel
injury relative to during the newborn period. When preoperative catheterization is performed, usually at a few
months of age, CT images guide the procedure, which focuses on identifying aortopulmonary collateral stenosis and
dual sources of pulmonary blood flow. Fewer angiograms
can be performed, limiting radiation and contrast exposure.
Some centers have also started to use CT angiograms to
create 3D digital and printed models, which can be sterilized and placed on the surgical field as an intraoperative
guide [55, 56].
In TOF patients who undergo staged repair and are
initially palliated with aortopulmonary shunts, CT is often
obtained prior to full repair to evaluate shunt patency and
assess for branch pulmonary artery stenoses or aneurysms.
In repaired TOF patients, CT can assess for right ventricular to pulmonary artery (RV to PA) conduit patency and
calcification, pseudoaneurysms, and pulmonary artery stenosis [57, 58]. In patients with contraindications to cardiac
MRI, functional CT has been used to assess RV volume
and function. Most reports demonstrate relatively
Curr Cardiovasc Imaging Rep (2019) 12: 30
Page 5 of 14 30
Fig. 2 Cardiac CT volume
renderings in a 2-day-old boy
with prenatally diagnosed TOF
with pulmonary atresia and
MAPCAs, demonstrating
aortopulmonary collaterals
(asterisk) arising from the
descending thoracic aorta and
confluent but severely
hypoplastic native branch
pulmonary arteries (short arrow).
Long arrow denotes the
connection of an aortopulmonary
collateral to the left pulmonary
artery
equivalent volume assessment between CT and MRI if a
CT scanner with appropriate temporal resolution is used,
but mild overestimation of RV end-diastolic volumes has
also been reported [59, 60].
CT can also demonstrate the proximity of the outflow tract
to the coronary arteries (Fig. 3), which recent TOF management guidelines for adult congenital CHD patients recommend assessing in all patients to help determine candidacy
for percutaneous pulmonary valve replacement [61]. CT was
also recently reported to identify thrombus and endocarditis,
not demonstrated on echocardiogram, as a cause of percutaneous pulmonary valve obstruction [62]. Before repeat surgical intervention, CT can identify the relationship of the RV
Fig. 3 Nineteen-year-old man
with double outlet RV and
pulmonary stenosis status postRastelli operation, with RV to PA
conduit stenosis. Cardiac CT
identified the LMCA coursing
just inferior to the conduit, a
contraindication to RV outflow
tract stenting
outflow tract to the sternum to minimize the risk of dissecting
an adherent or calcified pulmonary artery or conduit [27].
Several other cyanotic heart defects also involve pulmonary outflow obstruction or are repaired with RV to PA conduits, including variants of double outlet right ventricle with
RV outflow tract obstruction that are repaired with a Rastelli
procedure. Cardiac CT can play a similar role in these patients’
surveillance and management. In patients with PA/IVS, in
which the pulmonary valve is completely obstructed and the
RV is often smaller and hypertensive, cardiac CT has been
used in newborns to identify commonly associated coronary
abnormalities such as coronary stenoses and RV to coronary
artery fistulae [59, 60].
30 Page 6 of 14
Curr Cardiovasc Imaging Rep (2019) 12: 30
Transposition of the Great Arteries
Transposition of the great arteries refers to several types of
defects with ventriculoarterial discordance, but the most common is “simple” d-TGA without a ventricular septal defect and
with unobstructed outflow tracts [63]. In d-TGA, which represents 3% to 5% of congenital heart defects, patients typically
undergo an arterial switch operation (ASO) within the first
week of life [1]. Preoperative anatomy is usually determined
by echocardiogram, but dual-source cardiovascular CT in neonates has been reported to identify abnormal coronary artery
arrangements, which occur in up to one-third of d-TGA patients, with higher sensitivity than echocardiography [64–66].
Postoperatively, cardiovascular CT is often used to identify
complications such as coronary artery stenosis after reimplantation, which is reported in 8% to 10% of patients who have
undergone ASO [67–70]. CT is also commonly used to assess
for main and branch pulmonary artery obstruction after surgical LeCompte maneuver (Fig. 4) as well as neoaortic root
dilation [70]. While CT perfusion imaging may be useful to
evaluate for myocardial perfusion defects resulting from coronary artery obstruction, its use has been reported only in
conjunction with positron emission tomography (PET) in this
population [71]. In patients with more complex forms of dTGA with a ventricular septal defect and pulmonary outflow
obstruction, who undergo a Rastelli operation with RV to PA
conduit placement or a Nikaidoh operation with translocation
of the aorta and occasional reimplantation of the right coronary artery, CT is again a useful option to assess residual
lesions and guide reintervention (Fig. 5) [72].
Older patients with d-TGA who were repaired in the 1970s
and 1980s typically underwent a Mustard or Senning atrial
switch operation, in which systemic and pulmonary venous
drainage to the atria was redirected towards the appropriate
outflows. Atrial switch complications typically include baffle
leaks or obstruction and dysfunction of the systemic RV that
remains connected to the aorta [73]. Cardiac CT can be used to
assess baffle obstruction, patency of intrabaffle stents, and
Fig. 4 Two-month-old boy with
d-TGA status post arterial switch
operation. a Maximum intensity
projection (MIP) demonstrating
stenosis of the proximal branch
pulmonary arteries after
LeCompte maneuver. b Volume
rendering of the branch
pulmonary arteries viewed from
the cranial perspective
Fig. 5 Eleven-year-old girl with d-TGA status post-arterial switch
operation. Volume rendering of cardiac CT obtained for preoperative
planning prior to surgical reintervention demonstrates the left anterior
descending coronary artery (blue triangle) arising from the right
neoaortic sinus and coursing between the sternum (blue arrow) and
main pulmonary artery, posing risk of coronary injury during repeat
sternotomy
ventricular size and function [74]. While the Mustard and
Senning are typically no longer performed for d-TGA, the
atrial switch has reemerged in congenitally corrected or
levo-transposition of the great arteries (l-TGA), either in conjunction with the arterial switch as part of the double switch
procedure to address discordance at both the atrioventricular
and ventriculoarterial levels or along with a Rastelli procedure
in patients with l-TGA, VSD, and pulmonary stenosis (Fig. 6)
[75, 76]. Many patients with atrial switch repair of d-TGA,
who are prone to arrhythmias, and l-TGA, in which atrioventricular block is common, have also undergone pacemaker or
defibrillator placement. CT is therefore a more optimal
Curr Cardiovasc Imaging Rep (2019) 12: 30
Fig. 6 Two-year-old girl with l-TGA, ventricular septal defect, and a
hypoplastic left-sided RV, status post-hemi-Mustard, Rastelli, and
bidirectional Glenn operations. Asterisk denotes the pathway
connecting the pulmonary veins to the right-sided LV
modality than MRI to visualize anatomy and function and
guide EP interventions in these patients [73, 74].
Truncus Arteriosus
Truncus arteriosus is a relatively rare—0.5% of CHD—
defect in which the pulmonary and aortic outflow tracts
fail to separate during fetal development and remain as a
single outflow, allowing mixing of systemic and pulmonary venous blood [1]. CT is rarely used preoperatively for
this lesion, which is usually well delineated on echocardiogram. However, preoperative CT has been reported to
Fig. 7 Cardiac CT MIPs (a, b)
and volume rendering (c) in a 15year-old boy with truncus
arteriosus status post-RV-PA
conduit placement and
mechanical truncal (aortic) valve
replacement complicated by
infective endocarditis. A
diverticulum was seen on CT
arising from the left ventricular
outflow tract coursing between
the ascending aorta and RV-PA
conduit and compressing the
conduit. A small area of
hypoattenuation (asterisk)
suggestive of thrombus is also
seen within the diverticulum
Page 7 of 14 30
better demonstrate the branch pulmonary arteries and rule
out associated findings such as an interrupted aortic arch
[77, 78]. Postoperatively, CT can assess sequelae related to
both the left heart, which has an abnormal truncal valve
that is often eventually replaced with a mechanical valve,
and right heart, in which an RV to PA conduit is placed or
direct connection is created. Figure 7 demonstrates the left
ventricular outflow tract of a 15-year-old patient with
truncus arteriosus and recent infective endocarditis.
Echocardiogram could not clearly visualize the truncal
valve region due to mechanical valve artifact, but cardiovascular CT demonstrated a large diverticulum of the
truncal root that compressed his RV to PA conduit.
Total Anomalous Pulmonary Venous Return
In TAPVR, which makes up 1% to 2% of CHD, the pulmonary veins connect abnormally to various sites in the
systemic veins, including the superior vena cava
(supracardiac type), inferior vena cava (infracardiac), coronary sinus, or a combination of sites. Cyanosis is often
worsened by obstruction of the anomalous connection,
particularly in the infracardiac type, which may cause pulmonary congestion and severe hemodynamic instability.
Cardiovascular CT can be rapidly performed even in critically ill patients and can preoperatively identify the
course of the pulmonary veins, drainage sites, and areas
30 Page 8 of 14
of obstruction [79–82]. Post-repair, CT is a preferred modality to assess recurrent pulmonary venous obstruction,
which can occur in up to 18% of patients (Fig. 8) [83].
Cardiovascular CT is also useful for pulmonary vein imaging in patients with pulmonary venous abnormalities in
the setting of more complex defects, such as heterotaxy
syndrome or single ventricle disease, and can clearly delineate the sites of venous drainage and mechanisms of
obstruction (Fig. 9).
Single Ventricle Disease and Heterotaxy Syndrome
Single ventricle or functionally single ventricle defects cause
cyanosis secondary to right-to-left shunting, parallel systemic
and pulmonary circulations, and in some cases pulmonary
outflow obstruction. These defects include tricuspid atresia
and other forms of hypoplastic right heart syndrome, hypoplastic left heart syndrome, double inlet left ventricle (LV),
double outlet RV, unbalanced atrioventricular canal defects,
and severe forms of Ebstein anomaly. Most of these are palliated over the first 3 to 4 years of life by connecting the systemic venous return directly to the pulmonary arteries and
utilizing the single ventricle as the systemic ventricle,
pumping through an unobstructed native aorta or an augmented “neoaorta” constructed in part from the pulmonary artery.
As many of these defects are associated with abnormalities in
the great vessels and systemic and pulmonary venous connections, echocardiography does not always adequately determine complex anatomic details prior to palliation.
Cardiovascular CT is commonly used in these neonates for
preoperative planning and is often performed using a nonsedated “feed and wrap” method of swaddling and allowing
the patient to fall asleep after eating [84–86]. Some single
Curr Cardiovasc Imaging Rep (2019) 12: 30
ventricle disease occurs in the setting of heterotaxy syndrome,
in which the laterality of structures within the thorax and abdomen is arranged abnormally, with highly variable duplication of left- or right-sided structures known as isomerism. In
these patients, CT can assess not only intracardiac and
extracardiac abnormalities but can also identify bilateral left
or right atrial appendages and assess the situs of the lungs and
abdominal viscera [87].
Postoperatively, CT has been used to assess shunt patency, pulmonary artery stenosis, aortic arch obstruction,
and venovenous or aortopulmonary collateral vessels [57,
88]. It has also been used in pre-Glenn and pre-Fontan
procedural planning, in some cases as a noninvasive alternative to diagnostic cardiac catheterization. In a series of
32 single ventricle patients, half of whom underwent cardiovascular CT and half of whom underwent diagnostic
cardiac catheterization prior to second stage palliation,
CT had no diagnostic discrepancies compared to surgical
findings. Patients were exposed to much less radiation than
those who underwent catheterization (effective dose
1.1 mSv compared to 14 mSv), received less than half
the contrast of the catheterized cohort, and had only one
adverse event—increased cyanosis—compared to eight
events in the catheterized patients [89]. After palliation,
CT can evaluate Glenn and Fontan pathways for obstruction or thrombus (Fig. 10) [90]. Due to mixing of contrastopacified and non-opacified blood as the contrast enters
the pulmonary arteries from the SVC, injection protocols
should be optimized to achieve heterogeneous contrast
within the vessels and avoid false-positive diagnosis of
thrombus [57, 91]. A number of solutions have been proposed, including simultaneous arm and leg injection and
lower extremity injection, but many sources agree that delayed scanning ranging from 60 to 180 s after injection,
which may be guided by a timing bolus, yields homogeneous contrast opacification throughout the heart and
cavopulmonary pathways [92, 93].
Practical Considerations for Cardiovascular CT
in Cyanotic CHD
Fig. 8 Three-month-old girl with repaired supracardiac TAPVR and
discrete narrowing of the right lower pulmonary venous confluence and
left lower pulmonary vein as they enter the left atrium. The left upper
pulmonary vein (not shown) is occluded and drains instead via the
azygous (one asterisk) and hemiazygous (two asterisks) veins into the
superior vena cava. The right heart is dilated due to pressure and
volume overload
Performing cardiovascular CT in patients with cyanotic
cardiac defects, who span the spectrum of neonates to
adults and may be unrepaired, palliated, or fully repaired,
requires careful individualization of the exam to the patient, the physiology of the defect, and the clinical question
being addressed. Detailed guidelines addressing scan protocols and radiation dose reduction in pediatric and congenital heart disease have been published elsewhere, but
we will briefly review a few considerations pertinent to
cyanotic CHD patients [28, 29, 32, 94–96].
Curr Cardiovasc Imaging Rep (2019) 12: 30
Page 9 of 14 30
Fig. 9 Coronal MIP (a) and volume rendering from the posterior view (b)
obtained in a neonate with hypoplastic left heart syndrome to delineate
pulmonary venous drainage. The right-sided pulmonary veins (RPVs)
cross the midline to a left-sided vertical vein (VV) entering the
innominate vein. The left-sided pulmonary veins (LPVs) drain to a
tortuous channel that enters the coronary sinus (CS). DA = ductus
arteriosus, Ao = aorta
Sedation
when possible during the scan. For coronary artery imaging
in younger children, general anesthesia with suspended respiration is often required to minimize motion artifact. Although
this scenario is not ideal, these cases are often performed as a
lower-risk alternative to cardiac catheterization, which would
expose these patients to general anesthesia, an invasive procedure, and increased radiation.
Older 64-slice scanners that use prospectively ECGgated “step-and-shoot” technology, in which sequential
axial scans are obtained while the table moves along the
z-axis with each gantry rotation, may require up to 10 s to
scan the entire chest [94]. Resulting images may therefore
demonstrate “stair-step” artifact due to patient motion between sequential acquisitions. Patients being scanned with
this technology are more likely than those imaged with
high-pitch or wide coverage scanners to require sedation,
particularly when images are being used for 3D modeling
or printing. In ICU patients who are already intubated and
sedated, our team often requests paralysis and suspended
The need for sedation can vary depending on patient characteristics, CT scanner speed, and exam goals. For many CTs,
developmentally appropriate older children, typically over the
age of 4 years, can remain motionless in the scanner and by 6
or 7 years can cooperate with breath hold instructions [95]. Of
note, children with cyanotic CHD are well-documented to
have a high incidence of neurodevelopmental abnormalities
and genetic syndromes, which should be determined before
the study [22, 97]. Infants under 6 months can often utilize the
“feed and wrap” method outlined above. However, particularly in studies focusing more on extracardiac structures such as
the aortic arch or pulmonary arteries or veins, whose images
are less motion-sensitive, we find that even toddlers and
preschool-aged children may be scanned without sedation if
tightly wrapped or placed in a vacuum immobilizer device.
Child life therapists are often involved for support, and a parent wearing appropriate shielding can remain in the room
Fig. 10 Complex heterotaxy with dextrocardia, congenitally corrected
transposition of the great arteries with l-looped ventricles (RV is
anterior and leftward of the LV), and pulmonary valve atresia. a
Cardiac CT obtained post-Fontan palliation demonstrates compression
of the right-sided pulmonary veins (arrow) by an extracardiac Fontan
conduit coursing posterior to the left-sided atrium. b Subsequent CT
post-pulmonary venoplasty demonstrates persistent right pulmonary
vein narrowing (arrow) as well as focal hypoattenuation within the
Fontan conduit suggestive of thrombus
30 Page 10 of 14
respiration to optimize study quality without imposing significant additional clinical risk.
Intravenous Access and Contrast
Vascular access may be difficult in complex CHD patients
who have undergone multiple hospital admissions and interventions since birth. Intravenous (IV) placement should be
performed with attention to each patient’s systemic and pulmonary venous anatomy and any known occlusions or collateral vessels to ensure appropriate opacification of the heart
[57]. Typically, the speed of power injection is limited by IV
gauge, which may range from less than 1 ml/s using a 24gauge IV in a neonate to 5 ml/s using an 18-gauge IV in a
teenager or adult. As image contrast is generally optimized
when scanning at lower tube potentials (kVs) that generate
x-rays with energies closer to the k-edge of iodine, it is not
essential to push contrast particularly rapidly and risk extravasation [98]. Contrast dose may be determined more by the
desired duration of contrast opacification at a given injection
rate than by patient weight but typically ranges from 1.5 to
2 ml/kg.
Patients with any form of cyanotic CHD are known to
have a high prevalence—up to 50% by adulthood—of
chronic kidney disease resulting from insults related to
polycythemic hyperviscosity, diminished cardiac function,
nephrotoxic medication exposure, and cardiopulmonary
bypass runs [99, 100]. Therefore, care should be taken to
pre-hydrate if needed, avoid concomitant nephrotoxic
medications, and use low-osmolar or iso-osmolar contrast
such as iohexol or iodixanol [94].
As with all procedures in cyanotic CHD patients, even
small air bubbles should be carefully avoided during injection
to avoid crossing right-to-left shunts. When injecting contrast
in CHD patients, particularly those with complex defects who
may have unexpected flow dynamics or aortopulmonary or
venovenous collaterals, we carefully monitor the bolus tracker
and manually trigger the scan acquisition.
Radiation
The effective radiation dose is affected by x-ray tube potential
and current, scan mode, exposure time, z-axis coverage, and
imaging settings used when bolus tracking. The literature suggests that most congenital CHD scanning can be performed at
70 or 80 kV [32, 57]. While the use of iterative reconstruction
techniques allows lower tube currents to be selected for a
given patient size, the clinical question to be addressed should
primarily determine tube potential. If distal coronary artery
anatomy or other fine anatomic detail is not needed, tube
currents can be significantly reduced during both acquisition
and monitoring scans. Z-axis coverage should also be carefully chosen to cover only the area of interest. An infant’s
Curr Cardiovasc Imaging Rep (2019) 12: 30
cardiovascular anatomy can usually be imaged in 8 cm, while
adolescents and adults may require up to 16 cm or more.
Scanning throughout the cardiac cycle is less commonly
performed, but prospectively or retrospectively gated CT can
be used to assess ventricular function in patients who are unable to undergo cardiac MRI. Although low radiation doses
have been reported in dual-source high-pitch scanning, functional assessment with older scanners that have lower temporal resolution often produces undesirably high radiation exposure [32, 95, 101, 102].
Conclusion
Cardiovascular CT has developed into a key tool among congenital cardiac imaging modalities that complements the roles
of echocardiography and cardiac MRI in patients of all ages
with cyanotic CHD. It can provide information that informs
both preoperative planning and postoperative surveillance and
facilitates scanning even in critically ill patients or those with
implanted devices. In order to optimize imaging in cyanotic
CHD patients, the unique characteristics of each defect and
each patient must be carefully considered to develop an individualized protocol for each scan.
Compliance with Ethical Standards
Conflict of Interest Dr. Einstein receives consultancy fees from GE
Healthcare and receives grants from Roche Medical Systems, Canon
Medical Systems, and the International Atomic Energy Agency.
All other authors have nothing to disclose.
Human and Animal Rights and Informed Consent This article does not
contain any studies with human or animal subjects performed by any of
the authors.
Open Access This article is distributed under the terms of the Creative
Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the
Creative Commons license, and indicate if changes were made.
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Titanium Research Developments in the United Kingdom
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Abstract The paper presents highlights of the titanium alloy research developments since Ti-2015 (San Diego). The review
underlines the strong and collaborative fundamental research conducted at UK universities through strategic government
sponsored programmes. The role of advanced characterisation and modelling techniques in order to better understand the effects of
deformation, fatigue loading and environment on titanium alloys continues to be world leading. Researchers in the UK are also
continuing to develop a range of new alloys, methods of extraction and emerging near net shape processes via casting, powder and
wire-fed routes. 1. UK Ti Research Landscape Over the last 10 years, the £50 million Rolls-Royce / Engineering and Physical Sciences Research Council
(EPSRC) Strategic Partnership Flagship Programme in Structural Metallic Systems for Gas Turbine Applications has a clear aim:
through fundamental research, to provide the foundation for next-generation aero-engines. With respect to Ti research the joint-
funded initiative utilises and extends the capabilities of Cambridge, Oxford, Birmingham and Swansea Universities. A unique
feature of this Rolls-Royce / EPSRC Programme was the inclusion of a parallel training programme delivered by
an EPSRC Centre for Doctoral Training (CDT). Through this Centre, PhD and EngD students research industry-relevant issues
and contribute to the development of engine components destined to enter commercial service. Up until 2019, around 100 students
have joined the programme with over 60% of those graduating recruited to Rolls-Royce or the supply chain, many of whom are
working on Ti technical challenges. In 2019, the EPSRC Centre for Doctoral Training in Advanced Metallics Systems (The Univ. of Sheffield and
Manchester, partnering with I-Form the SFI Research Centre for Advanced Manufacturing in Dublin, received a further 5 years
funding and >£5M investment from EPSRC and a further £4M from industry for 70 studentships. Interestingly over the last 10
years, Ti projects have seen the largest growth of any metallic project, training graduates from a range of backgrounds into Ti
metallurgists. Over the last four years, the UK has continued to produce key discoveries at the fundamental level through to new
emerging near net shape processing methods. One of the most important EPSRC programmes since Ti-2015 was HexMat
(Heterogeneous Mechanics in Hexagonal Alloys across Length and Time Scales). This successful five-year collaborative
programme between Imperial College London, Univ. Manchester and Univ. Oxford has provided value insights into the
fundamental mechanisms in Ti and its performance in the aerospace sector. The programme has allowed UK experts to develop a
range of experimental, characterisation and modelling techniques for Ti. The key Ti industry partners during HexMat were Timet
and Rolls-Royce, who are also largely responsible for the majority of Ti alloy research in the UK. Titanium Research Developments in the United Kingdom Martin Jackson Department of Materials Science and Engineering, The University of Sheffield, Mappin Street, Sheffield, S1 MATEC Web of Conferences 321, 01006 (2020)
The 14th World Conference on Titanium MATEC Web of Conferences 321, 01006 (2020)
The 14th World Conference on Titanium https://doi.org/10.1051/matecconf/202032101006 2.1 HexMat Case Study 1 – Dwell Fatigue Modelling 2.1 HexMat Case Study 1 – Dwell Fatigue Modelling Dwell fatigue has been a long-standing problem in aero-engine Ti components, and particularly for discs. During late
stages of take-off and early cruise, the resulting high stress holds give rise to creep in crystallographic regions well-orientated for
slip, leading to stress redistribution on to adjacent crystallographic regions badly orientated which cyclically ratchets and leads to
basal facet formation and potentially subsequent crack growth. This results in a dwell fatigue debit (often a substantial reduction in
cyclic life compared to conventional fatigue). Fundamental property quantification has been carried out at Imperial for alpha and
beta phases respectively, in various alloys by integrating micro-pillar testing, quantitative characterisation and crystal plasticity
modelling (see Fig. 1). Interestingly, the basal, prismatic and slip systems show markedly differing strain rate sensitivities thereby
affecting local creep response and stress redistribution in dwell fatigue [1]. Crystal and dislocation plasticity models utilise the key
property data to facilitate microstructural analyses to understand and quantify the local creep and load shedding, the stress build up
and the facet nucleation process [2]. Figure 1: Micro-compression test on alpha-beta pillar and crystal plasticity modelling. Yellow arrows highlight active slip [1]. (Courtesy
of Fionn Dunne) Figure 1: Micro-compression test on alpha-beta pillar and crystal plasticity modelling. Yellow arrows highlight active slip [1]. (Courtesy
of Fionn Dunne) The modelling work permits the investigation of behaviour in virtual microstructures and in representative in-service
conditions so that rig spin tests carried out by Rolls-Royce may be interpreted in the context of the full thermomechanical loading
of in-service discs. These studies have demonstrated the role of temperature, alpha-beta microstructure, thermal alleviation, and
stress state in dwell fatigue [3], and have provided the pathway to dwell-resistant microstructures. Fig. 2 shows an example
IMI834 microstructure, together with the results of modelling studies to explain observed dwell failures in disc spin tests versus
safe operation for in-service thermomechanical conditions. A crucial over-arching conclusion is that while the chemistry and
processing in Ti alloys give rise to the microstructure, it is the latter which is crucial to controlling dwell debit (and not, for
example, Mo content, as has been believed for decades). Optimal dwell resistant microstructures are those which exploit
basketweave morphologies, and secondary alpha, ideally with high numbers of alpha variants, to inhibit soft grain region creep and
hence stress ratcheting in hard regions. 2. Fundamental research for aero-engine components © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution
License 4.0 (http://creativecommons.org/licenses/by/4.0/). MATEC Web of Conferences 321, 01006 (2020)
The 14th World Conference on Titanium MATEC Web of Conferences 321, 01006 (2020)
The 14th World Conference on Titanium https://doi.org/10.1051/matecconf/202032101006 2.1 HexMat Case Study 1 – Dwell Fatigue Modelling 2.2 HexMat Case Study 2 – Strain localisation in macrozones 2.2 HexMat Case Study 2 – Strain localisation in macrozones In tandem with the activities at Imperial College London, the Univ. Manchester have refined their high resolution digital
image correlation (HRDIC) technique in order to determine the effect of hard oriented macrozones on the local slip activity [5]. In
combination with grain orientation (EBSD) and strain (HRDIC), large area high resolution strain maps showed there is very
diffuse and homogeneous slip in the hard macrozone region compared to more planar and localised slip in the neighbouring
(softer) region occurring by prismatic or basal slip (Fig. 3). Relative Displacement Ratio (RDR) analysis was utilised in
combination with traditional slip trace analysis to identify the active slip mode in each grain which was validated by TEM
analysis. Figure 3: Strain localisation in Ti-6Al-4V UD rolled plate with hard oriented macrozones when loaded along RD. (a) IPF TD map, (b)
effective shear strain map at ~0.5% applied strain across an area of 0.36 x 1.08 mm2with (c) magnified view to highlight difference in slip
either side of the macrozone boundary and (d) TEM micrographs collected under various electron beam directions confirming slip in a
single grain lifted out from the hard oriented macrozone region. (Courtesy of João Quinta de Fonseca) Figure 3: Strain localisation in Ti-6Al-4V UD rolled plate with hard oriented macrozones when loaded along RD. (a) IPF TD map, (b)
effective shear strain map at ~0.5% applied strain across an area of 0.36 x 1.08 mm2with (c) magnified view to highlight difference in slip
either side of the macrozone boundary and (d) TEM micrographs collected under various electron beam directions confirming slip in a
single grain lifted out from the hard oriented macrozone region. (Courtesy of João Quinta de Fonseca) Figure 3: Strain localisation in Ti-6Al-4V UD rolled plate with hard oriented macrozones when loaded along RD. (a) IPF TD map, (b)
effective shear strain map at ~0.5% applied strain across an area of 0.36 x 1.08 mm2with (c) magnified view to highlight difference in slip
either side of the macrozone boundary and (d) TEM micrographs collected under various electron beam directions confirming slip in a
single grain lifted out from the hard oriented macrozone region. 2.1 HexMat Case Study 1 – Dwell Fatigue Modelling Figure 2: (a) IMI834 alpha-beta microstructure and (b) representative crystal plasticity modelling, and (c) hard grain stresses and
thermomechanical operational map of dwell fatigue sensitivity for spin tests and in-service conditions [3]. (Courtesy of Fionn Dunne) Figure 2: (a) IMI834 alpha-beta microstructure and (b) representative crystal plasticity modelling, and (c) hard grain stresses and
thermomechanical operational map of dwell fatigue sensitivity for spin tests and in-service conditions [3]. (Courtesy of Fionn Dunne) The dwell fatigue work within the EPSRC-funded programme grant HexMat [4] has had international impact with new
high-level TRL collaborations with Pratt & Whitney, Honeywell, GE, Rolls-Royce, IHI, AFRL and the FAA. Ref 4 also has a
weblink to the key journal papers from this programme. 2 MATEC Web of Conferences 321, 01006 (2020)
The 14th World Conference on Titanium MATEC Web of Conferences 321, 01006 (2020)
The 14th World Conference on Titanium https://doi.org/10.1051/matecconf/202032101006 2.4 Metastable Beta Alloy Development 2.2 HexMat Case Study 2 – Strain localisation in macrozones (Courtesy of João Quinta de Fonseca) 2.3 Small punch fatigue testing Following the successful application of small punch test techniques for the assessment of static and creep properties of
various aero-engine materials [6], recent attention has transferred to the development of small punch fatigue capability. In this
regard, the alloy Ti-6Al-4V in various conventional (cast and forged) and additive (electron beam melt) forms has been employed
to validate the technique [7,8] producing representative S-N curves in agreement with traditional uniaxial approaches
(evidenced through empirical data and FE modelling, Fig. 4 top). Classical fatigue mechanisms are replicated in small punch
samples, including cyclic plasticity (“cold creep”) and striated fatigue crack growth (Fig. 4). The ability to monitor constitutive
behaviour (load-displacement loops akin to stress-strain data, Fig. 4) and the sensitivity of the test to subtle microstructure
variations, offer an improved representation of the fatigue properties of additive materials. Small punch discs can usually be
extracted from the final built component, however complex or restricted in section size. This offers direct sampling of the
component, rather than the reliance on separately built test specimens where the combination of macroscopic sample geometry and
additive process parameters induce a non-representative microstructure. 3 MATEC Web of Conferences 321, 01006 (2020)
The 14th World Conference on Titanium https://doi.org/10.1051/matecconf/202032101006 Figure 4: (Top) Prediction of mean strain and maximum principal stress distributions across a SPF specimen after 100 fatigue cycles of
different applied load. (Bottom Left) Evidence of striations from a SPF test on Ti-6Al-4V, 20°C, R=0.1. (Bottom right) Load-
displacement hysteresis loops for a SPF test on a Ti-6Al-4V, 20°C, R=-1 [7]. (Courtesy of Rob Lancaster and Martin Bache) Figure 4: (Top) Prediction of mean strain and maximum principal stress distributions across a SPF specimen after 100 fatigue cycles of
different applied load. (Bottom Left) Evidence of striations from a SPF test on Ti-6Al-4V, 20°C, R=0.1. (Bottom right) Load-
displacement hysteresis loops for a SPF test on a Ti-6Al-4V, 20°C, R=-1 [7]. (Courtesy of Rob Lancaster and Martin Bache) 2.4 Metastable Beta Alloy Development 2.4 Metastable Beta Alloy Development Since the last World Titanium conference in 2015, research at the Univ. Cambridge has continued to focus on the
fundamental aspects of metastable beta Ti alloys and, in particular, on their associated phase transformations, both reconstructive
and diffusionless. Work investigating the effect of low temperature heat treatments on the microstructure of Ti-15Mo (wt.%) has discovered
the formation of a previously unreported B2 phase, see fig.5[9]. The formation mechanism of this phase remains under debate
although as the phase was only observed to form in thin foils, both hydrogen and a reduction in constraint have been postulated. Figure 5: Ti-15Mo (wt.%) prior to and following in situ heat treatment at 300°C in the TEM: (Left), SADP down the [001]β prior to
heating with corresponding BF image of the microstructure. (Right) SADP down the [001]β following 15 min at 300 °C and d)
corresponding BF image of the microstructure [9] (Courtesy of Nicholas Jones). Figure 5: Ti-15Mo (wt.%) prior to and following in situ heat treatment at 300°C in the TEM: (Left), SADP down the [001]β prior to
heating with corresponding BF image of the microstructure. (Right) SADP down the [001]β following 15 min at 300 °C and d)
corresponding BF image of the microstructure [9] (Courtesy of Nicholas Jones). The influence of Zr on the superelastic properties and the stability of the omega phase in Ti-Nb alloys have been
investigated using a combination of synchrotron X-ray radiation, advanced electron microscopy and atom probe tomography (Fig. 6). Zr was widely believed to be a strong omega suppressant commonly added to commercial alloys to mitigate the impact of this
phase. However, the recent work from Univ. Cambridge has shown that whilst this might be true for the athermal form, Zr has very 4 4 MATEC Web of Conferences 321, 01006 (2020)
The 14th World Conference on Titanium MATEC Web of Conferences 321, 01006 (2020) https://doi.org/10.1051/matecconf/202032101006 The 14th World Conference on Titanium little preventative effect on the formation of isothermal omega [10]. In situstudies investigating the transformational behaviour of
Ti-Nb-Zr alloys in response to variations in temperature and applied loads have also revealed some very interesting findings,
which require the mechanistic understanding of these transformations to be revisited. 2.4 Metastable Beta Alloy Development Figure 6: Graphical illustration of the role of Zr on ω phase formation in Ti-Nb alloys using X-ray diffraction, transmission electron
microscopy and atom probe tomography [10] (Courtesy of Nicholas Jones) Figure 6: Graphical illustration of the role of Zr on ω phase formation in Ti-Nb alloys using X-ray diffraction, transmission electron
microscopy and atom probe tomography [10] (Courtesy of Nicholas Jones) 2. Alternative extraction processes A new, novel method of Ti production via calciothermic reduction of TiO2, developed at the Univ. Bradford. The
Bradford Process is observed to be faster, cheaper and more environmentally friendly that competing methods – most notably the
industry standard Kroll process (Fig. 7). After proving the initial proof of concept the Univ. Bradford has developed the process to
the point it is ready for technical demonstrations (< 5kg CP Ti production) having built a bespoke furnace and secured all
appropriate IP relating to the process. Figure 7: (Left) Photograph of Ti product derived from “The Bradford Process”; (Right) Bespoke, small scale calciothermic reduction
cell at the Univ. Bradford (Courtesy of Russell Hodgetts). Figure 7: (Left) Photograph of Ti product derived from “The Bradford Process”; (Right) Bespoke, small scale calciothermic reduction
cell at the Univ. Bradford (Courtesy of Russell Hodgetts). duct derived from “The Bradford Process”; (Right) Bespoke, small scale calciothermic reduction
cell at the Univ. Bradford (Courtesy of Russell Hodgetts). Metalysis are continuing to scale up their operation now the process has been successfully demonstrated on the small
sale. Generation 4 is the first facility to take Metalysis’ solid-state, modular, electrochemical process to industrial scale (Fig. 8)
and can produce tens-to-hundreds of tonnes per annum of high value, niche and master alloys. In 2017, Metalysis expanded into
additional facilities at their Materials Discovery Centre, now sandwiched between state-of-the-art Rolls-Royce and McLaren
factories on the Advanced Manufacturing Park in Rotherham, South Yorkshire. 5 5 MATEC Web of Conferences 321, 01006 (2020)
The 14th World Conference on Titanium https://doi.org/10.1051/matecconf/202032101006 MATEC Web of Conferences 321, 01006 (2020)
The 14th World Conference on Titanium https://doi.org/10.1051/matecconf/202032101006 Figure 8: Photograph of (Left) Metalysis’ Generation4 industrial plant and (Right) Metalysis’ Materials Discovery Centre on Advanced
Manufacturing Park, Rotherham, South Yorkshire. (Courtesy of Ian Mellor) 4.1 Wire Arc Additive Manufacturing (WAAM) A team comprising of Thales Alenia Space, Cranfield Univ. and Glenalmond Technologies have successfully produced a
first full-scale prototype of a Ti pressure vessel to be used in future manned missions for space exploration. The vessel in Fig. 9
is made from Ti-6Al-4V and is approximately 1 m in height and 8.5 kg in mass. It has been deposited using the Wire + Arc
Additive Manufacturing (WAAM) process [11]. The Cranfield Univ. developed WAAM process provides an exciting opportunity
to go straight from a digital drawing to final structure. WAAM has integrated two individual pieces into a single part; eliminating
the need for long-lead-time forgings; and substantially reduced the amount of waste material removed by machining. It is
estimated that more than 200 kg of Ti-6Al-4V have been saved compared to a conventional forging machining route. Figure 9: A Ti-6Al-4V pressure vessel prototype manufactured by the Cranfield University WAAM process: (Left) post wire + arc AM
processing and (Right) final machinied component (Courtesy of Filomena Martina) Figure 9: A Ti-6Al-4V pressure vessel prototype manufactured by the Cranfield University WAAM process: (Left) post wire + arc AM
processing and (Right) final machinied component (Courtesy of Filomena Martina) 4.3 FAST-forge of near net shapes The Univ. Sheffield developed FAST-forge process [13] fully consolidates Ti powder and swarf into near net shapes in
two solid state steps using; (1) field assisted sintering technology (FAST) followed by (2) closed die forging (see Fig. 11). Since
Ti-2015, the process has picked up momentum and is now being developed through the Technology Readiness Levels in the
automotive and aerospace manufacturing sectors for applications such as connecting rods and rocker arms, etc. In March 2018,
the UK Secretary of State for Defence stated that “This ground-breaking method is not only faster and cheaper but could see a
huge expansion of titanium parts and equipment throughout the military”[14]. The FAST process also has been demonstrated to
effective enable intricate diffusion bonding (DB) of dissimilar Ti alloys prior to forging (known as FAST-DB [15]), opening
the opportunity of designing components with different alloys and specific properties in subcomponent regions (Fig. 11). These
developments are an example of the benefits of the EPSRC sponsored CDT in Advanced Metallics Systems, further underlining
the advantages to the UK and companies for developing new technologies as well as the unique graduate training in Ti
technologies. Figure 11: (Left) FAST-forge, field assisted sintering technology in combination with closed die forging is being developed to produce
near net shape components, such as engine rocker arms from a range of feedstocks including machined swarf; (Right) Closed die forged
I-bolt (by W.H. Tildesley, Wolverhampton, UK) from FAST preform of two dissimilar Ti alloys. Figure 11: (Left) FAST-forge, field assisted sintering technology in combination with closed die forging is being developed to produce
near net shape components, such as engine rocker arms from a range of feedstocks including machined swarf; (Right) Closed die forged
I-bolt (by W.H. Tildesley, Wolverhampton, UK) from FAST preform of two dissimilar Ti alloys. 4.2 Recycling of Powder and Swarf into wire feedstock With the increase in wire fed additive manufacturing technologies for large components, there is an increasing need to
reduce the cost of the Ti alloy wire feedstock. Work has continued at the Univ. Sheffield to develop continuous extrusion
processes such as ConformTM to consolidate commercial powder [12], machined swarf and surplus AM powder into wire. Fig. 10
shows surplus/oversized AM Ti powder being converted to wire form. Such wire has good ductility and can be coiled and further
cold drawn. Recently, work has been conducted with Airbus (Toulouse) to determine the effectiveness of recycling their waste Ti-
6Al-4V machined swarf into 10 mm diameter wire. 6 6 MATEC Web of Conferences 321, 01006 (2020)
The 14th World Conference on Titanium MATEC Web of Conferences 321, 01006 (2020) https://doi.org/10.1051/matecconf/202032101006 The 14th World Conference on Titanium Figure 10: (Left) Titanium wire derived from surplus AM powder exiting a BWE Conform machine; (Right) Ben Thomas, Univ. of
Sheffield with Ti-6Al-4V wire consolidated from powder. Figure 10: (Left) Titanium wire derived from surplus AM powder exiting a BWE Conform machine; (Right) Ben Thomas, Univ. of
Sheffield with Ti-6Al-4V wire consolidated from powder. 4.4 Near net shape investment castings One of the most cost effective near net shaping processes, is investment casting. In the UK, AMRC Castings has
now commissioned a large scale Ti casting facility centred around a 1 tonne VAR furnace with a large casting chamber, capable of
pouring moulds up to 2.5 m diameter and 2.5 m height (Fig. 12). The furnace itself has the capability to pour moulds statically or
centrifugally and has an active inert gas cooling system incorporated for optimisation the microstructure. This facility is supported
with a variety of additive and subtractive methods for pattern manufacture direct from CAD – eliminating the need for costly 7 7 MATEC Web of Conferences 321, 01006 (2020)
The 14th World Conference on Titanium https://doi.org/10.1051/matecconf/202032101006 tooling during development activities and offering the highest level of versatility. Fig. 12 also illustrates the results of
collaborative work with GE Aviation (Hamble, UK) to produce a flap interface part, which is currently machined from Al
alloys. An optimised final design can provide a 20% reduction in the overall weight of the assembly, by reducing the number of
fasteners and parts (reduced from nine to one as cast part). Only 20% of the as cast product is machined away to produce the final
part [16]. tooling during development activities and offering the highest level of versatility. Fig. 12 also illustrates the results of
collaborative work with GE Aviation (Hamble, UK) to produce a flap interface part, which is currently machined from Al
alloys. An optimised final design can provide a 20% reduction in the overall weight of the assembly, by reducing the number of
fasteners and parts (reduced from nine to one as cast part). Only 20% of the as cast product is machined away to produce the final
part [16] Figure 12: (Left) One tonne VAR investment casting facility at the Univ. Sheffield’s AMRC Castings, Advanced Manufacturing Park,
Rotherham; (Right) Flap Interface prototype investment cast Ti 6.7 kg blank [16]. (Courtesy of Matthew Cawood). Figure 12: (Left) One tonne VAR investment casting facility at the Univ. Sheffield’s AMRC Castings, Advanced Manufacturing Park,
Rotherham; (Right) Flap Interface prototype investment cast Ti 6.7 kg blank [16]. (Courtesy of Matthew Cawood). AMRC Castings have also provided net shaped components for the UK’s leading joining research institute, TWI Ltd, on a
European Space Agency (ESA) sponsored project to develop a low cost method to produce Ti-6Al-4V satellite propellant tanks
[17]. 4.5 Friction stir welding of castings 4.4 Near net shape investment castings TWI Ltd with technical support from Airbus Sapce and Defence initially conducted a detailed study on the effect of friction
stir welding (FSW) of cast Ti-6Al-4V product with rigorous mechanical testing. This has led to the manufacture of prototype tank
by the friction stir welding of two hemisphere domes with a cylindrical section of approximately 42 cm diameter (Fig. 13). Such
development has demonstrated a lower cost processing route, compared to the existing forging and forming routes. 8 8 MATEC Web of Conferences 321, 01006 (2020)
The 14th World Conference on Titanium MATEC Web of Conferences 321, 01006 (2020)
The 14th World Conference on Titanium https://doi.org/10.1051/matecconf/202032101006 Figure 13: The prototype propellant tank manufactured using stationary shoulder FSW of two hemispherical domes with a cylindrical
section; Photograph (Top left) before and (Top right) after FSW operation. The tank dimensions are approx. 75 cm in length by 42 cm
diameter; (Bottom left) Photograph of the tank mounted on the welding fixture; (Bottom right) stationary shoulder FSW tool setup. The
W-25Re probe rotates through a stationary shoulder mounted in a welding head. Channels for Ar flow are also visible [17]. (Courtesy of
Richard Freeman) Acknowledgements Martin Bache (Univ. Swansea), Matthew Cawood (AMRC Castings), Fionn Dunne (Imperial College London), Richard Freeman
(TWI Ltd.), Russell Hodgetts (Univ. Bradford), Nicholas Jones (Univ. Cambridge), Rob Lancaster (Univ. Swansea), Filomena
Martina (Univ. Cranfield), Ian Mellor (Metalysis Ltd.), João Quinta de Fonseca (Univ. Manchester). References [1] Z. Zhang et al.Jnl. Mech. Phys. Solids. 95 (2016) 393–410. [1] Z. Zhang et al.Jnl. Mech. Phys. Solids. 95 (2016) 393–410. [2] S. Joseph et al.Intl. Jnl. Plasticity, 110 (2018) 38-56. [2] S. Joseph et al.Intl. Jnl. Plasticity, 110 (2018) 38-56. [3] Z. Zheng et al.Intl. Jnl. Plasticity. 111 (2018) 234-252. [4] http://www.imperial.ac.uk/hexmat/about-us/ https://doi.org/10.1051/matecconf/202032101006 [4] http://www.imperial.ac.uk/hexmat/about-us/ [5] D. Lunt et al.Scripta Mater. 145 (2018) 45. [6] R.C. Hurst et al.Theor. Appl. Fract. Mech. 86 (2016), 69–77. [7] R.J. Lancaster et al.Mater. Sci. Eng. A 748 (2019) 21-29. R.J. Lancaster et al.Mater. Sci. Eng. A 748 (2019) 21-2 [8] D.T.S. Lewis et al.Mater. Sci. Eng. A 754 (2019) 719-727 [9] J.M. Bennett et al.Mater. Charact. 142 (2018) 523. [9] J.M. Bennett et al.Mater. Charact. 142 (2018) 523. [10] E.L. Pang et al.Acta Mater. 153 (2018) 62. [10] E.L. Pang et al.Acta Mater. 153 (2018) 62. [11] S.W. Williams et al.Mater. Sci. and Tech. 32 (2015) 641–647. [12] B. Thomas et al.Mater. Sci. and Tech. 33 (2017) 899-903. [12] B. Thomas et al.Mater. Sci. and Tech. 33 (2017) 899-903. [13] N.S. Weston, M. Jackson. J. Mater. Process. Tech. 243 (2017) 335-346. [14] https://www.gov.uk/government/news/porton-down-scientists-on-brink-of-titanium-revolution [15] J.J. Pope et al.J. Mater. Process. Tech. 269 (2019) 200-207. [16] A. Agarwal et al.The Royal Aeronautical Society’s 5th Aircraft Structural Design Conf. (Oct 2016) [16] A. Agarwal et al.The Royal Aeronautical Society’s 5th Aircraft Structural Design Conf. (Oct 2016)
[17] R. Freeman et al.ASM Intl. Conf. on Adv. Mater. and Processes (ADMAT 2017). [17] R. Freeman et al.ASM Intl. Conf. on Adv. Mater. and Processes (ADMAT 2017). 9 9
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https://figshare.com/articles/journal_contribution/A_Comparative_Investigation_of_Volatile_Organic_Compounds_of_Cattle_Rumen_Metabolites_using_HS-SPME_and_PoraPak-Q_Odor_Trapping_Methods/20448244/1/files/36590941.pdf
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A Comparative Investigation of Volatile Organic Compounds of Cattle Rumen Metabolites using HS-SPME and PoraPak-Q Odor Trapping Methods
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A Comparative Investigation of Volatile Organic Compounds
of Cattle Rumen Metabolites using HS-SPME and PoraPak-Q
Odor Trapping Methods Victor O Omondi 1,2, Geoffrey O Bosire 2, John M Onyari 2 and
Merid N Getahun 1*
1 International Centre of Insect Physiology and Ecology, P.O Box 30772-00100
Nairobi, Kenya
2 Department of Chemistry University of Nairobi P O Box 30197 00100 Nairobi Victor O Omondi 1,2, Geoffrey O Bosire 2, John M Onyari 2 and
Merid N Getahun 1*
1 International Centre of Insect Physiology and Ecology, P.O Box 30772-00100
Nairobi, Kenya
2 Department of Chemistry, University of Nairobi, P.O Box 30197-00100, Nairobi,
Kenya 2 Department of Chemistry, University of Nairobi, P.O Box 30197-00100, Nairo
Kenya * Corresponding Author: mgetahun@icipe.org (Merid N Getahun) Supplementary data A Comparative Investigation of Volatile Organic Compounds
of Cattle Rumen Metabolites using HS-SPME and PoraPak-Q
Odor Trapping Methods * Corresponding Author: mgetahun@icipe.org (Merid N Getahun) Validation of CO2 detection in cow rumen
samples Validation of CO2 detection in cow rumen
samples conditioned SPME was first introduced into the
GC/MS instrument and run as a blank and later
on the same was used to trap Volatile organic
compounds from the sample and analyzed
by GC/MS. Additionally, the respective CO2
peak areas for both the blank and sample were
compared (Fig. S1A-D & Table S1). conditioned SPME was first introduced into the
GC/MS instrument and run as a blank and later
on the same was used to trap Volatile organic
compounds from the sample and analyzed
by GC/MS. Additionally, the respective CO2
peak areas for both the blank and sample were
compared (Fig. S1A-D & Table S1). To ensure that CO2 was detected from our
samples as opposed to the residual air present in
the SPME protective needle, validation checks
was done to ascertain this. Before introducing
the SPME fiber into the sample headspace, a Anal. Chem. Lett. 2022, 12
DOI: 10.1080/22297928.2022.2100276
© 2022 Har Krishan Bhalla & Sons
Figure S1. Representative GC-MS chromatograms of cattle rumen CO2 presence validation comparison
between Blank SPME runs and Sample runs (A) 15 minutes (B) 5 minutes extraction times minutes Anal. Chem. Lett. 2022, 12
DOI: 10.1080/22297928.2022.2100276
© 2022 Har Krishan Bhalla & Sons
Figure S1. Representative GC-MS chromatograms of cattle rumen CO2 presence validation comparison
between Blank SPME runs and Sample runs (A) 15 minutes (B) 5 minutes extraction times minutes © 2022 Har Krishan Bhalla & Sons Figure S1 cont. Representative GC-MS chromatograms of cattle rumen CO2 presence validation
comparison between Blank SPME runs and Sample runs (C) 30 minutes & (D) 1 Hour extraction times Figure S1 cont. Representative GC-MS chromatograms of cattle rumen CO2 presence validation
comparison between Blank SPME runs and Sample runs (C) 30 minutes & (D) 1 Hour extraction times
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Retraction Note: Circulating plant miRNAs can regulate human gene expression in vitro
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www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Retraction: Circulating plant
miRNAs can regulate human gene
expression in vitro
Chiara Pastrello, Mike Tsay, Rosanne McQuaid, Mark Abovsky, Elisa Pasini,
Elize Shirdel, Marc Angeli, Tomas Tokar, Joseph Jamnik, Max Kotlyar, Andrea Jurisicova,
Joanne Kotsopoulos, Ahmed El-Sohemy & Igor Jurisica
Scientific Reports 6:32773; doi: 10.1038/srep32773; published online 08 September 2016; updated 22 May 2017
We are retracting this Article as we no longer have confidence in the data to support our central conclusion – the
detection of Brassica oleracea microRNAs in the bloodstream of humans who consumed broccoli. Upon concerns raised by a reader about the incorrect design of our microRNA primers, we checked the primer
OPEN Scientific Reports | 7:46826 | DOI: 10.1038/srep46826 Retraction: Circulating plant
miRNAs can regulate human gene
expression in vitro
OPEN Chiara Pastrello, Mike Tsay, Rosanne McQuaid, Mark Abovsky, Elisa Pasini,
Elize Shirdel, Marc Angeli, Tomas Tokar, Joseph Jamnik, Max Kotlyar, Andrea Jurisicova,
Joanne Kotsopoulos, Ahmed El-Sohemy & Igor Jurisica Chiara Pastrello, Mike Tsay, Rosanne McQuaid, Mark Abovsky, Elisa Pasini,
Elize Shirdel, Marc Angeli, Tomas Tokar, Joseph Jamnik, Max Kotlyar, Andrea Jurisicova,
Joanne Kotsopoulos, Ahmed El-Sohemy & Igor Jurisica Chiara Pastrello, Mike Tsay, Rosanne McQuaid, Mark Abovsky, Elisa Pasini,
Elize Shirdel, Marc Angeli, Tomas Tokar, Joseph Jamnik, Max Kotlyar, Andrea Jurisicova,
Joanne Kotsopoulos, Ahmed El-Sohemy & Igor Jurisica We are retracting this Article as we no longer have confidence in the data to support our central conclusion – the
detection of Brassica oleracea microRNAs in the bloodstream of humans who consumed broccoli. Upon concerns raised by a reader about the incorrect design of our microRNA primers, we checked the primer
sequences and detected antisense design of all the forward primers for broccoli microRNA detection, except for
miR824. This invalidates the results described in Figure 1 (except for miR824) and Figure 2 of the paper. We also
checked primers designed to detect human genes, and confirmed that all of them were in the correct orientation. 1 www.nature.com/scientificreports/ Figure 1. ANOVA comparison of different time points in broccoli feeding trial (Figure 2C in the paper). Shown are comparisons for raw Ct values of microRNAs (A), raw Ct values of reference genes (B), and (C)
microRNA values normalized only on one reference gene (listed on the left side of the figure). Figure 1. ANOVA comparison of different time points in broccoli feeding trial (Figure 2C in the paper). Shown are comparisons for raw Ct values of microRNAs (A), raw Ct values of reference genes (B), and (C)
microRNA values normalized only on one reference gene (listed on the left side of the figure). Figure 1. ANOVA comparison of different time points in broccoli feeding trial (Figure 2C in the paper). Shown are comparisons for raw Ct values of microRNAs (A), raw Ct values of reference genes (B), and (C)
microRNA values normalized only on one reference gene (listed on the left side of the figure). Following the primer analyses, we confirmed by sequencing that the primers for miR824 amplified correct
miRNA from broccoli. We then investigated the cause of the trend observed in Figure 2C. Retraction: Circulating plant
miRNAs can regulate human gene
expression in vitro
OPEN We first inspected raw
Ct values of miR160, miR2673 and miR21; as visible in Figure 1A below, there was no obvious dose response
in any of these PCR products (all p-values shown in Figure 1 come from ANOVA analysis). We then examined
raw Ct values of the 3 reference genes (ACTB, B2M and RNU6) that we used for normalization. As shown in
Figure 1B, ACTB, B2M and RNU6 were stable (except at T0). While in the paper we calculated expression using
geometric mean of reference genes, we also normalized the 3 microRNAs PCRs against the 3 reference genes Scientific Reports | 7:46826 | DOI: 10.1038/srep46826 2 www.nature.com/scientificreports/ separately, one at the time. As shown in Figure 1C, B2M is responsible for the trend observed in the paper, most
strongly in “T3 160 g” (consumption of 160 g of broccoli per day). Therefore, we also tested the stability of our 3 reference genes in experiments described in Figure 3 of the paper
(transfection of lung cancer cell lines) and Figure 4 (comparison of different treatments in a gastric cell line). We applied ANOVA to raw Ct values of each reference gene, and the resulting p-values are listed in Table 1. All
reference genes were stable across lung cancer cell lines, confirming results presented in Figure 3; however, some
effect (albeit not significant) was observed on the stability of RNU6 in the gastric cell line when transfected with
miR160 or treated with sulphoraphane (Figure 4). Figure 3
miR160 vs AllStar
Siport vs AllStar
NT vs AllStar
ACTB
0.9748
0.9705
0.9977
B2M
0.9887
0.9923
0.9832
RNU6
0.9968
0.9825
0.9997
Figure 4/Supplementary Table 7
miR160 vs NT
Sulphoraphane vs NT
DIM vs NT
ACTB
0.7293
0.8815
0.6318
B2M
0.9945
0.8172
0.8856
RNU6
0.0792
0.0866
0.9908
Table 1. P-values for ANOVA testing across comparisons for each reference gene. Figure 3
miR160 vs AllStar
Siport vs AllStar
NT vs AllStar
ACTB
0.9748
0.9705
0.9977
B2M
0.9887
0.9923
0.9832
RNU6
0.9968
0.9825
0.9997
Figure 4/Supplementary Table 7
miR160 vs NT
Sulphoraphane vs NT
DIM vs NT
ACTB
0.7293
0.8815
0.6318
B2M
0.9945
0.8172
0.8856
RNU6
0.0792
0.0866
0.9908
Table 1. P-values for ANOVA testing across comparisons for each reference gene. Table 1. P-values for ANOVA testing across comparisons for each reference gene. Lastly, we investigated whether repeating our experiments with correct primers targeting broccoli miRNAs could
provide evidence for the original finding of this paper. Retraction: Circulating plant
miRNAs can regulate human gene
expression in vitro
OPEN To this aim, we used two protocols: (a) the same protocol
used in the published version of the article (with correct and specific forward primers, one set targeting only the
microRNA and one set extending to the polyA immediately next to the microRNA, listed in Table 2), and (b) a
stem-loop protocol1. The stem-loop protocol has been described as more efficient in comparison to the ligation
method we used originally2. We tested two broccoli flower samples, the transfected cell line samples and eight
plasma samples from the controlled broccoli feeding trial. Using both protocols we were unable to confirm spe-
cific amplification of these miRNAs in human blood. Thus, we were not able to validate the central hypothesis of
this paper. 3
6826 | DOI: 10.1038/srep46826
In conclusion, the most appropriate course of action is to retract this paper. We apologize for this error and regret
any inconvenience this may have caused. All authors agree to the retraction of the paper. References
1. Varkonyi-Gasic, E. In Methods in molecular biology (Clifton, N.J.) 1456, 163–175 (2017). 2. Adhikari, S. et al. Hairpin priming is better suited than in vitro polyadenylation to generate cDNA for plant miRNA qPCR. Mol. Plant 6, 229–31 (2013). This work is licensed under a Creative Commons Attribution 4.0 International License. The images
or other third party material in this article are included in the article’s Creative Commons license,
unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license,
users will need to obtain permission from the license holder to reproduce the material. To view a copy of this
license, visit http://creativecommons.org/licenses/by/4.0/
© The Author(s) 2017
Mirna
Stem-loop primer
Forward primer
miR160
GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACTGGCATACAGG
CCACGTATGCCTGGCTC
miR2673
GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACACGAAGAGGAA
CGGCGCGTCTCTTCCTC
miR21
GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACTCAACATCAGT
TGCGCGGTAGCTTATCAG
Qiagen primers
Mirna
Primer miRNA specific
Primer with A tail
miR160
TGCCTGGCTCCCTGTATGCCA
TGCCTGGCTCCCTGTATGCCAA
miR2673
TCTCTTCCTCTTCCTCTTCGT
TCTCTTCCTCTTCCTCTTCGTAAA
miR21
TAGCTTATCAGACTGATGTTGA
TAGCTTATCAGACTGATGTTGAA
Table 2. Primers used in the 2 protocols to test validity of our hypothesis. Mirna
Stem-loop primer
Forward primer
miR160
GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACTGGCATACAGG
CCACGTATGCCTGGCTC
miR2673
GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACACGAAGAGGAA
CGGCGCGTCTCTTCCTC
miR21
GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACTCAACATCAGT
TGCGCGGTAGCTTATCAG
Qiagen primers
Mirna
Primer miRNA specific
Primer with A tail
miR160
TGCCTGGCTCCCTGTATGCCA
TGCCTGGCTCCCTGTATGCCAA
miR2673
TCTCTTCCTCTTCCTCTTCGT
TCTCTTCCTCTTCCTCTTCGTAAA
miR21
TAGCTTATCAGACTGATGTTGA
TAGCTTATCAGACTGATGTTGAA Table 2. Primers used in the 2 protocols to test validity of our hypothesis. n conclusion, the most appropriate course of action is to retract this paper. We apologize for this error and regre
ny inconvenience this may have caused. All authors agree to the retraction of the paper. References
1. Varkonyi-Gasic, E. In Methods in molecular biology (Clifton, N.J.) 1456, 163–175 (2017). 2. Adhikari, S. et al. Hairpin priming is better suited than in vitro polyadenylation to generate cDNA for plant miRNA qPCR. Mol. Plant 6, 229–31 (2013). This work is licensed under a Creative Commons Attribution 4.0 International License. The images
or other third party material in this article are included in the article’s Creative Commons license,
unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license,
users will need to obtain permission from the license holder to reproduce the material. To view a copy of this
license, visit http://creativecommons.org/licenses/by/4.0/
© The Author(s) 2017 References References
1. Varkonyi-Gasic, E. In Methods in molecular biology (Clifton, N.J.) 1456, 163–175 (2017). 2. Adhikari, S. et al. Hairpin priming is better suited than in vitro polyadenylation to generate cDNA for plant miRNA qPCR. Mol. Plant 6, 229–31 (2013). Thi
k i li
d
d
C
i
C
A
ib
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l Li
Th i 1. Varkonyi-Gasic, E. In Methods in molecular biology (Clifton, N.J.) 1456, 163–175 (2017). 2. Adhikari, S. et al. Hairpin priming is better suited than in vitro polyadenylation to generate cDNA for plant miRNA qPCR. Mol. Plant 6, 229–31 (2013). This work is licensed under a Creative Commons Attribution 4.0 International License. The images
or other third party material in this article are included in the article’s Creative Commons license,
unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license,
users will need to obtain permission from the license holder to reproduce the material. To view a copy of this
license, visit http://creativecommons.org/licenses/by/4.0/ © The Author(s) 2017 Scientific Reports | 7:46826 | DOI: 10.1038/srep46826 3
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Microdrop Deposition Technique: Preparation and Characterization of Diluted Suspended Particulate Samples
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Received: 23 May 2018; Accepted: 11 July 2018; Published: 16 July 2018 Abstract: The analysis of particulate matter (PM) in dilute solutions is an important target for
environmental, geochemical, and biochemical research. Here, we show how microdrop technology
may allow the control, through the evaporation of small droplets, of the deposition of insoluble
materials dispersed in a solution on a well-defined area with a specific spatial pattern. Using this
technology, the superficial density of the deposited solute can be accurately controlled. In particular,
it becomes possible to deposit an extremely reduced amount of insoluble material, in the order of
few µg on a confined area, thus allowing a relatively high superficial density to be reached within a
limited time. In this work, we quantitatively compare the microdrop technique for the preparation
of particulate matter samples with the classical filtering technique. After having been optimized,
the microdrop technique allows obtaining a more homogeneous deposition and may limit the sample
amount up to a factor 25. This method is potentially suitable for many novel applications in different
scientific fields such as demanding spectroscopic studies looking at the mineral fraction contained in
ice cores or to pollution investigations looking at the detection of heavy metals present in ultra-trace
in water. Keywords: ultra-dilution; droplets; water; evaporation; X-ray fluorescence Article Salvatore Macis 1,2,*, Giannantonio Cibin 3, Valter Maggi 4,5, Giovanni Baccolo 4,5 ID ,
Dariush Hampai 2 ID , Barbara Delmonte 4, Alessandro D’Elia 6 ID and Augusto Marcelli 2,7 ID 1
Department of Mathematics and Physics, Università di Roma Tor Vergata, via della Ricerca Scientific
00133 Rome, Italy 1
Department of Mathematics and Physics, Università di Roma Tor Vergata, via della Ricerca Scientifica 1,
00133 Rome, Italy 2
Laboratori Nazionali di Frascati, Istituto Nazionale di Fisica Nucleare, 00044 Frascati, Italy;
dariush.hampai@lnf.infn.it (D.H.); augusto.marcelli@lnf.infn.it (A.M.) g
3
Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, UK;
giannantonio.cibin@diamond.ac.uk g
4
Dipartimento di Scienze dell’Ambiente e della Terra, Università degli Studi di Milano Bicocca, Piazza della
Scienza, 1-20126 Milano, Italy; valtermaggi@gmail.com (V.M.); giovanni.baccolo@mib.infn.it (G.B.);
barbara.delmonte@unimib.it (B.D.) 5
Sezione di Milano-Bicocca, Istituto Nazionale di Fisica Nucleare, Piazza della Scienza, 3-20126 Milano, Italy
6
Department of Physics, University of Trieste, Via A. Valerio 2, 34127 Trieste, Italy; ale9149@gmail.com
7
Istituto Officina dei Materiali, Consiglio Nazionale delle Ricerche, Basovizza SS-14, km 163.5,
34149 Trieste, Italy 5
Sezione di Milano-Bicocca, Istituto Nazionale di Fisica Nucleare, Piazza della Scienza, 3-20126 Milano, Italy
6
Department of Physics, University of Trieste, Via A. Valerio 2, 34127 Trieste, Italy; ale9149@gmail.com
7
Istituto Officina dei Materiali, Consiglio Nazionale delle Ricerche, Basovizza SS-14, km 163.5,
34149 Trieste Italy y
6
Department of Physics, University of Trieste, Via A. Valerio 2, 34127 Trieste, Italy; ale9149@gmail.com
7
Istituto Officina dei Materiali, Consiglio Nazionale delle Ricerche, Basovizza SS-14, km 163.5, *
Correspondence: salvatore.macis@roma2.infn.it; Tel.: +39-067-259-4523 www.mdpi.com/journal/condensedmatter 1. Introduction Th
ti
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ith t
diff
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ti
p
g
p
presenting a quite large surface (about 1 cm2). In this case a critical issue is the low superficial density
of the PM deposited on top of it. The evaporation process, based on the deposition of micro-droplets, may overcome both issues. Indeed, the microdrop technique consists in the deposition of PM in association to liquid drops
presenting a diameter between 70 and 200 μm. After the evaporation of the solvent, the PM
depositional pattern resembles the one of the original droplets. The main disadvantages of this
method are the inhomogeneity of the deposition and the long time required for the evaporation of
the solvent. In fact, the evaporation of large drops (few mm of diameter) can last several hours. At
normal conditions of temperature and humidity, full evaporation of drops of water can require more
than 10 h per mL, and the density fluctuation of the deposited material is up to 80% as a consequence
of the so-called coffee-stain effect [1]. To optimize the process, we deposit micro-droplets and control
the deposition area with motorized translational stages. To this purpose, a study of the evaporation
dynamics is necessary to investigate the uniformity of deposition and to evaluate the evaporation rate of
micro-droplets presenting different sizes. When dealing with densely-packed deposition patterns, our
results show that is necessary to consider the interaction between different evaporating droplets. The evaporation of a drop on a surface can be described with two different models: the evaporation
with a “constant contact area” and that with a “constant contact angle” [2] (see Figure 1). results show that is necessary to consider the interaction between different evaporating droplets. The evaporation of a drop on a surface can be described with two different models: the evaporation
with a “constant contact area” and that with a “constant contact angle” [2] (see Figure 1). Figure 1. Deposition and evaporation processes for two different models: “constant contact area” (left)
and “constant contact angle” (right). The main difference is represented by the shape of materials
deposited on the substrate: pinned at the edge for the “constant contact area” model (bottom left) and
concentrated in the center for the “constant contact angle” model (bottom right). Figure 1. Deposition and evaporation processes for two different models: “constant contact area” (left)
and “constant contact angle” (right). 1. Introduction When dealing with complex samples, one of the main bottlenecks from the practical point of
view is their preparation. A suitable sample preparation method should allow saving time during
data acquisition, enhancing the S/N ratio, improving the detection limits, etc. Considering particulate
matter (PM) analysis of solutions, the most common preparation technique is filtration: it is easy,
reliable, and fast, but at the same time, it presents some drawbacks. Indeed, it requires a considerable Condens. Matter 2018, 3, 21; doi:10.3390/condmat3030021 www.mdpi.com/journal/condensedmatter 2 of 9 Condens. Matter 2018, 3, 21 sample consumption and PM whose size is smaller than the one of the filtration pores, which are not
retained on the filter. To avoid this point, the original solution could be deposited on a membrane
presenting a quite large surface (about 1 cm2). In this case a critical issue is the low superficial density
of the PM deposited on top of it. Condens. Matter 2018, 3, x FOR PEER REVIEW
2 of 9
retained on the filter. To avoid this point, the original solution could be deposited on a membrane The evaporation process, based on the deposition of micro-droplets, may overcome both issues. Indeed, the microdrop technique consists in the deposition of PM in association to liquid drops
presenting a diameter between 70 and 200 µm. After the evaporation of the solvent, the PM depositional
pattern resembles the one of the original droplets. The main disadvantages of this method are the
inhomogeneity of the deposition and the long time required for the evaporation of the solvent. In fact,
the evaporation of large drops (few mm of diameter) can last several hours. At normal conditions
of temperature and humidity, full evaporation of drops of water can require more than 10 h per
mL, and the density fluctuation of the deposited material is up to 80% as a consequence of the
so-called coffee-stain effect [1]. To optimize the process, we deposit micro-droplets and control the
deposition area with motorized translational stages. To this purpose, a study of the evaporation
dynamics is necessary to investigate the uniformity of deposition and to evaluate the evaporation rate
of micro-droplets presenting different sizes. When dealing with densely-packed deposition patterns,
our results show that is necessary to consider the interaction between different evaporating droplets. 1. Introduction The main difference is represented by the shape of materials
deposited on the substrate: pinned at the edge for the “constant contact area” model (bottom left) and
concentrated in the center for the “constant contact angle” model (bottom right). Figure 1. Deposition and evaporation processes for two different models: “constant contact area” (left)
and “constant contact angle” (right). The main difference is represented by the shape of materials
deposited on the substrate: pinned at the edge for the “constant contact area” model (bottom left) and
concentrated in the center for the “constant contact angle” model (bottom right). Figure 1. Deposition and evaporation processes for two different models: “constant contact area” (left)
and “constant contact angle” (right). The main difference is represented by the shape of materials
deposited on the substrate: pinned at the edge for the “constant contact area” model (bottom left) and
concentrated in the center for the “constant contact angle” model (bottom right). liquid drop and the surface. The shape of the drop remains almost “spherical” [2], but the contact
angle decreases. In accordance to the second model, the contact angle of the edge of the drop is
constant during the evaporation [2], thus, the shape of the drop remains “spherical”, but the contact
area between liquid and surface continuously decreases. The “constant contact area model” is
suitable when a strong interaction between the liquid and the substrate takes place, e.g., when
considering water and a hydrophilic surface [3,4]. This is clear in the work of Peschel et al., where at
higher concentration, the evaporation occurs with the constant area mode, i.e., during the
evaporation, the droplet does not shrink [5]. On the opposite, the second model is more appropriate when the drop-substrate interactions are
weak, as in the case of water on a hydrophobic substrate. As studied by Fittschen et al. [6], an ultra-
dil
d
l
i
i
h
i
d b
l
l
i
b
i
i
d
h
ff
i
In the first case, the evaporation takes place, maintaining a constant contact area between the
liquid drop and the surface. The shape of the drop remains almost “spherical” [2], but the contact
angle decreases. In accordance to the second model, the contact angle of the edge of the drop is
constant during the evaporation [2], thus, the shape of the drop remains “spherical”, but the contact
area between liquid and surface continuously decreases. 2. Experimental Using a micro-dispenser, micro-sized droplets can be deposited, controlling the spatial distribution
of drops, their size, and the deposition rate. The Microdrop technology [8] allows spreading of
extremely small amounts of a liquid solution. Moreover, thanks to dedicated devices, it is possible to
deposit single droplets whose volume is in the sub-nanoliter range. Such small droplets present an
interaction with the substrate, which can be considered negligible. The micro-dispenser is composed
of a head with a nozzle and a pumping chamber working with the same piezoelectric technology
used for inkjet printers. The head is controlled by a driver, i.e., a pulse generator, which controls
the piezoelectric actuator and sends pulses to the head. The device we used is the MD-K-130 (©
Microdrop Technologies GmbH, Norderstedt, Germany). With an inner nozzle diameter of 70 µm,
we may produce, with 1% volume repeatability, droplets with a minimum nominal volume of 180 pl
and at a drop rate from 1 to 2000 Hz [8,9]. To prepare a sample with a specific microdrop pattern, it is
necessary to control the number of drops on the substrate, releasing each of them at a well-defined and
repeatable distance. In our experimental setup, to generate the pattern, the head is maintained fixed
while the substrate translates under the stream of droplets released by the head. The motion of the
substrate is realized with two precision PI Micos (Physik Instrumente GmbH Karlsruhe, Karlsruhe,
Germany) Translational Stage VT-80 stages, assembled perpendicularly with respect to each other. This setting allows positioning the substrate with a nominal accuracy of 1 µm per axis. The desired
spatial patterns are obtained controlling the two translation stages with a LabView©-based code and
properly setting the Microdrop dispenser [9]. The characterization of the evaporation time and of the drop pattern was made with the Zeiss
Axio Imager M1 optical microscope (Carl Zeiss Microscopy LLC, Oberkochen, Germany) using 5×,
10×, and 20× magnification optics. The video camera had a x–y spatial resolution of 0.13 µm at 5×,
0.26 µm at 10×, and 0.52 µm at 20× magnification and allowed us to measure the droplet volume with
an uncertainty of ~3% of the measured volume. 1. Introduction The “constant contact area model” is suitable
when a strong interaction between the liquid and the substrate takes place, e.g., when considering water
and a hydrophilic surface [3,4]. This is clear in the work of Peschel et al., where at higher concentration,
the evaporation occurs with the constant area mode, i.e., during the evaporation, the droplet does not
shrink [5]. diluted solution is characterized by a low solution–substrate interaction, and the coffee stain
phenomenon is reduced. As a matter of fact, the insoluble components deposed by the evaporation
of the droplets have to be characterized in order to achieve the most homogeneous deposition starting
from defined droplet size. As shown by Sparks et al., droplet dimension and solution concentration
On the opposite, the second model is more appropriate when the drop-substrate interactions
are weak, as in the case of water on a hydrophobic substrate. As studied by Fittschen et al. [6],
an ultra-diluted solution is characterized by a low solution–substrate interaction, and the coffee stain
phenomenon is reduced. As a matter of fact, the insoluble components deposed by the evaporation of
the droplets have to be characterized in order to achieve the most homogeneous deposition starting 3 of 9 Condens. Matter 2018, 3, 21 from defined droplet size. As shown by Sparks et al., droplet dimension and solution concentration are
the main parameters that control the deposition [7]. Thus, the deposition method we presented can be
a fast, simple, and direct way to achieve a homogeneous deposition of insoluble materials considering
only the droplet size and the concentration of the solution. In this contribution, we will describe the optimization process of an experimental setup based on
the evaporation of water micro-droplets in accordance to the constant angle model. The uniformity of
the deposition will be determined, and results will be compared to those obtained through the standard
filtration method. Indeed, the microdrop method may offer many opportunities and dedicated
preparations in different scientific fields. One particular demanding application is the homogeneous
deposition onto specific substrates of inorganic components, e.g., coarse particles, mineral fractions,
and particulate matter for spectroscopic characterizations. Research that could benefit from this
particular type of sample preparation are ice core studies or detection of heavy metals present in
ultra-trace elements. 3. Results
achiev The analysis of the images of the droplets (see Figure 2) and of the measured parameters clearly
points out that when using a low concentration solution and a hydrophobic substrate such as Kapton,
the evaporation occurs in accordance to the “constant angle” model, well described by the theoretical
model introduced by Picknett and Bexon in 1977 [2]. The result is confirmed in Figure 3, where our
data are compared to the “constant angle” and “constant contact area” models. The deposition rate, i.e.,
the volume of the liquid deposited vs. time, was optimized considering the velocity of the stages and
the spatial pattern parameters of the deposited droplets. Indeed, a fundamental parameter that drives
the evaporation is the saturation of the atmosphere surrounding the droplets’ selves, which is directly
related to the mutual position of the droplets. Different droplet sizes and different distances between
successive droplets were considered to this aim. In Figure 4, we show the correlated parameters of the
deposition tests we performed using the translation stages combined with the microdrop. We defined
the area to cover (simulating an experimental condition, e.g., the deposition of 1 mL of solution onto
an area of ~35 mm2) and then showed data using the distance between two consecutive drops along
the deposition line, and the diameter of the droplet to be deposed as the two axes. The third axis
represents the evaporation rate density, i.e., the amount that can be deposed on the surface area
(defined above) per unit/area. Data presented in Figure 4 point out that the highest evaporation rate
at 20 ◦C (~0.7 mL/h cm2) is obtained setting the droplet diameter at 240 µm and the distance between
two consecutive droplets at 170 µm. process, all images and experimental parameters were analyzed and used to write an approximate model. 3. Results
The analysis of the images of the droplets (see Figure 2) and of the measured parameters clearly
points out that when using a low concentration solution and a hydrophobic substrate such as Kapton,
the evaporation occurs in accordance to the “constant angle” model, well described by the theoretical
model introduced by Picknett and Bexon in 1977 [2]. The result is confirmed in Figure 3, where our
data are compared to the “constant angle” and “constant contact area” models. The deposition rate,
i.e., the volume of the liquid deposited vs. 3. Results
achiev time, was optimized considering the velocity of the stages
and the spatial pattern parameters of the deposited droplets. Indeed, a fundamental parameter that
drives the evaporation is the saturation of the atmosphere surrounding the droplets’ selves, which is
directly related to the mutual position of the droplets. Different droplet sizes and different distances
between successive droplets were considered to this aim. In Figure 4, we show the correlated
parameters of the deposition tests we performed using the translation stages combined with the
microdrop. We defined the area to cover (simulating an experimental condition, e.g., the deposition
of 1 mL of solution onto an area of ~35 mm2) and then showed data using the distance between two
consecutive drops along the deposition line, and the diameter of the droplet to be deposed as the two
axes. The third axis represents the evaporation rate density, i.e., the amount that can be deposed on
the surface area (defined above) per unit/area. Data presented in Figure 4 point out that the highest
evaporation rate at 20 °C (~0.7 mL/h cm2) is obtained setting the droplet diameter at 240 μm and the
distance between two consecutive droplets at 170 μm. Figure 2. Optical microscope image taken at 20× magnification of the microdrop deposition of
multiple 180 pl droplets with the ink solution deposed on the kapton substrate (left). On the right two
microscope images taken at 5× magnification of a line of distilled water droplets: one at the beginning
of the evaporation (top right) and the second after 10 s (bottom right). Closer drops would determine a higher relative humidity in the air layers which surround the
Figure 2. Optical microscope image taken at 20× magnification of the microdrop deposition of multiple
180 pl droplets with the ink solution deposed on the kapton substrate (left). On the right two microscope
images taken at 5× magnification of a line of distilled water droplets: one at the beginning of the
evaporation (top right) and the second after 10 s (bottom right). Figure 2. Optical microscope image taken at 20× magnification of the microdrop deposition of
multiple 180 pl droplets with the ink solution deposed on the kapton substrate (left). On the right two
microscope images taken at 5× magnification of a line of distilled water droplets: one at the beginning
of the evaporation (top right) and the second after 10 s (bottom right). 2. Experimental We used a solution of ink and water (Gullor® ink
diluted at 50% with high purity milliQ water) for the optical analysis of the deposition, and high purity
milliQ water for the evaporation dynamic measurements, i.e., the diameter of the drop and the angle
of contact. The substrates we used for the evaporation analysis were Kapton® polyimide films from
DuPont of 0.005’ (125 µm) thickness, while we used polycarbonate Nucleopore© membrane filters
(pore size 0.45 µm and filter area diameter ~20 mm) for the analysis of morphology on evaporated
samples morphology by X-Ray Fluorescence. The evaluation of the PM deposition uniformity has been performed using the X-ray fluorescence
technique at the beamline B18 at the Diamond Light Source facility (Harwell, United Kingdom) [10],
using a monochromatic X-ray beam at 8 keV, with a focus of 100 µm radius and a detection limit of
100 ng/mm2. 4 of 9 Condens. Matter 2018, 3, 21 The evaluation of the evaporation was carried out measuring the diameter and height of droplets
as a function of time using the optical microscope and the camera. We measured the parameters of the
single droplets and of groups of droplets to evaluate the evaporation rate as a function of the size and of
the distance between several droplets. We then identified the minimum deposited area achievable with
the evaporation of small droplets, in the shortest time. To optimize the deposition process, all images
and experimental parameters were analyzed and used to write an approximate model. Condens. Matter 2018, 3, x FOR PEER REVIEW
4 of 9 3. Results
achiev Cl
d
ld d t
i
hi h
l ti
h
idit
i
th
i l
hi h
d th
Figure 2. Optical microscope image taken at 20× magnification of the microdrop deposition of multiple
180 pl droplets with the ink solution deposed on the kapton substrate (left). On the right two microscope
images taken at 5× magnification of a line of distilled water droplets: one at the beginning of the
evaporation (top right) and the second after 10 s (bottom right). drops selves, slowing the evaporation process. At variance, depositing drops at larger distance will
limit the amount of deposited liquid actually reducing the integrated efficiency of this process. To
further increase the evaporation rate, experiments conducted heating the substrate were also carried
out. As expected, as temperature increases, the evaporation rate increases exponentially, with
exponential value of the temperature of ~2.5. For example, at 80 °C, the evaporation rate increases
about 30 times [9]. However, it is worth noting that increasing temperature could not be always
feasible, since increasing the temperatures may affect the chemical stability of the samples. To avoid
the problem, thermal radiation focused on droplets may be considered. By selecting specific
Closer drops would determine a higher relative humidity in the air layers, which surround
the drops selves, slowing the evaporation process. At variance, depositing drops at larger distance
will limit the amount of deposited liquid actually reducing the integrated efficiency of this process. To further increase the evaporation rate, experiments conducted heating the substrate were also
carried out. As expected, as temperature increases, the evaporation rate increases exponentially,
with exponential value of the temperature of ~2.5. For example, at 80 ◦C, the evaporation rate 5 of 9 Condens. Matter 2018, 3, 21 increases about 30 times [9]. However, it is worth noting that increasing temperature could not be
always feasible, since increasing the temperatures may affect the chemical stability of the samples. To avoid the problem, thermal radiation focused on droplets may be considered. By selecting specific
wavelengths or frequencies, it would be possible to selectively increase the temperature of the solvent
and not the one of the PM contained in the solution. Another parameter, which could be modified to
fasten the deposition, is the speed of the two motorized stages. 3. Results
achiev Comparison among the droplet mass behavior for the evaporation at “constant contact area”
(blue line), at “constant contact angle” (black line), and experimental data (red squares) of a droplet of
a bi-distilled water solution with an initial diameter of 1 mm, at room temperature. Figure 3. Comparison among the droplet mass behavior for the evaporation at “constant contact area”
(blue line), at “constant contact angle” (black line), and experimental data (red squares) of a droplet
of a bi-distilled water solution with an initial diameter of 1 mm, at room temperature. Figure 4. This graph shows the evaporation rate density (volume evaporated per hour and area) at 20 °C
vs. drop diameter and distance between two consecutive drops. Here, the speed has also been
considered, i.e., the time associated with the motion of the translational stages used to map with
droplets the area to cover. To better understand the deposition pattern of the PM contained in a liquid solution deposited
Figure 4. This graph shows the evaporation rate density (volume evaporated per hour and area) at
20 ◦C vs. drop diameter and distance between two consecutive drops. Here, the speed has also been
considered, i.e., the time associated with the motion of the translational stages used to map with
droplets the area to cover. Figure 3. Comparison among the droplet mass behavior for the evaporation at “constant contact area”
(blue line), at “constant contact angle” (black line), and experimental data (red squares) of a droplet
of a bi-distilled water solution with an initial diameter of 1 mm, at room temperature. Figure 3. Comparison among the droplet mass behavior for the evaporation at “constant contact area”
(blue line), at “constant contact angle” (black line), and experimental data (red squares) of a droplet of
a bi-distilled water solution with an initial diameter of 1 mm, at room temperature. Figure 3. Comparison among the droplet mass behavior for the evaporation at “constant contact area”
(blue line), at “constant contact angle” (black line), and experimental data (red squares) of a droplet
of a bi-distilled water solution with an initial diameter of 1 mm, at room temperature. Figure 4. This graph shows the evaporation rate density (volume evaporated per hour and area) at 20 °C
vs. drop diameter and distance between two consecutive drops. 3. Results
achiev Here, the speed has also been
considered, i.e., the time associated with the motion of the translational stages used to map with
droplets the area to cover. To better understand the deposition pattern of the PM contained in a liquid solution deposited
with a Microdrop dispenser we prepared a suspension consisting in high purity water (MilliQ
Figure 4. This graph shows the evaporation rate density (volume evaporated per hour and area) at 20 °C
vs. drop diameter and distance between two consecutive drops. Here, the speed has also been
considered, i.e., the time associated with the motion of the translational stages used to map with
droplets the area to cover. To better understand the deposition pattern of the PM contained in a liquid solution deposited
Figure 4. This graph shows the evaporation rate density (volume evaporated per hour and area) at
20 ◦C vs. drop diameter and distance between two consecutive drops. Here, the speed has also been
considered, i.e., the time associated with the motion of the translational stages used to map with
droplets the area to cover. vs. drop diameter and distance between two consecutive drops. Here, the speed has also been
considered, i.e., the time associated with the motion of the translational stages used to map with
droplets the area to cover. To better understand the deposition pattern of the PM contained in a liquid solution deposited
h
d
d
d
h
h
(
ll Q
Figure 4. This graph shows the evaporation rate density (volume evaporated per hour and area) at 20 °C
vs. drop diameter and distance between two consecutive drops. Here, the speed has also been
considered, i.e., the time associated with the motion of the translational stages used to map with
droplets the area to cover. T
b tt
d
t
d th d
iti
tt
f th PM
t i
d i
li
id
l ti
d
it d
Figure 4. This graph shows the evaporation rate density (volume evaporated per hour and area) at
20 ◦C vs. drop diameter and distance between two consecutive drops. Here, the speed has also been
considered, i.e., the time associated with the motion of the translational stages used to map with
droplets the area to cover. technology) and a given amount (50 μg/mL) of a reference material: the NIST standard 2709a. 3. Results
achiev Our experiments were all conducted at
the maximum allowed speed (i.e., 13 mm·s−1), but faster motorized stages exist that would cover the
same area within a shorter time, i.e., just reducing the deposition time between two consecutive drops. Condens. Matter 2018, 3, x FOR PEER REVIEW
5 of 9
at the maximum allowed speed (i.e., 13 mm·s−1), but faster motorized stages exist that would cover the
same area within a shorter time, i.e., just reducing the deposition time between two consecutive drops. Condens. Matter 2018, 3, x FOR PEER REVIEW
5 of 9
at the maximum allowed speed (i.e., 13 mm·s−1), but faster motorized stages exist that would cover the
same area within a shorter time, i.e., just reducing the deposition time between two consecutive drops. Figure 3. Comparison among the droplet mass behavior for the evaporation at “constant contact area”
(blue line), at “constant contact angle” (black line), and experimental data (red squares) of a droplet
of a bi-distilled water solution with an initial diameter of 1 mm, at room temperature. Figure 3. Comparison among the droplet mass behavior for the evaporation at “constant contact area”
(blue line), at “constant contact angle” (black line), and experimental data (red squares) of a droplet of
a bi-distilled water solution with an initial diameter of 1 mm, at room temperature. Figure 3. Comparison among the droplet mass behavior for the evaporation at “constant contact area”
(blue line), at “constant contact angle” (black line), and experimental data (red squares) of a droplet
of a bi-distilled water solution with an initial diameter of 1 mm, at room temperature. Figure 3. Comparison among the droplet mass behavior for the evaporation at “constant contact area”
(blue line), at “constant contact angle” (black line), and experimental data (red squares) of a droplet
of a bi-distilled water solution with an initial diameter of 1 mm, at room temperature. Figure 4. This graph shows the evaporation rate density (volume evaporated per hour and area) at 20 °C
vs. drop diameter and distance between two consecutive drops. Here, the speed has also been
considered, i.e., the time associated with the motion of the translational stages used to map with
droplets the area to cover. To better understand the deposition pattern of the PM contained in a liquid solution deposited
with a Microdrop dispenser we prepared a suspension consisting in high purity water (MilliQ
Figure 3. 3. Results
achiev The
latter is a well-known soil reference material with a well-characterized composition [11] and grain size
distribution. To avoid the clog of the Microdrop nozzle, we selected a reference material whose particles
didn’t exceed 30 μm, i.e., about the half of the aperture of the nozzle orifice, as shown in Figure 5. with a Microdrop dispenser, we prepared a suspension consisting in high purity water (MilliQ
technology) and a given amount (50 μg/mL) of a reference material: the NIST standard 2709a. The
latter is a well-known soil reference material with a well-characterized composition [11] and grain size
distribution. To avoid the clog of the Microdrop nozzle, we selected a reference material whose particles
didn’t exceed 30 μm, i.e., about the half of the aperture of the nozzle orifice, as shown in Figure 5. To better understand the deposition pattern of the PM contained in a liquid solution deposited
with a Microdrop dispenser, we prepared a suspension consisting in high purity water (MilliQ
technology) and a given amount (50 µg/mL) of a reference material: the NIST standard 2709a. The latter is a well-known soil reference material with a well-characterized composition [11] and
grain size distribution. To avoid the clog of the Microdrop nozzle, we selected a reference material 6 of 9 Condens. Matter 2018, 3, 21 whose particles didn’t exceed 30 µm, i.e., about the half of the aperture of the nozzle orifice, as shown
in Figure 5. Condens. Matter 2018, 3, x FOR PEER REVIEW
6 of 9 Figure 5. The particle size distribution of the NIST standard reference soil material 2709a. Data were
obtained through the Coulter counter technique. Details can be found in [12]. Si
th
t
d
d
t i
F
it
ibl t
th X
fl
(XRF) t
h i
t
Figure 5. The particle size distribution of the NIST standard reference soil material 2709a. Data were
obtained through the Coulter counter technique. Details can be found in [12]. Condens. Matter 2018, 3, x FOR PEER REVIEW
6 of 9 Figure 5. The particle size distribution of the NIST standard reference soil material 2709a. Data were
obtained through the Coulter counter technique. Details can be found in [12]. S
h
d
d
bl
h
fl
h
Figure 5. The particle size distribution of the NIST standard reference soil material 2709a. Data were
obtained through the Coulter counter technique. Details can be found in [12]. 3. Results
achiev investigate its spatial distribution after deposition. In Figure 6, we compare two XRF profiles collected
on two deposited samples, measured along a straight line, with a spatial resolution limited by the
spot size of the beam (~200 μm). The red curve is the profile relative to the deposition of a single large
drop of ~1 mL volume of the NIST solution. The black curve refers to a deposition obtained with the
microdrop, made by multiple patterns of 240 μm diameter droplets with 170 μm between them. The
same total volume and the same solution were considered. The two profiles show significant
differences and it is remarkable that the homogeneous deposition was obtained with the latter
technique, despite the reduced amount of solution. Indeed, the large drop shows density fluctuations
from 50% to 80% of the fluorescence intensity, in particular at the edge due to the coffee-stain effect [1]. At
variance, the variability observed along the microdrop profile is around 10%, and the deposited soil
is uniformly distributed. Since the standard contains Fe, it was possible to use the X-ray fluorescence (XRF) technique
to investigate its spatial distribution after deposition. In Figure 6, we compare two XRF profiles
collected on two deposited samples, measured along a straight line, with a spatial resolution limited
by the spot size of the beam (~200 µm). The red curve is the profile relative to the deposition of
a single large drop of ~1 mL volume of the NIST solution. The black curve refers to a deposition
obtained with the microdrop, made by multiple patterns of 240 µm diameter droplets with 170 µm
between them. The same total volume and the same solution were considered. The two profiles
show significant differences and it is remarkable that the homogeneous deposition was obtained
with the latter technique, despite the reduced amount of solution. Indeed, the large drop shows
density fluctuations from 50% to 80% of the fluorescence intensity, in particular at the edge due to the
coffee-stain effect [1]. At variance, the variability observed along the microdrop profile is around 10%,
and the deposited soil is uniformly distributed. Figure 5. The particle size distribution of the NIST standard reference soil material 2709a. Data were
obtained through the Coulter counter technique. Details can be found in [12]. Since the standard contains Fe, it was possible to use the X-ray fluorescence (XRF) technique to
investigate its spatial distribution after deposition. 3. Results
achiev In Figure 6, we compare two XRF profiles collected
on two deposited samples, measured along a straight line, with a spatial resolution limited by the
spot size of the beam (~200 μm). The red curve is the profile relative to the deposition of a single large
drop of ~1 mL volume of the NIST solution. The black curve refers to a deposition obtained with the
microdrop, made by multiple patterns of 240 μm diameter droplets with 170 μm between them. The
same total volume and the same solution were considered. The two profiles show significant
differences and it is remarkable that the homogeneous deposition was obtained with the latter
technique, despite the reduced amount of solution. Indeed, the large drop shows density fluctuations
from 50% to 80% of the fluorescence intensity, in particular at the edge due to the coffee-stain effect [1]. At
variance, the variability observed along the microdrop profile is around 10%, and the deposited soil
is uniformly distributed. Figure 6. Comparison of two representative X-ray fluorescence profiles at the Fe K-edge: microdrop
5 × 7 mm2 pattern with drops of 2.9 μL (240 μm diameter—black line) and a single large drop
deposition (red line). Both samples have been obtained with a total volume deposition of 1 ± 0.1 mL
of a NIST solution 50 μg/mL concentration on a kapton film. Figure 6 compares a microdrop deposition, which clearly shows the homogeneity and the
overall quality of the samples prepared with the microdrop increase. The only disadvantage of this
Figure 6. Comparison of two representative X-ray fluorescence profiles at the Fe K-edge: microdrop
5 × 7 mm2 pattern with drops of 2.9 μL (240 μm diameter—black line) and a single large drop
deposition (red line). Both samples have been obtained with a total volume deposition of 1 ± 0.1 mL
of a NIST solution 50 μg/mL concentration on a kapton film. Figure 6 compares a microdrop deposition, which clearly shows the homogeneity and the
Figure 6. Comparison of two representative X-ray fluorescence profiles at the Fe K-edge: microdrop
5 × 7 mm2 pattern with drops of 2.9 µL (240 µm diameter—black line) and a single large drop
deposition (red line). Both samples have been obtained with a total volume deposition of 1 ± 0.1 mL of
a NIST solution 50 µg/mL concentration on a kapton film. 3. Results
achiev arison of two representative X-ray fluorescence profiles at the Fe K-
ern with drops of 2.9 μL (240 μm diameter—black line) and a s
ine). Both samples have been obtained with a total volume deposit of a NIST solution 50 μg/mL concentration on a kapton film. Figure 6 compares a microdrop deposition, which clearly shows the homogeneity and the
overall quality of the samples prepared with the microdrop increase. The only disadvantage of this
Figure 6. Comparison of two representative X-ray fluorescence profiles at the Fe K-edge: microdrop
5 × 7 mm2 pattern with drops of 2.9 μL (240 μm diameter—black line) and a single large drop
deposition (red line). Both samples have been obtained with a total volume deposition of 1 ± 0.1 mL
of a NIST solution 50 μg/mL concentration on a kapton film. Figure 6. Comparison of two representative X-ray fluorescence profiles at the Fe K-edge: microdrop
5 × 7 mm2 pattern with drops of 2.9 µL (240 µm diameter—black line) and a single large drop
deposition (red line). Both samples have been obtained with a total volume deposition of 1 ± 0.1 mL of
a NIST solution 50 µg/mL concentration on a kapton film. 7 of 9 Condens. Matter 2018, 3, 21 Figure 6 compares a microdrop deposition, which clearly shows the homogeneity and the overall
quality of the samples prepared with the microdrop increase. The only disadvantage of this method
is the deposition time. Indeed, as it can be inferred from Figure 4, the optimal configuration of this
micro-deposition allows us to deposit and to evaporate liquid samples with a rate of less than 1 mL
per hour. A simple and alternative way to reduce the deposition time can be obtained by combining
the microdrop deposition with a simultaneous filtering technique. In this way, the droplet is deposited
onto a wettable filter, and the liquid is pumped out by a vacuum pump. Condens. Matter 2018, 3, x FOR PEER REVIEW
7 of 9
method is the deposition time. Indeed, as it can be inferred from Figure 4, the optimal configuration
of this micro-deposition allows us to deposit and to evaporate liquid samples with a rate of less than
1 mL per hour. 3. Results
achiev Image of the X-ray Fluorescence at the Fe K-edge of the microdrop deposition area on the
filter (left); comparison of two XRF profiles (right): the microdrop deposition (black) and the pipette
deposition (blue) both achieved with a simultaneous vacuum filtration technique. Figure 7. Image of the X-ray Fluorescence at the Fe K-edge of the microdrop deposition area on the
filter (left); comparison of two XRF profiles (right): the microdrop deposition (black) and the pipette
deposition (blue) both achieved with a simultaneous vacuum filtration technique. In Figure 7 (right), one microdrop deposition profile of the image on the left is compared with
an analogous profile of the deposition of the same solution using a micro-pipette on the same type of
filter. From the analysis of the data, we may point out that by using the microdrop deposition, ~80%
of the material is distributed quite homogeneously (with a fluorescence intensity fluctuation of ~15%)
inside an area of ~8 mm2. Using the pipette deposition, the same amount of material is deposited
within an area six times larger (~50 mm2). A simple evaluation points out that the microdrop filtration
procedure covers an area 6.25 times smaller than the area wetted with the pipette, but the intensity
of the fluorescence signal, proportional to the deposed material is four times higher. In this case, the
total gain we achieved is ~25. 4. Conclusions
In Figure 7 (right), one microdrop deposition profile of the image on the left is compared with
an analogous profile of the deposition of the same solution using a micro-pipette on the same type of
filter. From the analysis of the data, we may point out that by using the microdrop deposition, ~80% of
the material is distributed quite homogeneously (with a fluorescence intensity fluctuation of ~15%)
inside an area of ~8 mm2. Using the pipette deposition, the same amount of material is deposited
within an area six times larger (~50 mm2). A simple evaluation points out that the microdrop filtration
procedure covers an area 6.25 times smaller than the area wetted with the pipette, but the intensity of
the fluorescence signal, proportional to the deposed material is four times higher. In this case, the total
gain we achieved is ~ 25. 3. Results
achiev A simple and alternative way to reduce the deposition time can be obtained by
combining the microdrop deposition with a simultaneous filtering technique In this way the droplet In Figure 7 (left), the 2 × 5 mm2 image shows a portion of the original 4 × 4 mm2 microdrop
deposition area of 1 mL NIST solution (50 µg/mL concentration) on a polycarbonate Nucleopore©
membrane filter (pore size 0.45 µm and filter area diameter ~20 mm), which is a hydrophilic substrate. This type of substrate absorbs the liquid solution, and the liquid is continuously pumped out through
the filter. The color map was obtained collecting the XRF signal at the Fe K-edge. Colors highlight an
area of approximately 2 × 3 mm2 where a controlled distribution is achieved. The internal variability
is probably also related to the spatial pattern of the pores characterizing the filtration membrane. combining the microdrop deposition with a simultaneous filtering technique. In this way, the droplet
is deposited onto a wettable filter, and the liquid is pumped out by a vacuum pump. In Figure 7 (left), the 2 × 5 mm2 image shows a portion of the original 4 × 4 mm2 microdrop
deposition area of 1 mL NIST solution (50 μg/mL concentration) on a polycarbonate Nucleopore©
membrane filter (pore size 0.45 μm and filter area diameter ~20 mm), which is a hydrophilic substrate. This type of substrate absorbs the liquid solution, and the liquid is continuously pumped out through
the filter. The color map was obtained collecting the XRF signal at the Fe K-edge. Colors highlight an
area of approximately 2 × 3 mm2 where a controlled distribution is achieved. The internal variability
is probably also related to the spatial pattern of the pores characterizing the filtration membrane Figure 7. Image of the X-ray Fluorescence at the Fe K-edge of the microdrop deposition area on the
filter (left); comparison of two XRF profiles (right): the microdrop deposition (black) and the pipette
deposition (blue) both achieved with a simultaneous vacuum filtration technique. Figure 7. Image of the X-ray Fluorescence at the Fe K-edge of the microdrop deposition area on the
filter (left); comparison of two XRF profiles (right): the microdrop deposition (black) and the pipette
deposition (blue) both achieved with a simultaneous vacuum filtration technique. Figure 7. The micr
granular matte
4. Conclusions it is necessary to measure accurately the evaporation rate of the pattern depositions. We show here
how to optimize such parameters, increasing the S/N ratio and the deposition homogeneity, reducing
at the same time the sample consumption. Indeed, S/N ratio is related to the XRF signal; increasing
the density and the homogeneity of the deposition, the S/N ratio of the XRF signal that outlines the
distribution of the material in the sample improves. In terms of deposition uniformity, with the
microdrop technique, the S/N ratio may improve ~4 times with respect to a classical filtration method. At the same time, it is possible to deposit and evaporate a solution onto any substrate and 5 times
more homogeneously than a large drop evaporation. These features make this technique effective
and competitive with respect to many and diverse applications. Among the many, we cite the
The microdrop technology is suitable to prepare homogeneous deposition of particulate and
granular matter samples. In order to establish a reliable procedure, reducing also the deposition
time, it is necessary to measure accurately the evaporation rate of the pattern depositions. We show
here how to optimize such parameters, increasing the S/N ratio and the deposition homogeneity,
reducing at the same time the sample consumption. Indeed, S/N ratio is related to the XRF signal;
increasing the density and the homogeneity of the deposition, the S/N ratio of the XRF signal that
outlines the distribution of the material in the sample improves. In terms of deposition uniformity,
with the microdrop technique, the S/N ratio may improve ~4 times with respect to a classical filtration
method. At the same time, it is possible to deposit and evaporate a solution onto any substrate and 8 of 9 Condens. Matter 2018, 3, 21 5 times more homogeneously than a large drop evaporation. These features make this technique
effective and competitive with respect to many and diverse applications. Among the many, we cite
the characterization of aerosols for pollution monitoring purposes, studies of metallic contaminants
in polluted water or the investigation of biological diluted materials at ultra-trace concentrations. Besides homogeneity, another important feature of the method proposed here, is the possibility
to concentrate in a very reduced area the material contained in an extremely diluted solution or
suspension. This makes it possible to consider impurities whose concentrations span from few ppb
to hundreds ppm. The micr
granular matte
4. Conclusions As an example, the method is suited for the characterization of the inorganic
insoluble particulate matter contained in ice core samples for paleoclimatic reconstructions. Ice core
dust samples prepared with a microdrop device could be successfully used for both X-rays absorption
and X-ray fluorescence measurements [13,14] and under particular experimental conditions also for
XRD experiments [15]. Certainly, the microdrop technique has to be improved for applications where
beams with spot size well below one micron will be available, as to ultra low brilliance synchrotron
radiation facilities and/or Free Electron Lasers. However, due to the low frequency rate of the existing
Free Electron Lasers (10–100 Hz), the technique could already be useful to control the deposition of
small samples such as small crystals, clusters, or organic systems delivered to the beam inside droplets. Author Contributions: S.M., G.C., V.M. and A.M. conceived and designed the experiment; D.H. and S.M. built the
experimental setup; S.M. and G.C. performed the experiments and analyzed data; G.B., B.D. and A.D. provided
materials and contributed to the data analysis; S.M., G.C., and A.M. wrote the paper. All authors have read and
approved the final manuscript. Funding: This research has been performed in cooperation among INFN-Laboratori Nazionali di Frascati, Milano
Bicocca University and the Diamond Light Source facility. The development of the microdrop instrumentation is a
project managed by A.M. with the support of DARST—Department of the Presidenza del Consiglio dei Ministri,
which is gratefully acknowledged. Part of the preparation was done in the EuroCold Laboratory at University
of Milano Bicocca, founded by Italian National Science Foundation NextData Project. Part of this research was
developed at Diamond, the UK national synchrotron radiation facility. One of us (S.M.) acknowledges the Roma
Tre University for financial support during his stage at Diamond. Conflicts of Interest: The authors declare no conflict of interest. References 1. Innocenzi, P.; Malfatti, L.; Piccinini, M.; Grosso, D.; Marcelli, A. Stain Effects Studied by Time-Resolved
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Meyer W Mikroverklebungen aus dem Tintenstrahldrucker PLUS; Eugen G Ed ; Leuze Verlag: Norderstedt 7. Sparks, C.M.; Fittschen, U.E.A.; Havrilla, G.J. Picoliter solution deposition for total reflection X-ray
fluorescence analysis of semiconductor samples. Spectrochim. Acta Part B 2010, 65, 805–811. [CrossRef] 8. Meyer, W. Mikroverklebungen aus dem Tintenstrahldrucker PLUS; Eugen, G., Ed.; Leuze Verlag: Nord
Germany, 2013; pp. 593–598. References (In German) 9. Macis, S. Preparation and Characterization of Ultra-Diluted Samples via Micro-Deposition of Droplets. Master’s Thesis, Roma Tre University, Roma, Italy, 2014. 9 of 9 Condens. Matter 2018, 3, 21 10. Dent, J.; Cibin, G.; Ramos, S.; Parry, S.A.; Gianolio, D.; Smith, A.D.; Scott, S.M.; Varandas, L.; Patel, S.;
Pearson, M.R.; et al. Performance of B18, the Core EXAFS Bending Magnet beamline at Diamond. J. Phys. Conf. Ser. 2013, 430, 012023. [CrossRef] 11. Mackey, E.A.; Christopher, S.J.; Lindstrom, R.M.; Long, S.E.; Marlow, A.F.; Murphy, K.E.; Paul, R.L.;
Popelka-Filcoff, R.S.; Rabb, S.A.; Sieber, J.R.; et al. Nebelsick, Certification of Three NIST Renewal Soil Standard
Reference Materials for Element Content: SRM 2709a San Joaquin Soil, SRM 2710a Montana Soil I, and SRM
2711a Montana Soil II; NIST Special Publication 260-172; National Institute of Standards and Technology:
Gaithersburg, MD, USA, 2010; p. 39. 12. Ruth, U.; Barbante, C.; Bigler, M.; Delmonte, B.; Fischer, H.; Gabrielli, P.; Gaspari, V.; Kaufmann, P.;
Lambert, F.; Maggi, V.; et al. Proxies and Measurement Techniques for Mineral Dust in Antarctic Ice
Cores. Environ. Sci. Technol. 2008, 42, 5675–5681. [CrossRef] [PubMed] 13. Marcelli, A.; Cibin, G.; Hampai, D.; Giannone, F.; Sala, M.; Pignotti, S.; Maggi, V.; Marino, F. XANES
characterization of deep ice core insoluble dust in the ppb range. J. Anal. At. Spectrom. 2012, 27, 33–37. [CrossRef] 14. Marcelli, A.; Maggi, V. Aerosols in Snow and Ice. Markers of Environmental Pollution and Climatic Changes:
European and Asian Perspectives; Superstripes Press: Rome, Italy, 2017; ISBN 9788866830771. 15. D’Elia, A.; Cibin, G.; Robbins, P.E.; Maggi, V.; Marcelli, A. Design and characterization of a mapping
device optimized to collect XRD patterns from highly inhomogeneous and low density powder samples. Nucl. Instrum. Methods Phys. Res. B 2017, 411, 22–28. [CrossRef] © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
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Use of Bladder Filling to Prevent Urinary System Complications in the Management of Placenta Percreta: a Randomized Prospective Study
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GebFra Science | Original Article
Use of Bladder Filling to Prevent Urinary System Complications in the
Management of Placenta Percreta: a Randomized Prospective Study
Blasenfüllung zur Verhinderung von Komplikationen
des Urogenitalsystems in Frauen mit Placenta percreta:
eine randomisierte prospektive Studie
Authors
Hüseyin Çağlayan Özcan 1, Özcan Balat 1, Mete Gurol Uğur 1, Seyhun Sucu 1, Neslihan Bayramoğlu Tepe 1,
Tanyeli Güneyligil Kazaz 2
Affiliations
1 Gaziantep University, School of Medicine, Dept. of
Obstetrics and Gynecology, Gaziantep, Turkey
2 Gaziantep University, School of Medicine, Dept. of
Biostatistics, Gaziantep, Turkey
Key words
bladder injury, placental implantation abnormality,
placenta percreta, urinary system injury
Schlüsselwörter
Blasenverletzung, plazentare Implantationsstörung,
Placenta percreta, Verletzung des Urogenitalsystems
received
revised
accepted
29. 9. 2017
27. 12. 2017
28. 12. 2017
Bibliography
DOI https://doi.org/10.1055/s-0044-100039
Geburtsh Frauenheilk 2018; 78: 173–178 © Georg Thieme
Verlag KG Stuttgart · New York | ISSN 0016‑5751
Obstetrics and Gynecology of Gaziantep University Hospital
between January 2015 and July 2016. Bladders were filled
with 300 ml saline solution to determine surgical borders better and enable dissection of the lower uterine segment without excessive bleeding or unintended injury.
Results A total of 66 women were included in the study: 32
women whose bladders were filled during surgery (filled-bladder group) and 34 women whose bladders were not filled (not
filled-bladder group). Comparisons of demographic and obstetrical data, surgical parameters, the need for transfusion, and
bladder injury rates revealed no significant differences between
the two groups. We did not observe any beneficial effect of filling the bladder on preventing urinary complications compared
with the women whose bladders were not filled (p = 0.339).
Conclusions Filling the bladder with saline solution and mobilization of the bladder from the lower uterine segment did not
have a statistically significant beneficial effect on preventing
complications of the genitourinary system. But although the
beneficial effects were not significant, shorter operation times,
shorter postoperative hospital stays, and fewer bladder injuries
were noted in patients whose bladders were filled.
ZU SAM ME N FA SS UN G
Correspondence
Hüseyin Çağlayan Özcan, MD, Assist. Prof.
Gaziantep University, Faculty of Medicine,
Department of Obstetrics and Gynecology, Sahinbey
En route to Kilis, 27800 Gaziantep, Turkey
ozcan.caglayan8@hotmail.com
AB STR AC T
Introduction The aim of our study was to evaluate the effect
of filling the bladder on peripartum genitourinary injuries (especially bladder complications) in women with placenta percreta and to compare patient characteristics.
Material and Methods Our prospective cohort study consisted of pregnant women with placenta percreta who underwent planned cesarean hysterectomy at the Department of
Özcan HÇ et al. Use of Bladder …
Geburtsh Frauenheilk 2018; 78: 173–178
Einleitung Ziel dieser Studie war es, die Auswirkungen einer
Blasenfüllung auf peripartale Verletzungen des Urogenitalsystems (insbesondere auf Blasenverletzungen) bei Frauen mit
Placenta percreta zu untersuchen und die Patientendaten zu
vergleichen.
Material und Methoden Unsere prospektive Kohortenstudie untersuchte Frauen mit Placenta percreta, die sich zwischen Januar 2015 und Juli 2016 einer geplanten Hysterektomie nach erfolgter Sectio caesarea in der Abteilung für Geburtshilfe und Gynäkologie des Gaziantep Universitätsklinikums unterzogen. Die Harnblase wurde jeweils mit 300 ml
Kochsalzlösung gefüllt, um sie während des chirurgischen Eingriffs besser abzugrenzen und eine Dissektion des unteren
Teils der Gebärmutter ohne übermäßigen Blutverlust oder
ungewollte Verletzung zu ermöglichen.
173
GebFra Science | Original Article
Ergebnisse Es wurden insgesamt 66 Frauen in die Studie eingeschlossen; bei 32 Frauen wurde die Harnblase vor dem Eingriff gefüllt (gefüllte-Blase-Gruppe) und bei 34 Frauen wurde
die Harnblase nicht gefüllt (nicht-gefüllte-Blase-Gruppe). Ein
Vergleich der demografischen, gynäkologischen und chirurgischen Daten sowie der Menge an Bluttransfusionen und Harnverletzungen ergab keine wesentlichen Unterschiede zwischen den beiden Gruppen. Die Füllung der Harnblase hatte
keine Auswirkungen auf die Verhinderung von Komplikationen des Urogenitalsystems verglichen mit den Frauen, bei de-
Introduction
Placenta percreta is a placental adhesive disorder defined by abnormal trophoblastic invasion through the uterine serosa layer
and potentially beyond [1]. The reported incidence is approximately 5–7 % of pregnancies with placental adhesive disorders
[2]. Increased cesarean section (CS) rates may be one reason for
this high incidence. Previous cesarean delivery is the most important risk factor, followed by placenta previa and advanced maternal age [3]. Antenatal diagnosis is helpful to reduce intraoperative
urologic complications and the need for blood transfusions [4].
Sonographic and color Doppler examination is generally accepted
as the first choice of imaging method for the diagnosis of
placental adhesive disorders [5]. In addition to the risk of severe
hemorrhage, there is also a risk of unintentional urinary tract
(UT) injury, which may result in urinary fistula, ureteral transection, and bladder laceration necessitating partial or total cystectomy [6]. To date, there is no prospective randomized study
in the English literature on the effect of filling the bladder during
the surgical management of placenta percreta for the prevention
of genitourinary system complications.
nen die Harnblase vor dem Eingriff nicht gefüllt wurde
(p = 0,339).
Schlussfolgerungen Das Füllen der Harnblase mit Kochsalzlösung und die Mobilisierung der Blase von dem unteren Teil
der Gebärmutter aus hatte keine statistisch signifikante positive Auswirkung auf die Verhinderung von Komplikationen
des Urogenitalsystems. Aber obwohl die positiven Auswirkungen nicht signifikant waren, waren die Operationszeiten und
der postoperative Krankenhausaufenthalt kürzer und es gab
weniger Harnblasenverletzungen in der Gruppe mit gefüllter
Harnblase.
The aim of our study was to evaluate the effect of filling the
bladder on peripartum genitourinary injuries (especially bladder
complications) in women with placenta percreta who underwent
planned cesarean hysterectomy and to compare patient characteristics.
Patients and Methods
Study design
This prospective cohort study included pregnant women with
placenta percreta who underwent CS at the Department of Obstetrics and Gynecology of Gaziantep University Hospital between
January 2015 and August 2017. All women included in the study
gave their written informed consent.
Diagnostic features of Doppler ultrasonography
Patients were diagnosed by Doppler ultrasonography (▶ Fig. 1) in
the antenatal period, and the diagnosis was confirmed during surgery performed by the same clinicians. Turbulent flow, the presence of lacunae, increased vascularity at the bladder–placenta
▶ Fig. 1 a Ultrasonographic image of placenta percreta in a 35-week-old pregnancy. Lacunar structures are indicated by arrows. Note the thin
border between uterus and bladder (pl = placenta, cx = cervix, bl = bladder); b Doppler ultrasonography image of the same patient with increased
vascularity at the vesicouterine junction and lacunar structures.
174
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Geburtsh Frauenheilk 2018; 78: 173–178
border, and damage to myometrial integrity are the standard
diagnostic criteria in ultrasonography (▶ Fig. 1).
Surgical characteristics
All specimens obtained were sent for pathological examination.
Placenta percreta is defined as the penetration of trophoblastic
tissue into all layers of the uterus. All operations were performed
under general anesthesia in the dorsal lithotomy position. We included only cases with placenta percreta in whom we performed
planned cesarean hysterectomy between the 34th and 36th week
of gestation. The decision of the surgeons regarding the appropriate surgical approach for cesarean hysterectomy depended on the
severity of disease. Their assessment was based on the presence
of increased vascularity in the lower uterine segment/bladder
zone and the extent of invasion of the uterine artery.
Bladder filling as a method to determine
the vesicouterine border
Patients were randomized either into the filled-bladder or the not
filled-bladder group. The first step in all surgeries was a midline
periumbilical incision. The infant was delivered through a fundal
vertical uterine incision. The bladder was filled with 300 ml saline
(this surgical technique was first defined by the head gynecologic
oncologist Prof. Dr. Ozcan Balat) to help determine the borders
better and enable dissection of the lower uterine segment without excessive bleeding or unintended injury (▶ Fig. 2). At the end
of the surgical procedure we filled the bladder of all patients with
diluted methylene blue to check for the presence of any missed
bladder injuries.
▶ Fig. 2 Intraoperative image of placenta percreta in which the
bladder was filled with 300 ml saline (asterisk, arrows, and plus sign
indicate the uterus, vesicouterine border and bladder, respectively).
Multidisciplinary approach to surgical management
Bladder complications without ureter involvement were treated
by the obstetrician alone or a consulting urologist. A urologist
was consulted when patients experienced complications that included ureter involvement. All patients with bladder injuries received antibiotic prophylaxis (cefazolin sodium 1 g 2 × 1 i. v.). All
patients were managed by a multidisciplinary approach which included an obstetrician, a gynecological oncologist, an anesthetist,
an intensive care physician, and a urologist. The same surgical
team carried out all operations. The surgical team that participated in this study has the skills to treat the retroperitoneal space,
bladder, and ureter, and are experienced in the surgical management of placenta percreta. We planned to prospectively include
women into the study for 20 months. The Institutional Ethics
Committee approved our study.
Statistical analysis
Normality of distribution of continuous variables was tested with
the Shapiro-Wilk test. Studentʼs t-test was used to compare two
independent groups of normally distributed variables. χ2 test was
used to assess the relationships between categorical variables.
Statistical analysis was performed using SPSS for Windows, version 11.5®. A p-value < 0.05 was considered statistically significant.
Özcan HÇ et al. Use of Bladder …
Geburtsh Frauenheilk 2018; 78: 173–178
Results
Patient characteristics
A total of 66 women were included in the study: 32 women whose
bladders were filled with saline before surgery (filled-bladder
group) and 34 women whose bladders were not filled before surgery (not filled-bladder group). The mean age of all women was
32.62 ± 4.49 years (range: 23–41 years). Mean body mass index
(kg/m2) was 28.38 ± 1.52 (range: 25–31). Parity, gravidity and
gestational age (weeks) at surgery were 3.41 ± 1.12 (range: 1–7),
4.86 ± 1.65 (range: 2–10) and 35.35 ± 3.27 (range: 22–40), respectively. Six women (9.1 %) had had 2 previous cesarean sections and 60 women (90.9 %) had had > 3 previous CS.
Operative outcomes
Mean preoperative and postoperative hemoglobin levels of women were 10.91 ± 1.63 g/dl (range: 5.2–13.7) and 10.47 ± 1.69 g/dl
(range: 2.4–13.8), respectively. Erythrocyte suspensions and fresh
frozen plasma replacements of 2.97 ± 1.93 units (range: 0–8) and
2.58 ± 1.82 units (range: 0–7) were transfused, respectively. The
mean operation time was 91.36 ± 33.78 minutes (range: 60–
210). The mean length of hospital stay for all women was
5.09 ± 4.22 days (range: 1–19).
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GebFra Science | Original Article
▶ Table 1 Comparison of patient groups with filled and unfilled bladders.
Filled bladder (n = 32)
Not filled bladder (n = 34)
p
▪ Age (years)*
32.31 ± 4.25
32.91 ± 4.74
0.591
▪ BMI (kg/m2)*
28.28 ± 1.53
28.47 ± 1.52
0.616
Maternal characteristics
▪ Gravidity (n)*
4.66 ± 1.15
5.06 ± 1.69
0.265
▪ Parity (n)*
3.34 ± 0.97
3.47 ± 1.26
0.650
▪ Previous cesarean section (n)*
3.25 ± 0.8
3.15 ± 0.66
0.570
▪ Gestational age (weeks)*
35.5 ± 2.72
35.47 ± 4.06
0.973
▪ Operation time (minutes)*
85.31 ± 35.01
97.06 ± 32.05
0.160
Postoperative stay in hospital (days)*
4.97 ± 3.86
5.21 ± 4.59
0.822
ICU admission, n (%)
8 (25)
8 (23.5)
0.889
Blood transfusion (units)*
▪ Erythrocytes
2.97 ± 2.09
2.97 ± 1.82
0.997
▪ Fresh frozen plasma
2.47 ± 1.83
2.68 ± 1.84
0.647
Bladder injury, n (%)
7 (21.9)
11 (32.4)
0.339
* mean ± SD
Influence of bladder filling on outcomes
Comparisons of demographic, obstetrical and surgical data, the
need for transfusion and bladder injury rates for the two groups
(filled-bladder group vs. not filled-bladder group) revealed no significant differences. Data are shown in ▶ Table 1. We did not observe any effect of filling the bladder on intraoperative or postoperative complications when compared with cases whose bladders were not filled (p = 0.339). The complications reported for
the women in our study, listed from the most common to the
least common, were bladder injury (69.2 %), pelvic hematoma
leading to re-laparotomy (15.3 %), ureteral injury (3.8 %), external
iliac vein injury (3.8 %), pelvic hematoma follow-up (3.8 %), and
ileus (3.8 %) (▶ Table 2).
Discussion
The incidence of placental invasion abnormalities is increasing and
presents surgical and diagnostic challenges for obstetricians. Our
hospital is the biggest regional tertiary referral center for complicated obstetrical surgery in southeastern Turkey. The high rate of
cases with placenta percreta can therefore be explained by the
high rates of referral. A history of multiple CS or placenta previa
increases the risk of placental invasion abnormalities [7]. A high
number (> 3) of previous cesarean sections is an obvious risk factor for complications; in addition, the complication rate for patients with placenta percreta is much higher due to deep placental
and vascular infiltration.
We did not detect any significant difference in the number of
previous CS on the presence or absence of complications. Given
the high morbidity and mortality rates associated with placental
adhesive disorders, antenatal diagnosis improves the outcome
[8]. The sensitivity and specificity for a diagnosis of placental invasion abnormalities diagnosed using ultrasound is 80–90 % in spe-
176
▶ Table 2 Number and percentage of intraoperative complications.
Filled
bladder (%)
Not filled
bladder (%)
▪ Bladder injury
7 (21.9)
11 (32.4)
▪ Ureteral injury
1 (3.1)
0
▪ External iliac vein injury
0
1 (2.9)
▪ Pelvic hematoma on
follow-up
1 (3.1)
0
▪ Pelvic hematoma leading
to re-laparatomy
2 (6.3)
2 (5.9)
▪ Ileus
0
1 (2.9)
Intraoperative complication
Postoperative complication
cialized centers [9], but these rates are likely to be much lower for
standard providers of obstetrical care. In our study, we did not include the records of patients with a false-positive diagnosis of
placenta percreta based on antenatal ultrasonography examination. Analysis of the sensitivity and specificity of ultrasonography
is therefore beyond the scope of our study. Poor access to ultrasound and limited experience on the part of the obstetrician are
factors reducing the likelihood of making an antenatal diagnosis.
Turbulent flow and the presence of lacunae, increased vascularity
at the bladder-placenta border, and damage to myometrial integrity are the standard diagnostic criteria in ultrasonography [10]. A
planned hysterectomy carried out by an experienced team is the
recommended procedure for the management of placental adhesive disorders [11]. Segment resection of the lower uterine seg-
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Geburtsh Frauenheilk 2018; 78: 173–178
ment, hysterectomy, or leaving the placenta in situ to be resorbed
are alternative approaches for the management of placental invasion anomalies [12]. In most patients with placenta percreta aggressive treatment is a must. Massive blood transfusions, urological injuries, and infections are major issues in this complicated
treatment [13].
Massive and/or persistent hemorrhage, bladder injury, ureteral
damage, and bowel injury are the most common complications
resulting in admission to the intensive care unit [14]. Eller et al.,
reporting on 76 cases, found that blood transfusion was required
in more than 80 % of cases [15]. This rate is comparable with the
figures in our study (91 %). Maternal and prenatal fetal deaths
from massive hemorrhage are the worst scenarios in women with
placenta percreta. There was one maternal death in our study
(1.5 %). Bladder injury, ureteral dilation, ureteral transection, and
fistula between the bladder and cervix are known urological complications of placenta percreta [16].
The incidence of unintentional genitourinary injuries with
placental adhesive disorders is reported to be as high as 29 %
[17]. Bladder injury is the most common surgical complication in
cases with adhesive placental disorders and this finding is related
to the uncertain borders between the bladder and the adhesive
tissue that occur as a result of placental invasion [18]. Ureteral injury rates can vary from 10 to 15 % [17]. There are many reports in
the literature aiming to decrease these serious urological complications. Preserving the bladder border is an important step in the
surgical approach to treat invasive placental disorders. Additional
procedures such as filling the bladder with saline, administering
intravenous dye, or instilling sterilized milk are used to preserve
the bladder or determine bladder injury [19]. In our study, bladder
and ureteral injury rates were 27.2 and 1.5 %, respectively. The
enormously increased risk of bladder injury in placenta percreta
cases became much more obvious when we retrospectively analyzed the rate of bladder injuries after normal CS in our clinic,
which was around 0.26 % (12/4801) over a period of five years.
In the majority of placental adhesive disorders, the placenta invades the lower uterine segment and/or the cervix. There are a
number of approaches to treat placental invasive disorders. The
treatment of choice is primarily surgical and consists of hysterectomy [18]. Results of a questionnaire study issued to members of
the Society of Perinatal Obstetricians stated that 93 % of cases
with placenta percreta were managed by total hysterectomy
[20]. In line with this finding we preferred total hysterectomy as
the surgery of choice for all hysterectomies in our study.
Maternal morbidity is a significant problem for women with
placenta percreta. Urinary tract injury is more common during
hysterectomies for placental invasive disorders than for hysterectomies performed for gynecological reasons (29 vs. 4.8 %) [21].
Performing different types of procedures can prevent inadvertent
genitourinary injuries. Subtotal hysterectomy, delayed hysterectomy, and placing ureteric catheters before CS are the most significant surgical approaches [22]. We used a specific surgical approach in our study that involved both filling the bladder with saline and mobilization of the bladder following delivery of the fetus. Statistical analysis revealed that, on average, 14.3 women
would have to undergo filling of their bladder (rather than no filling) to prevent one additional patient suffering an additional blad-
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Geburtsh Frauenheilk 2018; 78: 173–178
der injury. It is imperative on any obstetric surgeon treating
placenta percreta to develop an individualized protocol that includes management of maternal hemorrhage. The most important step is detecting pregnant women at risk for placenta percreta and directing them to appropriate centers in case of emergency. There is also an evident need to define the most appropriate surgical techniques and approaches. Our study is just another
such step.
As far as we know, our study is the largest prospective study in
the English literature on filling the bladder as a surgical approach
in the management of placenta percreta. The limitations of our
study include the relatively small number of women treated, given the fact that the number needed to treat to prevent further
bladder injury is 14.3. Because of the absence of conclusive data,
the big question remains on to how to choose the appropriate surgical approach for a specific placental invasive disorder.
Conclusion
Filling the bladder with saline and mobilization of the bladder
from the lower uterine segment did not have a statistically significant beneficial effect on preventing complications of the urinary
system (especially of the bladder). But although the results were
not significant, shorter operation times, shorter postoperative
stays in hospital, and fewer bladder injuries were observed in patients whose bladders were filled prior to surgery. Depending on
his or her surgical experience, the surgeon may prefer to fill the
bladder prior to surgery, and this approach may be considered,
especially in cases with placenta percreta where anatomical landmarks are unclear.
Funding
Our study did not receive any funding.
Conflict of Interest
The authors declare that they have no conflict of interest.
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https://openalex.org/W2080059844
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https://fr.copernicus.org/articles/16/111/2013/fr-16-111-2013.pdf
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A new Middle Jurassic caddisfly (Trichoptera, Hydrobiosidae) from China
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Mitteilungen aus dem Museum für Naturkunde in Berlin. Geowissenschaftliche Reihe
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cc-by
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Fossil Record 16 (1) 2013, 111–116 / DOI 10.1002/mmng.201300005 Fossil Record 16 (1) 2013, 111–116 / DOI 10.1002/mmng.201300005 A new Middle Jurassic caddisfly (Trichoptera, Hydrobiosidae)
from China Yan Gao1, Yunzhi Yao*, 1, 2 and Dong Ren1 1 Key Lab of Insect Evolution and Environmental Changes, Capital Normal University, Beijing 100048, China. E-mail: yaoyz100@gmail.com
2 State Key Laboratory of Palaeobiology and Stratigraphy (Nanjing Institute of Geology and Palaeontology, CAS), Nanjing 210008, China Key Words Jiulongshan Formation
Inner Mongolia
Philopotamidae Abstract Received 28 March 2012
Accepted 26 October 2012
Published 20 February 2013 Received 28 March 2012
Accepted 26 October 2012
Published 20 February 2013 Material belonging to a new fossil genus and species of caddisfly, Pulchercylindratus
punctatus n. gen., n. sp., was collected from the Daohugou locality (Middle Jurassic,
Jiulongshan Formation; Inner Mongolia, China). The new species is assigned to the
Hydrobiosidae according to subcylindrical shape of the 2nd segment of maxillary palp,
the forked R1 (in the forewing, located near apex), and long anal cells (in the fore-
wing). In addition, we propose to transfer the genus Juraphilopotamus Wang, Zhao &
Ren 2009, known from the same locality, to the family Hydrobiosidae, based on the
1st and 2nd segments of the maxillary palp being cylindrical, shorter than the 3rd seg-
ment. A Middle Jurassic origination of family Hydrobiosidae can be established based
on the new discovery. Material and methods Pulchercylindratus gen. nov. is characterized by the
second segment of maxillary palps being subcylindrical
(as opposed to globose), discoidal and medial cells
closed, forewing with all F1–F5 present, and long anal
cells. The new genus appears similar to the extant
genus Rhyacophila Pictet, 1834 (in the family Rhyaco-
philidae; Fig. 4C), but it can be distinguished easily
from the latter by its maxillary palps with the 2nd seg-
ments being globose (in contrast to subcylindrical), and
the anal cells short (in contrast to long). All specimens described here were collected from the Daohugou lo-
cality (approximately Bathonian age, or slightly older, ca. 165 Ma;
Wang & Ren 2009; Ren et al. 2009, 2010a, 2010b; Rasnitsny &
Zhang 2010; Zhao et al. 2011; Shi et al. 2011; Gao et al. 2012; Gu
et al. 2012); Middle Jurassic, Jiulongshan Formation; Shantou Town-
ship, Ningcheng County, Inner Mongolia, China), and are housed at
the Key Laboratory of Insect Evolution & Environmental Changes,
Capital Normal University (CNU, Beijing, China). Body length was measured from the head apex to the abdomen
apex. Interpretation and terminology used herein follow Holzenthal
et al. (2007): C, Costa; Sc, Subcosta; R, Radius; R1a and R1b, anterior
and posterior branches of anterior Radius, respectively; Rs, posterior
branch of R (composed of R2, R3, R4, and R5); M, media; M1 þ 2,
anterior branch of Media, composed on M1 and M2; M3 þ 4, posterior
branch of Media, composed on M3 and M4; Cu, Cubitus; Cu1, anterior
branch of Cubitus (composed of Cu1a and Cu1b); Cu2, posterior
branch of Cubitus; 1A, 2A, and 3A, first, second, and third branches
of anal vein; the forks giving rise to R2 and R3, R4 and R5, M1 and
M2, M3 and M4, CuA1a and CuA1b, are referred to as F1, F2, F3, F4,
and F5, respectively; the discoidal cell (dc) is the cell formed by the
branching of Rs into R2 þ 3 and R4 þ 5 and is closed apically by the
sectorial crossvein (s); the medial cell (mc) is formed by the branch-
ing of M into M1 þ 2 and M3 þ 4 and is closed apically by the medial
crossvein (m); anal cells delimited by 1A, 2A, and 3A. The new taxon is to be compared with known fossil
Hydrobiosidae. Introduction particular, four families of Trichoptera (including nine
genera and 15 species) have been documented from
China (Hong 1983; Lin 1986; Ren et al. 1995; Wang
et al. 2009a, 2009b; Davis et al. 2010). The order Trichoptera, so-called “hairy wings”, con-
tains three suborders, namely Annulipalpia, Integripal-
pia, and Spicipalpia, with over 13,000 known extant
species. The order is widely distributed around the
world, except for the polar regions (Morse 2012), and
has an extensive fossil record, with 37 families, 193
genera, and more than 660 species reported to date
(Handlirsch 1906–1908, 1939; Meunier 1918; Marty-
nova 1958; Sukastcheva 1968, 1973, 1982, 1990; Erick-
son 1983; Novokshonov 1993; Botosaneanu 1995; Jar-
zembowski 1995; Novokshonov et al. 1995; Ansorge
2002; Ivanov & Sukatsheva 2002; Ivanov & Melnitsky
2005; Wichard 2007; Wichard et al. 2009, 2011). In Among caddisflies, the Spicipalpia consists of five
extant families, namely Rhyacophilidae, Hydrobiosidae,
Glossosomatidae,
Hydroptilidae
and
Ptilocolepidae
(Ross 1956; Schmid 1970; Morse 1997; Malicky 2001,
2005; Ward et al. 2004). The family Hydrobiosidae was
first erected by Ulmer (1905) as a subfamily of Rhya-
cophilidae, and granted with family rank by Schmid
(1989). This is a large family of approximately 50 gen-
era (Holzenthal et al. 2007). So far, only three genera
and three species of the fossil Hydrobiosidae have been
reported (Table 1). Table 1. Fossil species currently ascribed to the family Hydrobiosidae. Species
Age
Locality
1
Bullivena grandis Novokshonov, Ivanov & Sukatsheva, 1995
Late Jurassic
Gobi Altai aymak, Mongolia
2
Palaeohydrobiosis siberambra Botosaneanu & Wichard, 1983
Late Cretaceous
East Taymyr, Maimetsha River. Russia
3
Atopsyche perlucida Wichard, 2007
Tertiary
Dominican amber
4
Juraphilopotamus lubricus Wang, Zhao & Ren, 2009
Middle Jurassic
Daohugou, Inner Mongolia, China
5
Pulchercylindratus punctatus n. gen., n. sp. Middle Jurassic
Daohugou, Inner Mongolia, China Table 1. Fossil species currently ascribed to the family Hydrobiosidae. # 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Yan Gao et al.: New Middle Jurassic caddisfly from China 112 Material and methods Compared with the genus Bullivena No-
vokshonov et al., 1995, the new genus has no particular
thickening of R, lacks the crossvein connecting R5 with
M1 þ 2, and has a closed discoidal cell (in contrast to an
open discoidal cell). Compared with the genus Palaeo-
hydrobiosis Botosaneanu et al., 1983, the new genus
has a closed discoidal cell, and F1 longer than F2 (as
opposed to F1 shorter than F2). Compared with the
genus Atopsyche Wichard, 2007, the new genus has
maxillary palps with the terminal segment being not
particularly elongate (as opposed to very elongate), a
median cell open, M fork located at the first third of Order Trichoptera Kirby, 1813 Order Trichoptera Kirby, 1813
Suborder Spicipalpia Weaver, 1983
Family Hydrobiosidae Ulmer, 1905 museum-fossilrecord.wiley-vch.de Systematic paleontology Figure 1. Pulchercylindratus punctatus n. gen., n. sp. Line
drawing of holotype, CNU-Tri-NN2011003. # 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim museum-fossilrecord.wiley-vch.de Pulchercylindratus n. gen. Holotype, <, CNU-Tri-NN2011003; paratypes, CNU-Tri-
NN2011004PC (dorsoventrally compressed, part and counterpart),
CNU-Tri-NN2011005 (laterally compressed). Wings (Fig. 4A): forewings, Sc straight, with an oblique cross
reaching anterior wing margin before half of wing length; Rs fo
basal to wing mid-length; stem of Rs nearly twice as long as disco
cell; F1 parallel to F2; M fork located opposite the first thir
forewing length; medial cell longer than discoidal cell; Cu1 and
forked at the same level; Cu2 and anal vein A1 þ 2 þ 3 distinct. Measurements
(in mm). Holotype,
<,
CNU-
NN2011003 (Figs 1, 2A): Body length 10.47, maxi
width of body 8.24; head length 0.88, width 1
length antennal segments (scapus, pedicelli, flage
meres): 0.29, 0.18, 3.88; Maxillary palp I–V: 0
0.29, 0.35, 0.35, 0.41; compound eye length 0.53, 0
thorax length 1.35, width 1.53; forewing length 9
width 3.47, Sc 5.47, R1 6.17, Rs 3.17, R2 þ 3 0
R
1 17 M 2 83 M
2 13 M
1 13 Cu 3
Figure
3. Juraphilopotamus
lubr
Wang, Zhao & Ren 2009. A–B. speci
CNU-Tri-NN–2007001 (holotype), ph
graph (A) and line drawing (B); C. M
illary palps (under ethyl alcohol). forewing length (as opposed to located at the forewing
id l
h)
d F
l
h
F
(
d
F
Wings (Fig. 4A): forewings, Sc straight, with an oblique crossvein,
reaching anterior wing margin before half of wing length; Rs forked
Figure
3. Juraphilopotamus
lubricus
Wang, Zhao & Ren 2009. A–B. specimen
CNU-Tri-NN–2007001 (holotype), photo-
graph (A) and line drawing (B); C. Max-
illary palps (under ethyl alcohol). forewing length (as opposed to located at the forewing
mid-length), and F1 longer than F2 (as opposed to F1
shorter than F2)
Wings (Fig. 4A): forewings
reaching anterior wing margi
basal to wing mid-length; stem
Figur
Wang
CNU-
graph
illary Figure
3. Juraphilopotamus
lubricus
Wang, Zhao & Ren 2009. A–B. specimen
CNU-Tri-NN–2007001 (holotype), photo-
graph (A) and line drawing (B); C. Max-
illary palps (under ethyl alcohol). Figure
3. Juraphilopotamus
lubricus
Wang, Zhao & Ren 2009. A–B. specimen
CNU-Tri-NN–2007001 (holotype), photo-
graph (A) and line drawing (B); C. Max-
illary palps (under ethyl alcohol). forewing length (as opposed to located at the forewing
mid-length), and F1 longer than F2 (as opposed to F1
shorter than F2). forewing length (as opposed to located at the forewing
mid-length), and F1 longer than F2 (as opposed to F1
shorter than F2). Wings (Fig. Pulchercylindratus n. gen. 4A): forewings, Sc straight, with an oblique crossvein,
reaching anterior wing margin before half of wing length; Rs forked
basal to wing mid-length; stem of Rs nearly twice as long as discoidal
cell; F1 parallel to F2; M fork located opposite the first third of
forewing length; medial cell longer than discoidal cell; Cu1 and Rs
forked at the same level; Cu2 and anal vein A1 þ 2 þ 3 distinct. Pulchercylindratus punctatus n. sp. Figures 1–3, 4A Figure 4. Forewing line drawings A. Pul-
chercylindratus punctatus n. gen., n. sp.; B.
Juraphilopotamus lubricus Wang, Zhao &
Ren 2009. F1–5 – apical forks 1–5; dc –
discoidal cell; mc – median cell; C. Rhya-
cophila chandleri Denning, 1956 (based on
Giersch 2002). Pulchercylindratus n. gen. Etymology. Genus name is a combination of the Latin pulcher (‘beau-
tiful’) and cylindratus (‘cylindrical’), gender feminine. Type species. Pulchercylindratus punctatus n. sp. Diagnosis. Head round, distinctly narrower than pronotum. Antennae
shorter than forewing, filiform, scapus and pedicelli broader than fla-
gellomeres. Maxillary palps five-segmented in both sexes, second
segment subcylindrical, all segments of subequal length. Ocelli pre-
sent. Anterior setal warts and posterolateral setal warts present on the
head. Prothorax narrow, a pair of pronotal setal warts visible on pro-
notum. Wing moderately broad and smoothly rounded distally; R5
reaching wing apex; R1 forked distally; discoidal and medial cells
closed in forewings, but discoidal cell open in hind-wings; Rs stem
nearly twice as long as discoidal cell; Rs and M four-branched, re-
spectively; F1–F5 present in forewings; anal cells long. Tibial spurs:
2, 4, 4. Remarks. The type species of the genus can be assigned
to the family Hydrobiosidae by the following combina-
tion of features: antenna with stout scapus, shorter than
head; maxillary palps 5-segmented, with 2nd segment
as short as 1st segment or longer, both segments shorter
than following ones; 4th and 5th segments as long as
3rd one, or longer, distally rounded (Fig. 2E); in fore-
wings R1 forked at apex (reduced or simple in few Hy-
drobiosidae genera only); long anal cells. The discoidal
being open or closed is not conclusive on the familial
assignment of the genus, because both states can be
found in the Hydrobiosidae. Figure 1. Pulchercylindratus punctatus n. gen., n. sp. Line
drawing of holotype, CNU-Tri-NN2011003. museum-fossilrecord.wiley-vch.de # 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Fossil Record 16 (1) 2013, 111–116 113 Figure 2. Photographs of Pulchercylindratus punctatus n. gen., n. sp.; A. CNU-Tri-NN2011003; B. CNU-Tri-NN2011005; C–D. CNU-Tri-NN2011004PC; E. Maxillary palps. Figure 2. Photographs of Pulchercylindratus punctatus n. gen., n. sp.; A. CNU-Tri-NN2011003; B. CNU-Tri-NN2011005; C–D. CNU-Tri-NN2011004PC; E. Maxillary palps. Figure 2. Photographs of Pulchercylindratus punctatus n. gen., n. sp.; A. CNU-Tri-NN2011003; B. CNU-Tri-NN2011005; C–D. CNU-Tri-NN2011004PC; E. Maxillary palps. museum-fossilrecord.wiley-vch.de Yan Gao et al.: New Middle Jurassic caddisfly from China 114 forewing length (as opposed to located at the forewing
mid-length), and F1 longer than F2 (as opposed to F1
shorter than F2). Pulchercylindratus punctatus n. sp. Figures 1–3, 4A
Etymology. Name derived from the Latin punctatus (‘spotted’). Locality and horizon. Daohugou Village, Shantou Township, Ning-
cheng County, Inner Mongolia, China (N 4118.9790, E 11914.3180);
Jiulongshan Formation, Middle Jurassic. Material. References Wang, Zhao & Ren (2009b) erected the species Jura-
philopotamus lubricus based on a single specimen from
the Daohugou locality, and placed it to the family Phi-
lopotamidae, based on wing venation characters. The
argument proposed by Wang et al. (2009b; Fig. 3) for
this assignment, based on family diagnosis in Carpenter
(1992, p. 363), was ‘discoidal cell and medial cell
closed’. It is worth mentioning that traditionally the
wing venation characters are widely used for the taxon-
omy of fossil Trichoptera. However these characters
can prove insufficient for family diagnoses, and should
ideally be complemented by body characters (Ivanov &
Melnitsky 2006; also, the polarity of wing characters
was never tested). Ansorge, J. 2002. Revision of the “Trichoptera” described by Geinitz
and Handlirsch from the Lower Toarcian of Dobbertin (Germany)
based new material. – Proceedings of the 10th International Sym-
posium on Trichoptera. – Nova Supplementa Entomologica, Kel-
tern: 55–74. Botosaneanu, L. & Wichard, W. 1983. Upper–Cretaceous Siberian
and Canadian Amber Caddisflies (Insecta: Trichoptera). – Bijdra-
gen tot de Dierkunde 53: 187–217. Botosaneanu,
L. 1995. Caddisflies
(Trichoptera)
from
Turonian
Upper-Cretaceous amber of New Jersey. – American Museum
Novitates 10024 (3140): 1–7. Carpenter, F. M. 1992. Treatise on Invertebrate Palaeontology. Part
R. Arthropoda 4 (3). Superclass Hexapoda. Geological Society
American and University Kansas, Baulder, Colorado and Law-
rence: pp. 1–617. Davis, S. R., Engel, M. S. & Ren Dong 2010. A pupal caddisfly from
the Early Cretaceous of China (Trichoptera). – Cretaceous Re-
search 31 (4): 396–399. Regarding the current case, the family Philopotami-
dae is characterized by 5-segmented maxillary palps in
both sexes, with the 1st segment being the shortest, the
2nd one being provided with a mesodistal brush of se-
tae, and the 5th segment long, annulate and flexible,
usually at least twice as long as preceding segment
(Neboiss 1991; Wiggins 2008). Reinvestigation of the
material described by Wang et al. (2009b) reveals that
only the basal three segments of one maxillary palp are
preserved, with the 1st and 2nd segments being cylind-
rical and shorter than the 3rd segment (Fig. 3C). This
suggests that the species does not belong to the Philo-
potamidae, but more likely to the family Hydrobiosidae
(in which the 1st and 2nd segments cylindrical and
shorter than the 3rd segment). Therefore we propose to
transfer J. lubricus from the family Philopotamidae to
the Hydrobiosidae. Erickson, J. M. 1983. Pulchercylindratus punctatus n. sp.
Figures 1–3, 4A Measurements
(in mm). Holotype,
<,
CNU-Tri-
NN2011003 (Figs 1, 2A): Body length 10.47, maximal
width of body 8.24; head length 0.88, width 1.06;
length antennal segments (scapus, pedicelli, flagello-
meres): 0.29, 0.18, 3.88; Maxillary palp I–V: 0.12,
0.29, 0.35, 0.35, 0.41; compound eye length 0.53, 0.35;
thorax length 1.35, width 1.53; forewing length 9.06,
width 3.47, Sc 5.47, R1 6.17, Rs 3.17, R2 þ 3 0.73,
R4 þ 5 1.17, M 2.83, M1 þ 2 2.13, M3 þ 4 1.13, Cu1 3.73,
Cu2 5.00 , 1A 1.77, 2A 1.40, 1A þ 2A 0.87; dc 1.43,
mc 2.23, F1–5 3.83, 4.33, 3.77, 3.53, 2.80, hind-wing
length 4.71, width 2.65; length fore leg: tibia 1.00, tar- Etymology. Name derived from the Latin punctatus (‘spotted’). Etymology. Name derived from the Latin punctatus (‘spotted’). Locality and horizon. Daohugou Village, Shantou Township, Ning-
cheng County, Inner Mongolia, China (N 4118.9790, E 11914.3180);
Jiulongshan Formation, Middle Jurassic. Locality and horizon. Daohugou Village, Shantou Township, Ning-
cheng County, Inner Mongolia, China (N 4118.9790, E 11914.3180);
Jiulongshan Formation, Middle Jurassic. Material. Holotype, <, CNU-Tri-NN2011003; paratypes, CNU-Tri-
NN2011004PC (dorsoventrally compressed, part and counterpart),
CNU-Tri-NN2011005 (laterally compressed). Diagnosis. Two pairs of setal warts in dorsal view, anterior setal warts
small and symmetrical; posterolateral setal warts prolate and obscure. Antennae with stout scapus and slender flagellomeres, many spines
distributed on inner side of antennae. Figure 4. Forewing line drawings A. Pul-
chercylindratus punctatus n. gen., n. sp.; B. Juraphilopotamus lubricus Wang, Zhao &
Ren 2009. F1–5 – apical forks 1–5; dc –
discoidal cell; mc – median cell; C. Rhya-
cophila chandleri Denning, 1956 (based on
Giersch 2002). museum-fossilrecord.wiley-vch.de
# 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Figure 4. Forewing line drawings A. Pul-
chercylindratus punctatus n. gen., n. sp.; B. Juraphilopotamus lubricus Wang, Zhao &
Ren 2009. F1–5 – apical forks 1–5; dc –
discoidal cell; mc – median cell; C. Rhya-
cophila chandleri Denning, 1956 (based on
Giersch 2002). Figure 4. Forewing line drawings A. Pul-
chercylindratus punctatus n. gen., n. sp.; B. Juraphilopotamus lubricus Wang, Zhao &
Ren 2009. F1–5 – apical forks 1–5; dc –
discoidal cell; mc – median cell; C. Rhya-
cophila chandleri Denning, 1956 (based on
Giersch 2002). museum-fossilrecord.wiley-vch.de # 2013 WILEY-VCH Verlag GmbH & Co. Acknowledgements We sincerely thank the reviewers of this paper (one anonymous and
Wilfried Wichard, Kln) for their improvement of our manuscript. We
are also grateful to Olivier Bthoux for useful discussion, and Yingy-
ing Cui for help with photographs. This research was funded by
grants from National Basic Research Program of China (973 Pro-
gram) (No. 2012CB821900); Fok Ying-Tong Education Foundation
for Young Teachers in the Higher Education Institutions of China
(No. 131021); the National Natural Science Foundation of China
(No. 31071964, 41272006, 31230065); the General Program of Science
and Technology Development Project of Beijing Municipal Education
Commission of China (No. KM201210028016); the PHR Project of
Beijing Municipal Commission of Education (No. 201107120) and the
State Key Laboratory of Palaeobiology and Stratigraphy (Nanjing Insti-
tute of Geology and Palaeontology, CAS) (No.123114). Pulchercylindratus punctatus n. sp.
Figures 1–3, 4A KGaA, Weinheim Fossil Record 16 (1) 2013, 111–116 115 someres I–V: 0.53, 0.41, 0.35, 0.29, 0.18; abdomen
length 4.24, width 1.18; ovipositor length 0.88; Para-
type,
CNU-Tri-NN2011004PC
(dorsoventrally
com-
pressed, part and counterpart (Figs 2C, D)) body length
6.04, maximal width of body 2.55; head length 0.94,
width 1.11; length antennae 3.74; Maxillary palp I–V:
0.12, 0.29, 0.35, 0.35, 0.41; thorax length 1.19, width
1.53; forewing length 7.40, width 1.96; length fore leg:
tibia 1.00, tarsomeres I–V: 0.53, 0.41, 0.35, 0.29, 0.18;
abdomen length 4.24, width 1.18; Paratype, CNU-Tri-
NN2011005 body length 5.78, maximal width of body
1.96; length pronotum 0.77, width 0.54; length fore
leg: tibia 2.55, tarsomeres 1.45; forewing length 7.82,
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(English edition) 85 (1): 75–80 of the Northeastern China. Science Press, Beijing. Ross, H. H. 1956. Evolution and Classification of the Mountain Cad-
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dae (Trichoptera). Memoires de la Socit Entomologique du Ca-
nada. # 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim museum-fossilrecord.wiley-vch.de
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The effect of cognitive task on postural stability in cervical dystonia
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Arquivos de Neuro-Psiquiatria
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| ERROR: type should be string, got "https://doi.org/10.1590/0004-282X20200038 https://doi.org/10.1590/0004-282X20200038 ARTICLE RESUMO Introdução: A distonia cervical (DC) é a forma mais comum de distonia focal. Não se sabe exatamente se posturas anormais da cabeça na \nDC causam problemas de equilíbrio. A execução de duas tarefas simultaneamente é situação comum da vida cotidiana. Objetivo: Avaliar \na estabilidade postural (EP) em pacientes com DC e o efeito da tarefa cognitiva na EP. Como objetivo secundário, avaliamos o efeito da \ntoxina onabotulínica A (BoNT) na EP. Métodos: Foram incluídos 24 pacientes com DC em tratamento com BoNT por pelo menos um ano e \n23 controles saudáveis. As análises posturográficas foram realizadas em todos os sujeitos na plataforma de posturografia estática sob \nquatro condições diferentes: olhos abertos, olhos fechados, postura tandem e tarefa cognitiva. Nos pacientes, a análise posturográfica \nfoi realizada imediatamente antes das injeções de BoNT e após quatro semanas. Resultados: Antes do tratamento, a oscilação ântero-\nposterior era significativamente maior nos pacientes com DC com os olhos abertos quando comparados aos controles (p=0,03). A tarefa \ncognitiva interferiu significativamente nas velocidades de oscilação. A postura tandem afetou significativamente muitos parâmetros de \noscilação, enquanto a condição de olhos fechados não. Após o tratamento, apenas dois parâmetros na posição tandem e um na tarefa \ncognitiva melhoraram no grupo de pacientes. Conclusões: O controle postural é prejudicado em pacientes com DC, provavelmente devido \nà comprometida integração proprioceptiva e sensório-motora. Em referência às teorias de dupla-tarefa, possivelmente devido à atenção \ndividida e à priorização de tarefas, a dupla-tarefa cognitiva e a tarefa postural mais difíceis perturbam o EP nesses pacientes. Palavras-chave: Toxinas Botulínicas; Torcicolo; Cognição; Postura; Equilíbrio Postural. ABSTRACT Background: Cervical dystonia (CD) is the most common form of focal dystonia. It is not known exactly whether abnormal head postures \nin cervical dystonia cause balance problems. Dual-tasking is a common every-day life situation. Objective: We aimed to evaluate postural \nstability (PS) in patients with CD and the effect of cognitive task on PS. As a secondary aim, we evaluated the effect of onabotulinum toxin \nA (BoNT) injection on PS. Methods: A total of 24 patients with CD who were on BoNT treatment for at least one year and 23 healthy controls \nwere included. Posturographic analyses were carried out in all the subjects on static posturography platform under four different conditions: \neyes open, eyes closed, tandem stance and cognitive task. In patients, posturographic analysis was carried out just before the BoNT \ninjections and was repeated four weeks later. Results: Before treatment, the anterior-posterior sway was significantly higher in CD patients \nwith the eyes open condition compared to the controls (p=0.03). Cognitive task significantly affected several sway velocities. Tandem stance \nsignificantly affected many sway parameters, whereas the eyes closed condition did not. After treatment, only two parameters in tandem \nstance and one in cognitive task improved within the patient group, in a pairwise comparison. Conclusions: Postural control is impaired in \nCD patients probably due to the impaired proprioceptive and sensorimotor integration. In reference to dual task theories possibly due to \ndivided attention and task prioritization, cognitive dual-task and harder postural task disturbes the PS in these patients. Keywords: Botulinum Toxin; Torticollis; Cognition; Posture; Postural Balance. Subjects Twenty-four patients with CD who were followed-up for at \nleast one year in our movement disorders unit were enrolled \nin this prospective study. Inclusion criteria required patients \nhaving received BoNT injection every three months regularly \nfor at least one year. Patients on any other treament for CD, \nwith mental retardation or dementia, with comorbid neuro-\nlogical disorders that may affect posture and balance, such \nas polyneuropathy, ataxia, cerebrovascular disease, multiple \nsclerosis, rheumatologic or orthopedic problems, vestibulop-\nathy or otological disease, severely blurred vision, on seda-\ntive drugs or substances were excluded. Written informed \nconsent was obtained from all participants before enroll-\nment. This study was approved by the Ethics Committee of \nÇukurova University, Faculty of Medicine. There are some studies on postural balance in CD. Barr \net al. compared the functional measures of mobility, gait, \nreaction time, and postural balance in ten people with CD \nand ten healthy controls. They reported that CD patient \nswayed more than controls, with poor postural control18. De Pauw et al. compared postural control during quiet sitting \nin 23 patients with CD and 36 healthy controls. They reported \nincreased postural sway and impaired PS in CD patients5. Bove et al. studied PS in 16 CD patients and 12 healthy con-\ntrols, CD patients were found to sway definitely more than \ncontrols7. Lekhel et al.19 and Moreu et al.20 reported no dif-\nferences in postural sway in stance between healthy controls \nand patients with CD. These studies all have different meth-\nodological approaches and the results were controversial. Furthermore, 23 healthy controls were included. The patients and the controls were similar in age and gen-\nder distribution. Neurological examination was performed \nin all subjects. Except for dystonia, CD patients were neu-\nrologically normal. Dystonia severity was assessed with \nthe Toronto Western Spasmodic Torticollis Rating Scale \n(TWSTRS); dystonia severity, disability and pain scores were \ndetermined24. The presence of dystonic head tremor was \nnoted. Patients received BoNT injections as needed. All of \nthem received onabotulinumtoxin A (BOTOX®, Allergan, Inc., \nIrvine, CA, USA), and the muscles to be treated were selected \nbased on the neurological examination and electromyogra-\nphy (EMG), according to each patient’s needs. Evaluations \nof patients including TWSTRS and static posturographyic \nmeasurements were repeated four weeks after the injection, \nwhich is when the highest treatment effect is expected25. Dual-tasking, a common every-day life situation, is the \nability to coordinate performance on two functional activ-\nity performing simultaneously21. METHODS Postural control and balance are provided by somato-\nsensory integrations as well as the integration of vestibular \nand visual inputs5. In CD, sensorimotor integration deficits \ndetected by motor-evoked potentials with transcranial stim-\nulation have been reported6. It is emphasized that sensorim-\notor integration deficits seen in these patients are related to \nimpaired neck proprioception, and impaired neck proprio-\nception leads to balance and posture impairments7,8,9,10,11. Mild deficits in vestibular reflexes have been defined in \npatients with CD12. The vestibular system has complex con-\nnections within the brain stem, thalamus and cortex. In CD, \nit is possible that there is a defect in these central connec-\ntions and in their relation with other modalities that control \nhead and eye movements in particular13. However, studies \nhave controversial results on this subject13,14,15,16,17. Subjects During daily activities, \nhumans involuntarily perform dual tasks without any prob-\nlem. Daily activities, such as listening to music while walking \nor running, writing while listening or answering questions, \nare examples of dual-tasking. Healthy people can provide \npostural control while performing another motor or cogni-\ntive task22. There are no studies in literature evaluating the \nPS under dual task condition in CD patients. Furthermore, \nto the best of our knowledge, there is only one study on the \neffect of BoNT treatment on PS in these patients. Müller et al. reported no beneficial effects of BoNT on dynamic balance \nin phasic CD23. Arq Neuropsiquiatr 2020;78(9):549-555 INTRODUCTION It causes involuntary contraction of neck muscles, leading to \ntonic or clonic head movements, subsequently resulting in \nsustained abnormal head postures. It has several types: torti-\ncollis, laterocollis, anterocollis and retrocollis. Most patients Cervical dystonia (CD) is the most common form of focal \ndystonia. Its prevelance ranges from 20‒4,100 cases/million1. 1Çukurova University, Faculty of Medicine, Department of Neurology, Adana, Turkey. 2Çukurova University, Faculty of Medicine, Movement Disorders Unit, Department of Neurology, Adana, Turkey. Turgay DEMİR \n https://orcid.org/0000-0002-7076-8571; Mehmet BALAL \n https://orcid.org/0000-0001-8320-6597; \nMeltem DEMİRKİRAN \n https://orcid.org/0000-0002-4649-5315\nCorrespondence: Turgay Demir; E-mails: drtdemir@gmail.com, tdemir@cu.edu.tr \nConflict of interest: There is no conflict of interest to declare. Ethical approval: Cukurova University, Faculty of Medicine Ethics Committee approved the study protocol (protocol number: 78-2016.06.01_32). Informed consent: Informed consent was obtained from all participants included in the study. Received on September 21, 2019; Received in its final form on November 29, 2019; Accepted on March 24, 2020. 1Çukurova University, Faculty of Medicine, Department of Neurology, Adana, Turkey. 2Çukurova University, Faculty of Medicine, Movement Disorders Unit, Department of Neurology, Adana, Turkey. Turgay DEMİR \n https://orcid.org/0000-0002-7076-8571; Mehmet BALAL \n https://orcid.org/0000-0001-8320-6597; \nMeltem DEMİRKİRAN \n https://orcid.org/0000-0002-4649-5315\nCorrespondence: Turgay Demir; E-mails: drtdemir@gmail.com, tdemir@cu.edu.tr \nConflict of interest: There is no conflict of interest to declare. Ethical approval: Cukurova University, Faculty of Medicine Ethics Committee approved the study protocol (protocol number: 78-2016.06.01_32). Informed consent: Informed consent was obtained from all participants included in the study. Received on September 21, 2019; Received in its final form on November 29, 2019; Accepted on March 24, 2020. 549 of cognitive task on PS in patients with CD. We also seek \nto analyze whether BoNT has any effect on the PS of these \npatients. Therefore, our secondary aim was to evaluate the \neffect of BoNT on PS, within the patient group. with CD have mixed type postures2. In addition to abnormal \npostures, some patients have clonic head movements result-\ning in dystonic tremor. Botulinum toxin (BoNT) treatment is \nthe first line treatment for CD and is reported to be effec-\ntive in 70‒92% of patients3,4. Whether abnormal head posture \ncause balance problems in CD is unknown. Participants Demographic features of the subjects are shown in \nTable 2. The most common CD type was torticollis and was \npresent in 83.3% (n=20) of patients. Dystonic head tremor \nwas present in 37.5% (n=9) of them (Table 3). RESULTS RESULTS Statistical analysis All analyses were performed using the SPSS® for Windows \nsoftware package (Version 20.0. Armonk, NY: IBM Corp.). Continuous variables were summarized as means and stan-\ndard deviation. The normality of distribution for continu-\nous variables was confirmed with the Shapiro Wilk test. For variables with a normal distribution, a parametric test, \nsuch as Student’s t-test was used. Variables without a nor-\nmal distribution were evaluated using the nonparamet-\nric Mann-Whitney U test. For comparison of two related \n(paired) continuous variables, paired samples Student’s \nt-test for variables with a normal distribution or Wilcoxon \nSigned Rank test for variables without a normal distribu-\ntion was used. Pearson’s coefficient was used in the correla-\ntion analysis of numeric variables with a normal distribu-\ntion, and Spearman’s correlation coefficient was used for \nthe analysis of numerical variables without normal distri-\nbution. The hypothesis tests were conducted to verify the Posturographic parameters With the baseline EO condition, only A-P sway and sway path \nvelocity were significantly higher in CD than in controls. EC did \nnot have any effect on PS, whereas TAN affected almost all sway \nparameters in CD significantly (Table 4). The effect of cognitive \ndual task was more prominent in the sway velocities (Table 4). BoNT treatment did not lead to a significant reduction in the \nsway parameters. Only a few parameters get better after the injec-\ntion. A-P sway in EO, lateral sway and sway path in TAN and lat-\neral sway velocity in cognitive dual tasking improved significantly \nafter BoNT injection (Table 5). Age, disease duration and TWSTRS \nscore did not correlate with any posturographic parameters. he \ny \nCD patients \n(n=24) \nControls \n(n=23)\nAge (years)\n48±14.5 (range 20‑72) 49.3±13.8 (range 22‑72)\nGender (F/M)\n14/10\n14/9\nDisease \nduration (years)\n13.1±9.9 (range 1‑32)\n_\nTWSTRS score\n12.3±6.1 (range 5‑27)\n_\nTable 2. Demographic and clinical characteristics \nof participants. CD: cervical dystonia; F: Female; M: Male; TWSTRS: Toronto Western \nSpazmodic torticollis scale\nn\n%\nDHT\nn (%)\nTWSTRS\nPure Type\n14\n58.3\n7 (29.4)\n11.7\nTorticollis\n10\n41.6\nLaterocollis\n3\n12.5\nAnterocollis\n1\n4.2\nMixed Type\n10\n41.6\n2 (8.3)\n21.3\nTorticollis-Laterocollis\n3\n12.5\nTorticollis-Retrocollis\n4\n16.6\nTorticollis-Anterocollis\n2\n8.3\nTorticollis-Laterocollis-\nAnterocollis\n1\n4.2\nTable 3. Types of cervical dystonia. DHT: dystonic head tremor; TWSTRS: Toronto Western Spasmodic \nTorticollis Scale CD patients \n(n=24) \nControls \n(n=23)\nAge (years)\n48±14.5 (range 20‑72) 49.3±13.8 (range 22‑72)\nGender (F/M)\n14/10\n14/9\nDisease \nduration (years)\n13.1±9.9 (range 1‑32)\n_\nTWSTRS score\n12.3±6.1 (range 5‑27)\n_\nTable 2. Demographic and clinical characteristics \nof participants. CD: cervical dystonia; F: Female; M: Male; TWSTRS: Toronto Western \nSpazmodic torticollis scale Table 2. Demographic and clinical characteristics \nof participants. Posturography protocol Patients and controls underwent static posturography \n(SPG). SPG was carried out just before a new injection of \nBoNT in CD and was repeated four weeks later in these \npatients. It was performed on a force platform (Lucerne II, \nOtopront®, Germany) in a quiet room. The subjects were \ntold to stand on the platform in an upright position as sta-\nble as possible, barefoot, with their feet 4 cm apart. The \narms were held alongside the body. Each recording lasted \n30 seconds. The first recording was done with the patient \nwith eyes open (EO); the second, with the eyes closed (EC); \nthe third, with tandem stance with eyes closed (TAN); \nand the last, with the EO producing words starting with “K” Considering the currently available data from the liter-\nature, we hypothesized that CD patients may have postural \ninstability due to abnormal head and neck postures and a \ncognitive dual-task may affect PS in these patients. In this \nsense, the primary aim of our study was to evaluate the effect 550 Arq Neuropsiquiatr 2020;78(9):549-555 nullity of the correlation coefficients. The statistical level of \nsignificance for all tests was considered as 0.05. nullity of the correlation coefficients. The statistical level of \nsignificance for all tests was considered as 0.05. (COG). The cognitive performance of participants was not \nevaluated during the cognitive task. In TAN, one foot is in \nfront of the other, and the arms lifted up and extended in \nfront of the body. Sway path, anterior-posterior (A-P) sway, \nlateral sway, sway area, as well as the velocity of all of these \nsway values (sway path/time, A-P sway/time, lateral sway/\ntime, sway area/time) were recorded. Definition of SPG \nparameters are shown in Table 1. Posturographic parameters Parameter\nDefinition\nSway path\nSum of the movements of the \ncentre of pressure during the whole \nexamination time (cm)\nWay velocity\nAverage speed of the movement \nof the centre of pressure (cm/sec) \n(way velocity)\nSway Area (SA)\nCalculation of the sway area \nacording to the formula of Diener/\nDichgans/Bacher (cm2), xi and y iare \nthe coordinates at the time i of the \ncentre of pressure)\n\t\n n-1\nSA = ½ Σ I xi+1 yi+1 xi I\n\t\n i = 1\nSA velocity\nArea divided by time (cm2/sec) (Sway \narea velocity)\nAnterior-posterior (A-P) \nSum of the y components of the \nmovement vectors (cm/s)\nΣ I yi+1-yi I\nA-P sway velocity\nQuotient of Antero-Posterior ant \ntime (cm/s) (Anterior-posterior sway \nvelocity)\nLateral \nSum of the x components of the \nmovement vectors (cm)\nΣ I yi+1-yi I\nLateral sway velocity\nQuotient of lateral and time (cm/s) \n(lateral sway velocity)\nTable 1. Definiton of the static posturography parameters. n\n%\nDHT\nn (%)\nTWSTRS\nPure Type\n14\n58.3\n7 (29.4)\n11.7\nTorticollis\n10\n41.6\nLaterocollis\n3\n12.5\nAnterocollis\n1\n4.2\nMixed Type\n10\n41.6\n2 (8.3)\n21.3\nTorticollis-Laterocollis\n3\n12.5\nTorticollis-Retrocollis\n4\n16.6\nTorticollis-Anterocollis\n2\n8.3\nTorticollis-Laterocollis-\nAnterocollis\n1\n4.2\nTable 3. Types of cervical dystonia. DHT: dystonic head tremor; TWSTRS: Toronto Western Spasmodic \nTorticollis Scale Demir T et al. Postural stability in cervical dystonia. 551 DISCUSSION A-P: antero-posterior; COG: cognitive task; EO: eyes open; EC: eyes closed; \nLS:lateral sway; SA:sway area; SP sway path; SD: standard deviation; \nTAN: tandem stance\nPosturographic \nparameters \nand TWSTRS\nBefore \nBoNT\n(mean±SD)\nAfter BoNT\n(mean±SD)\np-value\nTWSTRS score\n12.3±6.1\n8.8±4.6\n<0.001\nEO\nA-P sway (cm)\n28.2±11.7\n24.9±7.1\n0.012\nLS (cm)\n21.5±11.5\n20.1±8.2\n0.169\nSP (cm)\n40.3±17\n36.7±11\n0.071\nSA (cm2)\n6.6±6.2\n5.2±3.1\n0.146\nA-P sway velocity (cm/s)\n0.5±0.5\n1±0.001\n0.987\nLS velocity (cm/s)\n0.8±0.5\n0.5±0.5\n0.426\nSP velocity(cm/s) \n1.4±0.6\n1.2±0.4\n0.135\nSA velocity (cm2/s)\n0.008±0.2\n0.01±0.01\n0.162\nEC\nA-P sway (cm)\n43.8±31.2\n39.1±16.8\n0.153\nLS (cm)\n25.5±12.5\n23.7±10.5\n0.193\nSP (cm)\n56.6±34.3\n51.3±20.1\n0.149\nSA (cm2)\n12.2±13.4\n10.9±9.2\n0.810\nA-P sway velocity (cm/s)\n1±0.5\n1.3±0.5\n0.059\nLS velocity (cm/s)\n1.5±1.1\n0.7±0.5\n0.257\nSP velocity(cm/s) \n1.8±1.1\n1.7±0.6\n0.439\nSA velocity (cm2/s)\n0.2±0.5\n0.1±0.3\n0.083\nTAN\nA-P sway (cm)\n97.1±67.6\n96.1±60.7\n0.225\nLS (cm)\n80.7±44.2\n76.7±40.4\n0.038\nSP (cm)\n140±86.2\n136.9±78.4\n0.048\nSA (cm2)\n54.1±.60.1\n53.8±62.2\n0.478\nA-P sway velocity (cm/s)\n3.4±2.5\n3.1±2\n0.314\nLS velocity (cm/s)\n3.3±2.2\n2.6±1.3\n0.289\nSP velocity(cm/s) \n4.6±1.8\n4.5±2.6\n0.309\nSA velocity (cm2/s)\n7.5±9.1\n6.9±2.2\n0.582\nCOG\n A-P sway (cm)\n36.1±19.1\n35.1±16.5\n0.396\nLS (cm)\n25.2±15.3\n24.2±15\n0.073\nSP (cm)\n49.2±25.6\n48.1±23.9\n0.278\nSA (cm2)\n11.1±15.6\n12.9±16\n0.276\nA-P sway velocity (cm/s)\n0.8±0.6\n0.8±0.4\n0.655\nLS velocity (cm/s)\n1.2±0.6\n0.7±0.6\n0.046\nSP velocity(cm/s) \n1.6±0.9\n1.6±0.9\n0.987\nSA velocity (cm2/s)\n0.4±0.6\n0.2±0.5\n0.164\nTable 5. Pairwise comparisons of mean postural sways, \nvelocities and TWSTRS scores before and after BoNT. A-P: antero-posterior; BoNT: botulinum toxin; COG: cognitive task; EO: eyes \nopen; EC: eyes closed; LS:lateral sway; SA:sway area; SD: standard deviation; \nSP :sway path;TWSTRS:Toronto-Western Spazmodic Torticollis Rating Scale Table 5. Pairwise comparisons of mean postural sways, \nvelocities and TWSTRS scores before and after BoNT. DISCUSSION CD compared to the healthy controls under baseline EO con-\ndition. EC condition does not have any effect, whereas TAN, \nwhich is the harder postural task, affected PS of CD patients \nmore prominently than the other conditions. Cognitive task This is the first study to investigate PS in patients with \nCD under cognitive dual task condition. One of the results of \nour study is that postural control is impaired in patients with Posturographic \nparameters \nand TWSTRS\nBefore \nBoNT\n(mean±SD)\nAfter BoNT\n(mean±SD)\np-value\nTWSTRS score\n12.3±6.1\n8.8±4.6\n<0.001\nEO\nA-P sway (cm)\n28.2±11.7\n24.9±7.1\n0.012\nLS (cm)\n21.5±11.5\n20.1±8.2\n0.169\nSP (cm)\n40.3±17\n36.7±11\n0.071\nSA (cm2)\n6.6±6.2\n5.2±3.1\n0.146\nA-P sway velocity (cm/s)\n0.5±0.5\n1±0.001\n0.987\nLS velocity (cm/s)\n0.8±0.5\n0.5±0.5\n0.426\nSP velocity(cm/s) \n1.4±0.6\n1.2±0.4\n0.135\nSA velocity (cm2/s)\n0.008±0.2\n0.01±0.01\n0.162\nEC\nA-P sway (cm)\n43.8±31.2\n39.1±16.8\n0.153\nLS (cm)\n25.5±12.5\n23.7±10.5\n0.193\nSP (cm)\n56.6±34.3\n51.3±20.1\n0.149\nSA (cm2)\n12.2±13.4\n10.9±9.2\n0.810\nA-P sway velocity (cm/s)\n1±0.5\n1.3±0.5\n0.059\nLS velocity (cm/s)\n1.5±1.1\n0.7±0.5\n0.257\nSP velocity(cm/s) \n1.8±1.1\n1.7±0.6\n0.439\nSA velocity (cm2/s)\n0.2±0.5\n0.1±0.3\n0.083\nTAN\nA-P sway (cm)\n97.1±67.6\n96.1±60.7\n0.225\nLS (cm)\n80.7±44.2\n76.7±40.4\n0.038\nSP (cm)\n140±86.2\n136.9±78.4\n0.048\nSA (cm2)\n54.1±.60.1\n53.8±62.2\n0.478\nA-P sway velocity (cm/s)\n3.4±2.5\n3.1±2\n0.314\nLS velocity (cm/s)\n3.3±2.2\n2.6±1.3\n0.289\nSP velocity(cm/s) \n4.6±1.8\n4.5±2.6\n0.309\nSA velocity (cm2/s)\n7.5±9.1\n6.9±2.2\n0.582\nCOG\n A-P sway (cm)\n36.1±19.1\n35.1±16.5\n0.396\nLS (cm)\n25.2±15.3\n24.2±15\n0.073\nSP (cm)\n49.2±25.6\n48.1±23.9\n0.278\nSA (cm2)\n11.1±15.6\n12.9±16\n0.276\nA-P sway velocity (cm/s)\n0.8±0.6\n0.8±0.4\n0.655\nLS velocity (cm/s)\n1.2±0.6\n0.7±0.6\n0.046\nSP velocity(cm/s) \n1.6±0.9\n1.6±0.9\n0.987\nSA velocity (cm2/s)\n0.4±0.6\n0.2±0.5\n0.164\nTable 5. Pairwise comparisons of mean postural sways, \nvelocities and TWSTRS scores before and after BoNT. DISCUSSION A-P: antero-posterior; BoNT: botulinum toxin; COG: cognitive task; EO: eyes \nopen; EC: eyes closed; LS:lateral sway; SA:sway area; SD: standard deviation; \nSP :sway path;TWSTRS:Toronto Western Spazmodic Torticollis Rating Scale our study is that postural control is impaired in patients with \nPostural sways and \nvelocities\nCervical \nDystonia\n(mean±SD)\nControl\n(mean±SD)\np-value\nEO\nA-P sway (cm)\n28.2±11.7\n22.3±6.4\n0.039\nLS (cm)\n21.5±11.5\n19±5.7\n0.351\nSP (cm)\n40.3±17.0\n33.5±8.8\n0.095\nSA (cm2)\n6.6±6.2\n4.2±2\n0.089\nA-P sway velocity (cm/s)\n0.5±0.5\n0.7±0.4\n0.763\nLS velocity (cm/s)\n0.8±0.5\n0.9±0.2\n0.214\nSP velocity(cm/s) \n1.4±0.6\n1.1±0.3\n0.038\nSA velocity (cm2/s)\n0.008±0.2\n0.001±0.001\n0.162\nEC\nA-P sway (cm)\n43.8±31.2\n35.5±23\n0.090\nLS (cm)\n25.5±12.5\n21.5±8.4\n0.388\nSP (cm)\n56.6±34.3\n46.6±24.9\n0.166\nSA (cm2)\n12.2±13.4\n8±8.5\n0.126\nA-P sway velocity (cm/s)\n1±0.5\n0.7±0.5\n0.089\nLS velocity (cm/s)\n1.5±1.1\n1.2±0.7\n0.155\nSP velocity(cm/s) \n1.8±1.1\n1.5±0.8\n0.239\nSA velocity (cm2/s)\n0.2±0.5\n0.09±0.2\n0.250\nTAN\nA-P sway (cm)\n97.1±67.6\n35.4±19.7\n<0.001\nS (cm)\n80.7±44.2\n29.7±24.7\n<0.001\nSP (cm)\n140±86.2\n52.5±33.7\n<0.001\nSA (cm2)\n54.1±60.1\n11.3±15.5\n<0.001\nA-P sway velocity (cm/s)\n3.4±2.5\n2±0.7\n<0.001\nLS velocity (cm/s)\n3.3±2.2\n1±0.9\n0.086\nSP velocity(cm/s) \n4.6±1.8\n2.3±1.1\n<0.001\nSA velocity (cm2/s)\n7.5±9.1\n1.6±1.8\n<0.001\nCOG\nA-P sway (cm)\n36.1±19.1\n29.5±10.3\n0.163\nLS (cm)\n25.2±15.3\n19.2±6.1\n0.253\nSP (cm)\n49.2±25.6\n39.7±12.1\n0.221\nSA (cm2)\n11.1±15.6\n6.3±5\n0.234\nA-P sway velocity (cm/s)\n0.8±0.6\n0.8±0.3\n0.933\nLS velocity (cm/s)\n1.2±0.6\n0.2±0.5\n<0.001\nSP velocity(cm/s) \n1.6±0.9\n0.7±0.7\n<0.001\nSA velocity (cm2/s)\n0.4±0.6\n0.2±0.5\n<0.001\nTable 4. Comparison of mean postural sways and velocities \nbetween patients and controls. A-P: antero-posterior; BoNT: botulinum toxin; COG: cognitive task; EO: eyes \nopen; EC: eyes closed; LS:lateral sway; SA:sway area; SD: standard deviation; \nSP : sway path; TWSTRS: Toronto-Western Spazmodic Torticollis Rating Scale DISCUSSION Postural sways and \nvelocities\nCervical \nDystonia\n(mean±SD)\nControl\n(mean±SD)\np-value\nEO\nA-P sway (cm)\n28.2±11.7\n22.3±6.4\n0.039\nLS (cm)\n21.5±11.5\n19±5.7\n0.351\nSP (cm)\n40.3±17.0\n33.5±8.8\n0.095\nSA (cm2)\n6.6±6.2\n4.2±2\n0.089\nA-P sway velocity (cm/s)\n0.5±0.5\n0.7±0.4\n0.763\nLS velocity (cm/s)\n0.8±0.5\n0.9±0.2\n0.214\nSP velocity(cm/s) \n1.4±0.6\n1.1±0.3\n0.038\nSA velocity (cm2/s)\n0.008±0.2\n0.001±0.001\n0.162\nEC\nA-P sway (cm)\n43.8±31.2\n35.5±23\n0.090\nLS (cm)\n25.5±12.5\n21.5±8.4\n0.388\nSP (cm)\n56.6±34.3\n46.6±24.9\n0.166\nSA (cm2)\n12.2±13.4\n8±8.5\n0.126\nA-P sway velocity (cm/s)\n1±0.5\n0.7±0.5\n0.089\nLS velocity (cm/s)\n1.5±1.1\n1.2±0.7\n0.155\nSP velocity(cm/s) \n1.8±1.1\n1.5±0.8\n0.239\nSA velocity (cm2/s)\n0.2±0.5\n0.09±0.2\n0.250\nTAN\nA-P sway (cm)\n97.1±67.6\n35.4±19.7\n<0.001\nS (cm)\n80.7±44.2\n29.7±24.7\n<0.001\nSP (cm)\n140±86.2\n52.5±33.7\n<0.001\nSA (cm2)\n54.1±60.1\n11.3±15.5\n<0.001\nA-P sway velocity (cm/s)\n3.4±2.5\n2±0.7\n<0.001\nLS velocity (cm/s)\n3.3±2.2\n1±0.9\n0.086\nSP velocity(cm/s) \n4.6±1.8\n2.3±1.1\n<0.001\nSA velocity (cm2/s)\n7.5±9.1\n1.6±1.8\n<0.001\nCOG\nA-P sway (cm)\n36.1±19.1\n29.5±10.3\n0.163\nLS (cm)\n25.2±15.3\n19.2±6.1\n0.253\nSP (cm)\n49.2±25.6\n39.7±12.1\n0.221\nSA (cm2)\n11.1±15.6\n6.3±5\n0.234\nA-P sway velocity (cm/s)\n0.8±0.6\n0.8±0.3\n0.933\nLS velocity (cm/s)\n1.2±0.6\n0.2±0.5\n<0.001\nSP velocity(cm/s) \n1.6±0.9\n0.7±0.7\n<0.001\nSA velocity (cm2/s)\n0.4±0.6\n0.2±0.5\n<0.001\nTable 4. Comparison of mean postural sways and velocities \nbetween patients and controls. A-P: antero-posterior; COG: cognitive task; EO: eyes open; EC: eyes closed; \nLS:lateral sway; SA:sway area; SP sway path; SD: standard deviation; \nTAN: tandem stance Table 4. Comparison of mean postural sways and velocities \nbetween patients and controls. 552 Arq Neuropsiquiatr 2020;78(9):549-555 function is required to provide postural control and balance in \nsuch patients under dual task conditions. affected the sway velocities in patients significantly. There are \na few studies in literature investigating PS in CD, and the \nresults are controversial7,17,19,20,22. Lekhel et al.19 and Moreau \net al.20 reported that there was no significant difference in \nlateral and A-P sways between CD patients and controls. In contrast to these studies, Bove et al.7 and Wöber et al.26 \nreported significant increase in postural sway parameters \nsuch as sway path, sway area, medio-lateral and A-P sway val-\nues in CD compared to the controls. Barr et al. reported that \nCD patients swayed more than controls, had poor postural \ncontrol and walked more slowly than controls18. De Pauw \net al. reported increased postural sway and impaired PS in \npatients with CD5. DISCUSSION For this to happen, precise data from vestibular, \nvisual, body sensory systems should be taken, reconciled, \nunnecessary information should be eliminated and selected \nactions should be made appropriate. A robust neurological \nand skeletal system is required for all this to occur. However, \nour results suggest that visual input may not have much \neffect on PS in these patients. Therefore, we assume that \nvisual input probably has the least effect on postural balance \nin patients with CD. As suggested by some earlier studies, \nproprioceptive and sensorimotor integration are probably \nmore important components of PS in patients with CD7,8,9,10,11. Up to date, there are no studies evaluating the effect \nof dual-tasking on postural control in patients with CD. An important finding of the present study is the effect of cog-\nnitive dual-tasking in patients with CD. We found that cog-\nnitive task impaired PS in these patients more than controls. The effect was more prominent on the sway velocities in pos-\nturographic analyses. Some theories have been put forward \nto explain the difficulties in performing dual tasks. The most \nprevalent are capacity sharing, bottleneck (task switching), \nand cross talk27. According to capacity sharing theory, which is \nthe most widely accepted, performing two tasks at the same \ntime decreases the performance of each task due to the divi-\nsion of capacity for tasks27. In bottleneck (task-switching) \nmodel, parallel processing may be impossible for some men-\ntal operations27. Some operations may simply require a single \nmechanism to be dedicated to them for some period of time. When two tasks need the mechanism simultaneously, a bot-\ntleneck occurs, and one or both tasks will be delayed or oth-\nerwise impaired. The crosstalk model is being used to refer \nto conditions in which informational code overlaps across \ntasks27. Taleli et al.28 suggested that differential cortical activa-\ntion within the higher neural centers can affect task prioritiza-\ntion, further allowing increased conscious attention while car-\nrying out cognitive or motor tasks. Therefore, we can say that \nattention is an important variable to maintain PS under dual \ntask conditions in patients with CD and an adequate attention The present study has limitations. The absence of BoNT-\nnaive patient group is one of them. We included only the \npatients who were already under treatment. The number \nof patients with dystonic head tremor was low. DISCUSSION Differences in the study protocols includ-\ning patient selection and variations in the posturographic \nprotocols may explain these discrepancies. During dual task assessment, the two tasks performed \nsimultaneously in a dual task paradigm can be two cognitive, \ntwo motor, or a motor and a cognitive task29. Dual task costs, \nthat is, decline in performance of either or both tasks can be \ndue to age or illness-related conditions30,31. Hence, patients \nwith CD may experience reduction in performance when \nperforming two activities that require attention at the same \ntime, such as finding words starting with K and maintaining \nPS, leading to dual task costs32. They may have a tendency \nto prioritize the motor task, which is PS, in the presence of a \ncognitive or another challenging motor task33,34,35,36,37. In the literature, there are contradictory results on the \neffect of BoNT on PS in CD patients. In our study, BoNT injec-\ntion had little effect on PS of CD patients; in cognitive task, only \none sway velocity (lateral sway velocity), and in TAN stance \ntwo sway parameters (lateral sway and sway path) improved. Wöber et al.26 reported that the whole-body postural control \nwas impaired in more than 75% of idiopathic CD patients and \nhad improved with local injections with BoNT-A. On the other \nhand, De Pauw et al.38 reported that BoNT injection had little \nutility on cervical sensorimotor control, postural control, and \nvisual vertical perception. In our study, as seen in the improve-\nment of TWSTRS scores, BoNT is an effective treatment for CD, \nbut it did not have much effect on PS of these patients. It affects \nthe contraction of the muscles, leading to a better posture of \nthe head and neck, but this peripherally and improved posture \nprobably does not have any retrograde effects on central mecha-\nnisms which control the PS of patients with CD. In other words, \nthe PS of CD is the result of central mechanisms. Controlling the \nperipheral mechanism, such as improving the faulty contraction \nof neck muscles, probably does not provide additional help in \nthe maintenance of PS. More data in larger series is needed on \nthis subject to fully understand the effect of BoNT on PS in CD. hh The normal balance function provides the ability to \nmaintain the erect posture of the body in motion or stable \ncondition. Demir T et al. Postural stability in cervical dystonia. DISCUSSION Therefore, \nwe were unable to analyze the effect of dystonic head tremor \non these parameters in relation to dual-tasking. Different \ntypes of CD may have different impacts on PS. Nonetheless, \nthe number of patients are not enough to evaluate the effect \nof each type of CD. For standardization, we used the same \norder of tests in the posturographic analysis in every subject. This might have had some practice effects. In conclusion, PS is impaired in patients with CD. Vision \nmay not be as essential for PS in these patients like proprio-\nceptive and sensorimotor integration are. Cognitive dual \ntask disturbes PS in CD probably due to divided attention \nand task prioritization. BoNT injection does not seem to \nhave much effect on PS in patients with CD. More compre-\nhensive studies of posturography with complex motor or cog-\nnitive tasks may provide further information on this subject. 553 Demir T et al. Postural stability in cervical dystonia. References 1988 Feb;45(2):164-9. https://doi.org/10.1001/\narchneur.1988.00520260050019 1988 Feb;45(2):164-9. https://doi.org/10.1001/\narchneur.1988.00520260050019 1. 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Postural control and the relation with cervical sensorimotor \ncontrol in patients with idiopathic adult-onset cervical dystonia. Exp Brain Res. 2018 Mar;236(3):803-11. https://doi.org/10.1007/\ns00221-018-5174-x 22. Dault MC, Frank JS, Allard F. Influence of a visuo-spatial, verbal \nand central executive working memory task on postural control. Gait Posture. 2001 Oct;14(2):110-6. https://doi.org/10.1016/s0966-\n6362(01)00113-8 6. Abbruzzese G, Marchese R, Buccolieri A, Gasparetto B, \nTrompetto C. Abnormalities of sensorimotor integration in focal \ndystonia: a transcranial magnetic stimulation study. Brain. 2001 \nMar;124(Pt 3):537-45. https://doi.org/10.1093/brain/124.3.537 23. Müller J, Ebersbach G, Wissel J, Poewe W. Dynamic balance function \nin phasic cervical dystonia following Botulinum Mov Disord. 2001 \nSep;16(5):934-7. https://doi.org/10.1002/mds.1164 7. Bove M, Brichetto G, Abbruzzese G, Marchese R, Schieppati M. Postural responses to continuous unilateral neck muscle vibration \nin standing patients with cervical dystonia. Mov Disord. 2007 \nMar;22(4):498-503. https://doi.org/10.1002/mds.21357 24. Consky ES, Basinki A, Belle L, Ranawaya R, Lang AE. The \nToronto Western Spasmodic Torticollis Rating Scale (TWSTRS): \nassessment of validity and inter-rater reliability. Neurology. 1990;40(Suppl 1):S445. 8. De Pauw J, Mercelis R, Hallemans A, Michiels S, Truijen S, Cras P, \net al. Cervical sensorimotor control in idiopathic cervical dystonia: \na cross-sectional study. Brain Behav. 2017;7(9):e00735. https://doi. org/10.1002/brb3.735 25. Odergren T, Hjaltason H, Kaakkola S, Solders G, Hanko J, \nFehling C, et al. A double blind, randomised, parallel group study \nto investigate the dose equivalence of dysport and botox in the \ntreatment of cervical dystonia. J Neurol Neurosurg Psychiatry. 1998 \nJan;64(1):6-12. https://doi.org/10.1136/jnnp.64.1.6 9. Vuillerme N, Pinsault N. Experimental neck muscle pain impairs \nstanding balance in humans. Exp Brain Res. 2009 Feb;192(4):723-9. https://doi.org/10.1007/s00221-008-1639-7 26. Wöber E, Schnider P, Steinhoff N, Trattnig S, Zebenholzer K, Auff E. Posturographic findings in patients with idiopathic cervical dystonia \nbefore and after local injections with botulinum toxin. Eur Neurol. 1999;41(4):194-200. https://doi.org/10.1159/000008050 10. Treleaven J, Jull G, LowChoy N. The relationship of cervical joint \nposition error to balance and eye movement disturbances in \npersistent whiplash. Man Ther. 2006 May;11(2):99-106. https://doi. org/10.1016/j.math.2005.04.003 27. Pashler H. Dual-task interference in simple tasks: data and theory. Psychol Bull. 1994 Sep;116(2):220-44. https://doi.org/10.1037/0033-\n2909.116.2.220 11. Field S, Treleaven J, Jull G. Standing balance: a comparison between \nidiopathic and whiplash-induced neck pain. Man Ther. 2008 \nJun;13(3):183-91. https://doi.org/10.1016/j.math.2006.12.005 28. Talelli P, Ewas A, Waddingham W, Rothwell J, Ward N. Neural \ncorrelates of age-related changes in cortical neurophysiology. Neuroimage. 2008 May;40(4):1772-81. https://doi.org/10.1016/j. neuroimage.2008.01.039 12. Münchau A, Bronstein AM. DISCUSSION Role of the vestibular system in the \npathophysiology of spasmodic torticollis. J Neurol Neurosurg \nPsychiatry. 2001 Sep;71(3):285-8. https://doi.org/10.1136/\njnnp.71.3.285 29. Saxena S, Cinar E, Majnemer A, Gagnon I. Does dual tasking ability \nchange with age across childhood and adolescence? A systematic \nscoping review. Int J Dev Neurosci. 2017 May;58:35-49. https://doi. org/10.1016/j.ijdevneu.2017.01.012 13. Bronstein AM, Rudge P. Vestibular involvement in spasmodic \ntorticollis. J Neurol Neurosurg Psychiatry. 1986 Mar;49(3):290-5. https://doi.org/10.1136/jnnp.49.3.290 14. Stejskal L, Tománek Z. Postural laterality in torticollis and torsion \ndystonia. J Neurol Neurosurg Psychiatry. 1981 Nov;44(11):1029-34. https://doi.org/10.1136/jnnp.44.11.1029 30. Bekkers EMJ, Dockx K, Devan S, Van Rossom S, Verschueren SMP, \nBloem BR, et al. The ımpact of dual-tasking on postural stability in \npeople with Parkinson’s disease with and without freezing of gait. Neurorehabil Neural Repair. 2018 Feb;32(2):166-74. https://doi. org/10.1177/1545968318761121 15. Stell R, Bronstein AM, Marsden CD. Vestibulo-ocular abnormalities \nin spasmodic torticollis before and after botulinum toxin injections. J Neurol Neurosurg Psychiatry. 1989 Jan;52(1):57-62. https://doi. org/10.1136/jnnp.52.1.57 31. Liu YC, Yang YR, Tsai YA, Wang RY. Cognitive and motor dual task \ngait training improve dual task gait performance after stroke - A \nrandomized controlled pilot trial. Sci Rep. 2017;7(1):4070. https://doi. org/10.1038/s41598-017-04165-y 16. Huygen PL, Verhagen WI, Van Hoof JJ, Horstink MW. Vestibular \nhyperreactivity in patients with idiopathic spasmodic torticollis. J Neurol Neurosurg Psychiatry. 1989 Jun;52(6):782-5. https://doi. org/10.1136/jnnp.52.6.782 32. Krampe RT, Schaefer S, Lindenberger U, Baltes PB. Lifespan changes \nin multi-tasking: concurrent walking and memory search in children, \nyoung, and older adults. Gait Posture. 2011 Mar;33(3):401-5. https://\ndoi.org/10.1016/j.gaitpost.2010.12.012 17. Diamond SG, Markham CH, Baloh RW. Ocular counterrolling \nabnormalities in spasmodic torticollis. Arch Neurol. 554 Arq Neuropsiquiatr 2020;78(9):549-555 33. Brown LA, Sleik RJ, Polych MA, Gage WH. Is the prioritization \nof postural control altered in conditions of postural threat in \nyounger and older adults? J Gerontol A Biol Sci Med Sci. 2002 \nDec;57(12):M785-92. https://doi.org/10.1093/gerona/57.12.m785 36. Li KZH, Lindenberger U, Freund AM, Baltes PB. Walking while \nmemorizing: age-related differences in compensatory behavior. Psychol Sci. 2001 May;12(3):230-7. https://doi.org/10.1111/1467-\n9280.00341 37. Rapp M, Krampe RT, Baltes PB. Adaptive task prioritization in aging: \nselective resource allocation to postural control is preserved in \nAlzheimer disease. Am J Geriatr Psychiatry. 2006 Jan;14(1):52-61. https://doi.org/10.1097/01.JGP.0000192490.43179.e7 34. Doumas M, Rapp M, Krampe RT. Working memory and postural \ncontrol: adult age differences in potential for improvement, task-\npriority and dual-tasking. J Gerontol B Psychol Sci Soc Sci. 2009 \nMar;64B(2):193-201. https://doi.org/10.1093/geronb/gbp009 38. DISCUSSION De Pauw J, Cras P, Truijen S, Mercelis R, Michiels S, Saeys W, et al. The effect of a single botulinum toxin treatment on somatosensory \nprocessing in idiopathic isolated cervical dystonia: an observational \nstudy. J Neurol. 2018 Nov;265(11):2672-2683. https://doi. org/10.1007/s00415-018-9045-y 35. Doumas M, Smolders C, Krampe RT. Task prioritization in aging: \neffects of sensory information on concurrent posture and memory \nperformance. Exp Brain Res. 2008 May;187(2):275-81. https://doi. org/10.1007/s00221-008-1302-3 555 Demir T et al. Postural stability in cervical dystonia."
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https://www.biorxiv.org/content/biorxiv/early/2023/02/09/2023.02.08.527763.full.pdf
|
English
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Proteome-wide comparison of tertiary protein structures reveal extensive molecular mimicry in<i>Plasmodium</i>-human interactions
|
bioRxiv (Cold Spring Harbor Laboratory)
| 2,023
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Abstract
12 Molecular mimicry is a strategy used by parasites to escape the host immune system and successfully
13
transmit to a new host. To date, high-throughput examples of molecular mimicry have been limited
14
to comparing protein sequences. However, with advances in the prediction of tertiary structural
15
models, led by Deepmind’s AlphaFold, it is now possible to compare the tertiary structures of
16
thousands of proteins from parasites and their hosts, to identify more subtle mimics. Here, we present
17
the first proteome-level search for tertiary structure similarity between the proteins from Plasmodium
18
falciparum and human. Of 206 P. falciparum proteins that have previously been proposed as
19
mediators of Plasmodium-human interactions, we propose that seven evolved to molecularly mimic a
20
human protein. By expanding the approach to all P. falciparum proteins, we identified a further 386
21
potential mimics, with 51 proteins corroborated by additional biological data. These findings
22
demonstrate a valuable application of AlphaFold-derived tertiary structural models, and we discuss
23
key considerations for its effective use in other host-parasite systems. 24 .
CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 9, 2023.
;
https://doi.org/10.1101/2023.02.08.527763
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 9, 2023. ;
https://doi.org/10.1101/2023.02.08.527763
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 9, 2023. ;
https://doi.org/10.1101/2023.02.08.527763
doi:
bioRxiv preprint Proteome-wide comparison of tertiary protein structures reveal extensive
1
molecular mimicry in Plasmodium-human interactions
2
Viraj Muthye1,2, James D. Wasmuth1,2*
3
1 Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta, Canada
4
2 Host-Parasite Interactions Research Training Network, University of Calgary, Calgary, Alberta,
5
Canada
6
* Correspondence:
7
Corresponding Author: James D. Wasmuth (jwasmuth@ucalgary.ca)
8
9
Keywords: molecular mimicry, malaria, plasmodium, AlphaFold, tertiary structure, host-
10
parasite interactions
11
Abstract
12
Molecular mimicry is a strategy used by parasites to escape the host immune system and successfu
13
transmit to a new host. To date, high-throughput examples of molecular mimicry have been limite
14
to comparing protein sequences. However, with advances in the prediction of tertiary structural
15
models, led by Deepmind’s AlphaFold, it is now possible to compare the tertiary structures of
16
thousands of proteins from parasites and their hosts, to identify more subtle mimics. Here, we pres
17
the first proteome-level search for tertiary structure similarity between the proteins from Plasmodi
18
falciparum and human. Of 206 P. falciparum proteins that have previously been proposed as
19
mediators of Plasmodium-human interactions, we propose that seven evolved to molecularly mim
20
human protein. By expanding the approach to all P. falciparum proteins, we identified a further 38
21
potential mimics, with 51 proteins corroborated by additional biological data. These findings
22
demonstrate a valuable application of AlphaFold-derived tertiary structural models, and we discus
23
key considerations for its effective use in other host-parasite systems. 24
25
Introduction
26
Parasites encounter host defenses at various points in their life cycle and employ a wide range of
27
strategies for evading their host’s immune response and successfully transmitting to a new host
28
(Chulanetra & Chaicumpa, 2021). Keywords: molecular mimicry, malaria, plasmodium, AlphaFold, tertiary structure, host-
10
parasite interactions
11 Keywords: molecular mimicry, malaria, plasmodium, AlphaFold, tertiary structure, host-
10
parasite interactions
11 p
y p
molecular mimicry in Plasmodium-human interactions
2 1 Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta, Canada
4 2 Host-Parasite Interactions Research Training Network, University of Calgary, Calgary, Alberta,
5
Canada
6
* Correspondence:
7
Corresponding Author: James D. Wasmuth (jwasmuth@ucalgary.ca)
8
9
Keywords: molecular mimicry, malaria, plasmodium, AlphaFold, tertiary structure, host-
10
parasite interactions
11
Abstract
12 p
Corresponding Author: James D. Wasmuth (jwasmuth@ucalgary.ca)
8
9 .
CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 9, 2023.
;
https://doi.org/10.1101/2023.02.08.527763
doi:
bioRxiv preprint These host-parasite interactions may be mediated by parasite-
29
derived molecules—including proteins, lipids, sugars–that unexpectedly resemble host-derived
30
molecules. This is termed ‘molecular mimicry’, which was originally defined as the sharing of
31
antigens between parasite and host (Damian, 1964). One of the earliest reports of molecular mimic
32
came from the parasitic nematode Ascaris lumbricoides, which possesses A- and B-like blood gro
33
antigens in its polysaccharides (Oliver-González, 1944). The definition of molecular mimicry has
34
adapted to keep up with molecular and genomic technologies and is now widely considered to
35 Introduction
26 M128L shares significant sequence similarity with host CD47 and competes with it to
53
bind with its receptor SIRPα. Within eukaryotic pathogens, the apicomplexan Babesia microti
54
expresses the BmP53 protein which contains a domain that resembles thrombospondin (TSP1), a
55
component of platelet cells (Mousa et al., 2017). The BmP53 TSP-1 is immunologically cross-
56
reactive with human and it is proposed that BmP3 helps cloak the extra-cellular stages from the
57
immune system. 58 To the best of our knowledge, the first study to identify host-parasite molecular mimicry at a
59
genome-scale across multiple species was by Ludin and colleagues (Ludin et al., 2011). They
60
considered the protein sequences from eight species of eukaryotic parasites, the host (human), and
61
seven non-pathogenic, eukaryotic, negative control species. Their approach identified multiple
62
potential instances of mimicry in these parasites. For example, they detected a 14 amino acid motif in
63
multiple PfEMP1 proteins in Plasmodium falciparum that was identical to the heparin-binding
64
domain in human vitronectin, a protein with multiple roles in human including cell-adhesion. The
65
approach was repeated to find ninety-four potential mimicry proteins in a tapeworm-fish system
66
(Hebert et al., 2015). It was also adapted and expanded for use with 62 pathogenic bacteria and
67
identified approximately 100 potential mimics (Doxey & McConkey, 2013). These approaches rely
68
on two proteins sharing enough sequence similarity to be detected by the sequence alignment
69
software, e.g., BLAST (Altschul et al., 1990). However, proteins may share too little sequence
70
similarity. For instance, several viruses express proteins with tertiary structure similarity, but
71
undetectable sequence similarity to human Bcl-2, and interfere with regulation of apoptosis
72
(Kvansakul et al., 2007; Westphal et al., 2007). Similarly, in Plasmodium falciparum, a search of
73
parasite proteins targeted to host extracellular vesicles revealed that at least eight shared unexpected
74
and significant tertiary structure similarity with host proteins (Armijos-Jaramillo et al., 2021). 75 The opportunity to detect host-parasite mimicry at the level of tertiary structure has been limited by
76
the number of available tertiary protein structures. Even for a parasite as important as P. falciparum,
77
the protein databank (PDB) contains structures from less than 4% of the protein-coding genes in its
78
genome (Table 1). We expect that most, if not all, other bacterial and eukaryotic pathogen species
79
will have worse coverage. Introduction
26 Parasites encounter host defenses at various points in their life cycle and employ a wide range of
27
strategies for evading their host’s immune response and successfully transmitting to a new host
28
(Chulanetra & Chaicumpa, 2021). These host-parasite interactions may be mediated by parasite-
29
derived molecules—including proteins, lipids, sugars–that unexpectedly resemble host-derived
30
molecules. This is termed ‘molecular mimicry’, which was originally defined as the sharing of
31
antigens between parasite and host (Damian, 1964). One of the earliest reports of molecular mimicry
32
came from the parasitic nematode Ascaris lumbricoides, which possesses A- and B-like blood group
33
antigens in its polysaccharides (Oliver-González, 1944). The definition of molecular mimicry has
34
adapted to keep up with molecular and genomic technologies and is now widely considered to
35
similarity between proteins at the level of primary structure (amino acid sequence) and tertiary
36
structure (summarized in (Tayal et al., 2022)). An assumption is that molecular mimicry confers a
37
fitness benefit to the pathogen. However, in immunology research, the term molecular mimicry can
38 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 9, 2023. ;
https://doi.org/10.1101/2023.02.08.527763
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 9, 2023. ;
https://doi.org/10.1101/2023.02.08.527763
doi:
bioRxiv preprint be used to explain the cross-reactivity between exogenous and self-peptides and is the theoretical
39
framework for understanding autoimmunity (Getts et al., 2013). Related to both these definitions,
40
molecular mimicry might also result in heterologous immunity, in which the infection from one
41
parasite protects against infection by other parasites with similar antigenic molecules (Balbin et al.,
42
2023). 43
Here, our focus is molecular mimicry which likely confers a fitness advantage to the parasite, by
44
either co-opting or disrupting the function of the mimicked host protein. Examples of molecular
45
mimicry come from most branches of life. For instance, pathogenic bacterium Escherichia coli
46
injects the TccP protein into host cells, which targets the polymerization of host actin. Introduction
26 TccP contains
47
multiple repeated motifs that mimic an internal regulatory element present in host N-WASP (neural
48
Wiskott–Aldrich syndrome protein), which results in the activation of N-WASP (Sallee et al., 2008). 49
This promotion of actin polymerization results in the creation of structures on epithelial cells that
50
promote pathogen survival in the intestine. In another example, the myxoma virus decreases the
51
number of activated macrophages by expressing its M128L protein on the host cell surface (Cameron
52
et al., 2005). M128L shares significant sequence similarity with host CD47 and competes with it to
53
bind with its receptor SIRPα. Within eukaryotic pathogens, the apicomplexan Babesia microti
54
expresses the BmP53 protein which contains a domain that resembles thrombospondin (TSP1), a
55
component of platelet cells (Mousa et al., 2017). The BmP53 TSP-1 is immunologically cross-
56
reactive with human and it is proposed that BmP3 helps cloak the extra-cellular stages from the
57
immune system. 58
To the best of our knowledge, the first study to identify host-parasite molecular mimicry at a
59
genome-scale across multiple species was by Ludin and colleagues (Ludin et al., 2011). They
60
considered the protein sequences from eight species of eukaryotic parasites, the host (human), and
61
seven non-pathogenic, eukaryotic, negative control species. Their approach identified multiple
62
potential instances of mimicry in these parasites. For example, they detected a 14 amino acid motif in
63
multiple PfEMP1 proteins in Plasmodium falciparum that was identical to the heparin-binding
64
domain in human vitronectin, a protein with multiple roles in human including cell-adhesion. The
65
approach was repeated to find ninety-four potential mimicry proteins in a tapeworm-fish system
66
(Hebert et al., 2015). It was also adapted and expanded for use with 62 pathogenic bacteria and
67
identified approximately 100 potential mimics (Doxey & McConkey, 2013). These approaches rely
68
on two proteins sharing enough sequence similarity to be detected by the sequence alignment
69
software, e.g., BLAST (Altschul et al., 1990). However, proteins may share too little sequence
70
similarity. For instance, several viruses express proteins with tertiary structure similarity, but
71
undetectable sequence similarity to human Bcl-2, and interfere with regulation of apoptosis
72
(Kvansakul et al., 2007; Westphal et al., 2007). Introduction
26 For instance, pathogenic bacterium Escherichia coli
46
injects the TccP protein into host cells, which targets the polymerization of host actin. TccP contains
47
multiple repeated motifs that mimic an internal regulatory element present in host N-WASP (neural
48
Wiskott–Aldrich syndrome protein), which results in the activation of N-WASP (Sallee et al., 2008). 49
This promotion of actin polymerization results in the creation of structures on epithelial cells that
50
promote pathogen survival in the intestine. In another example, the myxoma virus decreases the
51
number of activated macrophages by expressing its M128L protein on the host cell surface (Cameron
52
et al., 2005). M128L shares significant sequence similarity with host CD47 and competes with it to
53
bind with its receptor SIRPα. Within eukaryotic pathogens, the apicomplexan Babesia microti
54
expresses the BmP53 protein which contains a domain that resembles thrombospondin (TSP1), a
55
component of platelet cells (Mousa et al., 2017). The BmP53 TSP-1 is immunologically cross-
56
reactive with human and it is proposed that BmP3 helps cloak the extra-cellular stages from the
57
immune system. 58 be used to explain the cross-reactivity between exogenous and self-peptides and is the theoretical
39
framework for understanding autoimmunity (Getts et al., 2013). Related to both these definitions,
40
molecular mimicry might also result in heterologous immunity, in which the infection from one
41
parasite protects against infection by other parasites with similar antigenic molecules (Balbin et al.,
42
2023). 43 Here, our focus is molecular mimicry which likely confers a fitness advantage to the parasite, by
44
either co-opting or disrupting the function of the mimicked host protein. Examples of molecular
45
mimicry come from most branches of life. For instance, pathogenic bacterium Escherichia coli
46
injects the TccP protein into host cells, which targets the polymerization of host actin. TccP contains
47
multiple repeated motifs that mimic an internal regulatory element present in host N-WASP (neural
48
Wiskott–Aldrich syndrome protein), which results in the activation of N-WASP (Sallee et al., 2008). 49
This promotion of actin polymerization results in the creation of structures on epithelial cells that
50
promote pathogen survival in the intestine. In another example, the myxoma virus decreases the
51
number of activated macrophages by expressing its M128L protein on the host cell surface (Cameron
52
et al., 2005). Introduction
26 Similarly, in Plasmodium falciparum, a search of
73
parasite proteins targeted to host extracellular vesicles revealed that at least eight shared unexpected
74
and significant tertiary structure similarity with host proteins (Armijos-Jaramillo et al., 2021). 75
The opportunity to detect host-parasite mimicry at the level of tertiary structure has been limited by
76
the number of available tertiary protein structures. Even for a parasite as important as P. falciparum,
77
the protein databank (PDB) contains structures from less than 4% of the protein-coding genes in its
78
genome (Table 1). We expect that most, if not all, other bacterial and eukaryotic pathogen species
79
will have worse coverage. Proteome-wide searches for host-parasite molecular mimicry at the level
80
of tertiary structure depended on in silico predictions that were of inconsistent quality (Armijos-
81
Jaramillo et al., 2021). The prediction of tertiary protein structures from amino acid sequences has
82
seen a much-publicised boon, in no small part to the development of AlphaFold (Ronneberger et al.,
83
2021). In an early large-scale application, the AlphaFold Protein Structure Database (AFdb) provided
84
. CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 9, 2023. ;
https://doi.org/10.1101/2023.02.08.527763
doi:
bioRxiv preprint be used to explain the cross-reactivity between exogenous and self-peptides and is the theoretical
39
framework for understanding autoimmunity (Getts et al., 2013). Related to both these definitions,
40
molecular mimicry might also result in heterologous immunity, in which the infection from one
41
parasite protects against infection by other parasites with similar antigenic molecules (Balbin et al.,
42
2023). 43 be used to explain the cross-reactivity between exogenous and self-peptides and is the theoretical
39
framework for understanding autoimmunity (Getts et al., 2013). Related to both these definitions,
40
molecular mimicry might also result in heterologous immunity, in which the infection from one
41
parasite protects against infection by other parasites with similar antigenic molecules (Balbin et al.,
42
2023). 43
Here, our focus is molecular mimicry which likely confers a fitness advantage to the parasite, by
44
either co-opting or disrupting the function of the mimicked host protein. Examples of molecular
45
mimicry come from most branches of life. Introduction
26 For instance, RIFIN, a prominent erythrocyte surface protein
99
expressed by P. falciparum, binds to human LILRB1 which inhibits stimulation of the immune
100
response. RIFIN does this by mimicking MHC Class I, the activating ligand of LILRBA (Harrison et
101
al., 2020). Meanwhile, the circumsporozoite protein (CSP), which promotes invasion of human liver
102
cells, has an 18 amino acid region that is similar to a cytoadhesive region in mammalian
103
thrombospondin (Cerami et al., 1992; Robson et al., 1988). 104 In this study, our goal was to identify P. falciparum proteins which share tertiary structure similarity
105
with human proteins but not detectable sequence similarity. First, we examined P. falciparum
106
proteins which are known or have been implicated to directly interact with human biomolecules. We
107
found new potential instances of molecular mimicry. Second, we extended our approach to consider
108
all P. falciparum proteins and leveraged experimental datasets to filter the candidate mimics. Overall,
109
our study highlights the advantages of using tertiary protein structures for identifying instances of
110
molecular mimicry. 111 Introduction
26 For instance, the PDB structure 7F9N is compo
123
of four chains (A to D), of which two chains (A and B) are from P falciparum Rifin (RIF,
124
PF3D7_1000500) and two chains (C and D) are from the human leukocyte-associated
125
immunoglobulin-like receptor 1 protein (LAIR1, Q6GTX8) (Figure 1A). We included only chain
126
and B for P. falciparum. Additionally, multiple structure chains were chimeric or ambiguous, i.e
127
mapping to multiple source organisms. For instance, the PDB structure 4O2X has two chains (A
128 Our parasitic species of study is Plasmodium falciparum. While our understanding of the mediators
92
of host-parasite interactions is limited, as the leading cause of severe malaria in humans, P. 93
falciparum might represent the current pinnacle of our knowledge. Furthermore, the protein tertiary
94
structures are complemented with a broad range of curated -omics datasets available on PlasmoDB,
95
which can help with candidate prioritization (Amos et al., 2022; Aurrecoechea et al., 2008). Proteins
96
expressed by P. falciparum mediate interactions with its human host at multiple stages in its life
97
cycle (Acharya et al., 2017). Molecular mimicry plays a role at both the liver and blood stages for
98
immune evasion and cytoadherence. For instance, RIFIN, a prominent erythrocyte surface protein
99
expressed by P. falciparum, binds to human LILRB1 which inhibits stimulation of the immune
100
response. RIFIN does this by mimicking MHC Class I, the activating ligand of LILRBA (Harrison et
101
al., 2020). Meanwhile, the circumsporozoite protein (CSP), which promotes invasion of human liver
102
cells, has an 18 amino acid region that is similar to a cytoadhesive region in mammalian
103
thrombospondin (Cerami et al., 1992; Robson et al., 1988). 104 Our parasitic species of study is Plasmodium falciparum. While our understanding of the mediators
92
of host-parasite interactions is limited, as the leading cause of severe malaria in humans, P. 93
falciparum might represent the current pinnacle of our knowledge. Furthermore, the protein tertiary
94
structures are complemented with a broad range of curated -omics datasets available on PlasmoDB,
95
which can help with candidate prioritization (Amos et al., 2022; Aurrecoechea et al., 2008). Proteins
96
expressed by P. falciparum mediate interactions with its human host at multiple stages in its life
97
cycle (Acharya et al., 2017). Molecular mimicry plays a role at both the liver and blood stages for
98
immune evasion and cytoadherence. Introduction
26 Proteome-wide searches for host-parasite molecular mimicry at the level
80
of tertiary structure depended on in silico predictions that were of inconsistent quality (Armijos-
81
Jaramillo et al., 2021). The prediction of tertiary protein structures from amino acid sequences has
82
seen a much-publicised boon, in no small part to the development of AlphaFold (Ronneberger et al.,
83
2021). In an early large-scale application, the AlphaFold Protein Structure Database (AFdb) provided
84 2 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 9, 2023. ;
https://doi.org/10.1101/2023.02.08.527763
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 9, 2023. ;
https://doi.org/10.1101/2023.02.08.527763
doi:
bioRxiv preprint tertiary structure predictions for 16 model organisms and 32 pathogen species of global health
85
concern (https://alphafold.com). Complementing the release of AlphaFold was Foldseek, a novel
86
approach to aligning tertiary protein structures (van Kempen et al., 2022). Comparisons showed that
87
FoldSeek was nearly 20,000 times faster than existing protein structure aligners while maintaining
88
accuracy (but see (Holm, 2022)). These two major advances in structural bioinformatics—AlphaFold
89
and Foldseek—have empowered us to investigate the usefulness of using tertiary protein structures
90
for identifying instances of host-parasite molecular mimicry. 91 (
p
p
)
p
g
p
,
approach to aligning tertiary protein structures (van Kempen et al., 2022). Comparisons showed t
87
FoldSeek was nearly 20,000 times faster than existing protein structure aligners while maintainin
88
accuracy (but see (Holm, 2022)). These two major advances in structural bioinformatics—Alpha
89
and Foldseek—have empowered us to investigate the usefulness of using tertiary protein structur
90
for identifying instances of host-parasite molecular mimicry. 91
Our parasitic species of study is Plasmodium falciparum. While our understanding of the mediato
92
of host-parasite interactions is limited, as the leading cause of severe malaria in humans, P. 93
falciparum might represent the current pinnacle of our knowledge. Introduction
26 Furthermore, the protein tertia
94
structures are complemented with a broad range of curated -omics datasets available on PlasmoD
95
which can help with candidate prioritization (Amos et al., 2022; Aurrecoechea et al., 2008). Prote
96
expressed by P. falciparum mediate interactions with its human host at multiple stages in its life
97
cycle (Acharya et al., 2017). Molecular mimicry plays a role at both the liver and blood stages fo
98
immune evasion and cytoadherence. For instance, RIFIN, a prominent erythrocyte surface protei
99
expressed by P. falciparum, binds to human LILRB1 which inhibits stimulation of the immune
100
response. RIFIN does this by mimicking MHC Class I, the activating ligand of LILRBA (Harriso
101
al., 2020). Meanwhile, the circumsporozoite protein (CSP), which promotes invasion of human l
102
cells, has an 18 amino acid region that is similar to a cytoadhesive region in mammalian
103
thrombospondin (Cerami et al., 1992; Robson et al., 1988). 104
In this study, our goal was to identify P. falciparum proteins which share tertiary structure simila
105
with human proteins but not detectable sequence similarity. First, we examined P. falciparum
106
proteins which are known or have been implicated to directly interact with human biomolecules. 107
found new potential instances of molecular mimicry. Second, we extended our approach to consi
108
all P. falciparum proteins and leveraged experimental datasets to filter the candidate mimics. Ov
109
our study highlights the advantages of using tertiary protein structures for identifying instances o
110
molecular mimicry. 111
Methods and Materials
112
2.1. Compiling the datasets of tertiary protein structures
113
We downloaded tertiary protein structures for Plasmodium falciparum 3D7 (parasite), human (ho
114
and 15 negative control species, i.e., species that are not infected by P. falciparum (Table 1, File
115
Protein structures for these species were downloaded from two sources - 1) the RCSB Protein Da
116
Bank (PDB) (experimentally-determined protein structures, last accessed 06/16/2022), and 2) the
117
AlphaFold Protein Structure Database (AFdb) (computationally-predicted protein structures,
118
https://alphafold.ebi.ac.uk/). The structures downloaded from both sources were processed before
119
analysis (explained below). 120
2.1.1. Processing the PDB structures: Several PDB structures were composed of chains from
121
multiple source organisms. We separated such structures into individual chains and extracted the
122
appropriate chains corresponding to each species. 2.1. Compiling the datasets of tertiary protein structures
113 We downloaded tertiary protein structures for Plasmodium falciparum 3D7 (parasite), human (host),
4
and 15 negative control species, i.e., species that are not infected by P. falciparum (Table 1, File S1). 5
Protein structures for these species were downloaded from two sources - 1) the RCSB Protein Data
6
Bank (PDB) (experimentally-determined protein structures, last accessed 06/16/2022), and 2) the
7
AlphaFold Protein Structure Database (AFdb) (computationally-predicted protein structures,
8
https://alphafold.ebi.ac.uk/). The structures downloaded from both sources were processed before
9
analysis (explained below). 0 3 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 9, 2023. ;
https://doi.org/10.1101/2023.02.08.527763
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 9, 2023. ;
https://doi.org/10.1101/2023.02.08.527763
doi:
bioRxiv preprint B) which map to both P. falciparum and Escherichia coli strain K12. Such chains were discarded
129
not confound downstream analysis. 130
2.1.2. Processing the AlphaFold structures: AlphaFold assigns a score to each residue in the
131
predicted structure called the ‘pLDDT’ score. This score is a measure of prediction confidence fo
132
that residue. The pLDDT scores range from 0 (low confidence of prediction) to 100 (high confid
133
of prediction). Regions with a pLDDT score above 90 are modeled with high accuracy, between
134
90 are modeled well, and 50-70 are modeled with low confidence. The AlphaFold database sugg
135
that regions with pLDDT scores less than 50 should not be interpreted as this low score could be
136
indicative of intrinsic protein disorder. For each AlphaFold structure, we calculated the proportio
137
the total residues with a pLDDT score of more than 70. We retained predicted structures with at
138
half the residues of the structure modeled with a pLDDT score above 70 (Figure 1B, File S1). 139
2.2. Identification of Plasmodium falciparum proteins known to interact with human molecu
140
We performed a literature survey to identify P. falciparum proteins that are known to interact wit
141
human molecules. We started with a review of P. 2.1. Compiling the datasets of tertiary protein structures
113 falciparum-human protein interactions (Achary
142
al., 2017). Then, we identified all abstracts on PubMed using the query - [‘plasmodium falciparu
143
AND ‘interact*’ AND ‘protein*’ AND ‘human*’] from 2017 to 2022. This resulted in 648 abstra
144
(as of 8/8/2022 1:36 PM). We read all 648 abstracts to identify the P. falciparum proteins of inte
145
The PlasmoDB ID for each protein was mapped to Uniprot IDs using PlasmoDB (Release 52, 30
146
August 2022). Three large gene families (PfEMP1, RIFIN, and STEVOR) play an important role
147
host-parasite interactions and pathogenesis in P. falciparum. Proteins belonging to these three
148
families were downloaded from PlasmoDB. 149
2.3. Identification of sequence and structure similarity between Plasmodium falciparum and
150
human proteins
151
2.3.1. Analysis of protein sequence similarity: We analyzed sequence similarity between the prot
152
from P. falciparum, human, and 15 negative control species. We determined sequence similarity
153
using three pairwise alignment search tools. SSEARCH36 implements the Smith-Waterman
154
algorithm guaranteeing the optimal alignment. We used the following parameters for SSEARCH
155
from Fasta36 - ‘m 8 -s BL62 -f 12 -g 1’. BLASTP (Altschul et al., 1990) and DIAMOND (Buchf
156
et al., 2021) implement heuristic algorithms that are faster than SSEARCH36 but do not guarante
157
the optimal alignment. We used BLASTP with an e-value cut-off of 1e-3 and performed an ultra-
158
sensitive DIAMOND BLASTP search with the same e-value cut-off. For both aligners, we search
159
using the BLOSUM45 and BLOSUM62 substitution matrices. All other parameters were left as
160
default. Additionally, we used OrthoFinder version 2.5.4 to identify groups of orthologous protei
161
between all the 17 species used in this study, using DIAMOND as the aligner (Emms & Kelly, 2
162
The results of this analysis were used to identify human proteins that had orthologs in only the ot
163
three vertebrates used in this study (mouse, rat, and zebrafish). 164
2.3.2. Analysis of protein structure similarity: We aligned all P. falciparum structures to a databa
165
consisting of human structures and control structures. The structural aligner used was Foldseek v
166
(easy-search -s 9.5 --max-seqs 1000). We used an e-value cut-off of 0.01. Foldseek was also used
167
visualize structural alignments using the option ‘format-mode 3’. 168
2.3.3. Expression analysis: Expression analysis of P. falciparum proteins was carried out using th
169
publicly available RNA-seq datasets available in PlasmoDB. 2.2. Identification of Plasmodium falciparum proteins known to interact with human molecules
140 We performed a literature survey to identify P. falciparum proteins that are known to interact with
141
human molecules. We started with a review of P. falciparum-human protein interactions (Acharya et
142
al., 2017). Then, we identified all abstracts on PubMed using the query - [‘plasmodium falciparum’
143
AND ‘interact*’ AND ‘protein*’ AND ‘human*’] from 2017 to 2022. This resulted in 648 abstracts
144
(as of 8/8/2022 1:36 PM). We read all 648 abstracts to identify the P. falciparum proteins of interest. 145
The PlasmoDB ID for each protein was mapped to Uniprot IDs using PlasmoDB (Release 52, 30
146
August 2022). Three large gene families (PfEMP1, RIFIN, and STEVOR) play an important role in
147
host-parasite interactions and pathogenesis in P. falciparum. Proteins belonging to these three
148
families were downloaded from PlasmoDB. 149 We performed a literature survey to identify P. falciparum proteins that are known to interact with
141
human molecules. We started with a review of P. falciparum-human protein interactions (Acharya et
142
al., 2017). Then, we identified all abstracts on PubMed using the query - [‘plasmodium falciparum’
143
AND ‘interact*’ AND ‘protein*’ AND ‘human*’] from 2017 to 2022. This resulted in 648 abstracts
144
(as of 8/8/2022 1:36 PM). We read all 648 abstracts to identify the P. falciparum proteins of interest. 145
The PlasmoDB ID for each protein was mapped to Uniprot IDs using PlasmoDB (Release 52, 30
146
August 2022). Three large gene families (PfEMP1, RIFIN, and STEVOR) play an important role in
147
host-parasite interactions and pathogenesis in P. falciparum. Proteins belonging to these three
148
families were downloaded from PlasmoDB. 149 2.1. Compiling the datasets of tertiary protein structures
113 We identified all P. falciparum prot
170
with expression in the 90th percentile in at least one of the stages in the intra-erythrocytic life cyc
171 B) which map to both P. falciparum and Escherichia coli strain K12. Such chains were discarded to
29
not confound downstream analysis. 30 B) which map to both P. falciparum and Escherichia coli strain K12. Such chains were discarded to
9
not confound downstream analysis. 0 B) which map to both P. falciparum and Escherichia coli strain K12. Such chains were discarded to
29
not confound downstream analysis. 30 129
130 2.1.2. Processing the AlphaFold structures: AlphaFold assigns a score to each residue in the
131
predicted structure called the ‘pLDDT’ score. This score is a measure of prediction confidence for
132
that residue. The pLDDT scores range from 0 (low confidence of prediction) to 100 (high confidence
133
of prediction). Regions with a pLDDT score above 90 are modeled with high accuracy, between 70-
134
90 are modeled well, and 50-70 are modeled with low confidence. The AlphaFold database suggests
135
that regions with pLDDT scores less than 50 should not be interpreted as this low score could be
136
indicative of intrinsic protein disorder. For each AlphaFold structure, we calculated the proportion of
137
the total residues with a pLDDT score of more than 70. We retained predicted structures with at least
138
half the residues of the structure modeled with a pLDDT score above 70 (Figure 1B, File S1). 139 3.1. Assembling and filtering the datasets of crystallised and computationally-predicted tertiary
177
protein structures
178 We compiled tertiary protein structures from Plasmodium falciparum 3D7 (parasite), human (host),
179
and 15 negative control species, those not infected by the parasite (Table 1, File S1). These structures
180
were downloaded from the RCSB Protein Data Bank (PDB) and the AlphaFold Protein Structure
181
database (AFdb). All AlphaFold-generated structures were filtered using the pLDDT score, a per-
182
residue metric of the confidence of prediction accuracy. In line with the AlphaFold documentation,
183
we considered structures to be high confidence if at least half their residues had a pLDDT score
184
above 70. Through this filtering, we retained 56% of P. falciparum structures, 74% of human
185
structures, and between 97% (E. coli) and 52% (Oryza sativa) for the control species (Table 1, Figure
186
1B, File S1). 187 2.3. Identification of sequence and structure similarity between Plasmodium falciparum and
150
human proteins
151 ;
https://doi.org/10.1101/2023.02.08.527763
doi:
bioRxiv preprint (young ring 8 hpi, late ring/early trophozoite 16 hpi, mid trophozoite 24 hpi, late troph
172
early schizont 40 hpi, schizont 44 hpi, late schizont 48 hpi, and purified merozoites 0 h
173
from (Wichers et al., 2019). We also identified all P. falciparum proteins with expressi
174
percentile in the ring and/or sporozoite stage using data from (Zanghì et al., 2018). 175
Results
176
3.1. Assembling and filtering the datasets of crystallised and computationally-pred
177
protein structures
178
We compiled tertiary protein structures from Plasmodium falciparum 3D7 (parasite), h
179
and 15 negative control species, those not infected by the parasite (Table 1, File S1). T
180
were downloaded from the RCSB Protein Data Bank (PDB) and the AlphaFold Protein
181
database (AFdb). All AlphaFold-generated structures were filtered using the pLDDT s
182
residue metric of the confidence of prediction accuracy. In line with the AlphaFold doc
183
we considered structures to be high confidence if at least half their residues had a pLDD
184
above 70. Through this filtering, we retained 56% of P. falciparum structures, 74% of
185
structures, and between 97% (E. coli) and 52% (Oryza sativa) for the control species (T
186
1B, File S1). 187
3.2. Investigating the effect of the source of tertiary structures on Foldseek alignm
188
We wanted to determine whether the source of the tertiary structure—crystallised (PDB
189
computationally-predicted (AlphaFold) —affected the Foldseek search results. Followi
190
filtering steps, 167 P. falciparum proteins were represented by structures from both PD
191
AlphaFold and 159 aligned to at least one structure from the host and/or negative contr
192
each of these 159 proteins, we compared the Foldseek results for their PDB and AlphaF
193
structures. For most of these proteins (107/159), the results for both PDB and AlphaFo
194
agreed between 90 and 100%. For almost 10% of these proteins (15/159), the agreemen
195
results was lesser than 50% (File S2, Figure S1). 196
3.3. Structural analysis of parasite proteins experimentally known to interact with
197
proteins
198
We performed a literature review and identified 74 P. falciparum proteins that interact
199
molecules at various stages in its life-cycle (File S3). We also included three large P. fa
200
gene families which are thought to play a role in parasite virulence—PfEMP1 (61 prot
201
(158 proteins), and STEVOR (32 proteins) (File S3). 3.2. Investigating the effect of the source of tertiary structures on Foldseek alignments
188 We wanted to determine whether the source of the tertiary structure—crystallised (PDB) or
189
computationally-predicted (AlphaFold) —affected the Foldseek search results. Following our
190
filtering steps, 167 P. falciparum proteins were represented by structures from both PDB and
191
AlphaFold and 159 aligned to at least one structure from the host and/or negative control species. For
192
each of these 159 proteins, we compared the Foldseek results for their PDB and AlphaFold
193
structures. For most of these proteins (107/159), the results for both PDB and AlphaFold queries
194
agreed between 90 and 100%. For almost 10% of these proteins (15/159), the agreement between the
195
results was lesser than 50% (File S2, Figure S1). 196 2.3. Identification of sequence and structure similarity between Plasmodium falciparum and
150
human proteins
151 2.3.1. Analysis of protein sequence similarity: We analyzed sequence similarity between the proteins
152
from P. falciparum, human, and 15 negative control species. We determined sequence similarity
153
using three pairwise alignment search tools. SSEARCH36 implements the Smith-Waterman
154
algorithm guaranteeing the optimal alignment. We used the following parameters for SSEARCH36
155
from Fasta36 - ‘m 8 -s BL62 -f 12 -g 1’. BLASTP (Altschul et al., 1990) and DIAMOND (Buchfink
156
et al., 2021) implement heuristic algorithms that are faster than SSEARCH36 but do not guarantee
157
the optimal alignment. We used BLASTP with an e-value cut-off of 1e-3 and performed an ultra-
158
sensitive DIAMOND BLASTP search with the same e-value cut-off. For both aligners, we searched
159
using the BLOSUM45 and BLOSUM62 substitution matrices. All other parameters were left as
160
default. Additionally, we used OrthoFinder version 2.5.4 to identify groups of orthologous proteins
161
between all the 17 species used in this study, using DIAMOND as the aligner (Emms & Kelly, 2019). 162
The results of this analysis were used to identify human proteins that had orthologs in only the other
163
three vertebrates used in this study (mouse, rat, and zebrafish). 164 2.3.2. Analysis of protein structure similarity: We aligned all P. falciparum structures to a database
165
consisting of human structures and control structures. The structural aligner used was Foldseek v4
166
(easy-search -s 9.5 --max-seqs 1000). We used an e-value cut-off of 0.01. Foldseek was also used to
167
visualize structural alignments using the option ‘format-mode 3’. 168 2.3.3. Expression analysis: Expression analysis of P. falciparum proteins was carried out using the
169
publicly available RNA-seq datasets available in PlasmoDB. We identified all P. falciparum proteins
170
with expression in the 90th percentile in at least one of the stages in the intra-erythrocytic life cycle
171 4 4 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 9, 2023. ;
https://doi.org/10.1101/2023.02.08.527763
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 9, 2023. 2.3. Identification of sequence and structure similarity between Plasmodium falciparum and
150
human proteins
151 Overall, 206 of these proteins we
202
by at least one structure in our database of PDB and high-quality AlphaFold structures
203
understand whether molecular mimicry plays a role in how these proteins interact with
204
asked the question: do these 206 P. falciparum proteins share sequence and/or tertiary
205
similarity with human proteins? 206
We found that 31 proteins (15%) shared structural similarity with at least one human p
207
these, three proteins aligned to human proteins which were restricted in vertebrates at t
208 (young ring 8 hpi, late ring/early trophozoite 16 hpi, mid trophozoite 24 hpi, late trophozoite 32 hpi,
172
early schizont 40 hpi, schizont 44 hpi, late schizont 48 hpi, and purified merozoites 0 hpi) using data
173
from (Wichers et al., 2019). We also identified all P. falciparum proteins with expression in the 90th
174
percentile in the ring and/or sporozoite stage using data from (Zanghì et al., 2018). 175 3.3. Structural analysis of parasite proteins experimentally known to interact with human
197
proteins
198 This
249
suggests that P38, in addition to binding to GLPA, functions in adhesion owing to its similarity to the
250
Ig-like domains. 251
CyPRA: The cysteine-rich protective antigen (CyPRA, PF3D7_0423800) interacts with two other
252
parasite proteins (PfRH5 and PfRipr) to form a complex on the surface of an invading merozoite and
253
is considered a potential vaccine target (Ragotte et al., 2020). CyPRA was represented by multiple
254
structures from both the PDB and AFdb The only human protein which shared tertiary structural
255 Next, from these 31 proteins, we removed all parasite proteins which shared sequence similarity with
214
human proteins – 15, 16, and 15 proteins aligned to human proteins by BLASTP, DIAMOND, and
215
SSEARCH36 respectively (Figure 2A). Only 11 of these 31 P. falciparum proteins shared structure
216
similarity but not sequence similarity to human proteins (Figure 2A). For these 11 proteins, we
217
visually inspected their structural alignments with human proteins (File S4) and here we present the
218
biological relevance of their interactions for seven proteins. 219 Three PfEMP1 proteins: P. falciparum erythrocyte membrane protein 1 (PfEMP1) proteins primarily
220
function in adhesion of infected erythrocytes to the vasculature. Here, our results suggest potential
221
novel functions for three members of this large gene-family. Of the 61 PfEMP1 family proteins
222
analyzed in this study, six shared structural similarity, but not sequence similarity, to human proteins. 223
Two PfEMP1 proteins (PF3D7_0100300 and PF3D7_0600400) were similar to the human tyrosine-
224
protein phosphatase non-receptor type 23 (PTPN23). Additionally, PTPN23 was the second-best
225
Foldseek alignment for one more PfEMP1 protein (PF3D7_0937600). Interestingly, PTPN23
226
interacts with six human proteins that function in cytokine signaling (PTPN4, PTPN9, PTPN13,
227
PTPN14, and GRAP2), suggesting that the three PfEMP1 proteins could be involved in immune
228
modulation of the host by interfering in cytokine signaling (Figure S2). 229 AMA1: The apical membrane antigen (AMA1, PF3D7_1133400) interacts with the host erythrocyte
230
membrane transport protein Kx protein (Kato et al., 2005) and is considered a potential vaccine target
231
(Remarque et al., 2008). AMA1 has multiple crystallised structures, in addition to its AlphaFold
232
prediction. AMA1 was aligned to human plasma kallikrein protein (KLKB1), a member of the
233
plasma kallikrein–kinin system (KKS) in human. KKS proteins are known to interact with immune
234
and complement systems (Wu, 2015). 3.3. Structural analysis of parasite proteins experimentally known to interact with human
197
proteins
198 KLKB1 has also been implicated in the renin-angiotensin
235
system (RAS), where it converts prorenin to renin (Schmaier, 2003). Some studies have reported an
236
anti-malarial effect for RAS components. For instance, angiotensin II (which is formed by the
237
cleavage of angiotensin to angiotensin I by renin and conversion to angiotensin II by angiotensin-
238
converting enzyme) has been shown to inhibit different stages of multiple Plasmodium species and
239
also reduce cerebral malaria pathogenesis (reviewed in (De et al., 2022)). We propose that AMA1
240
mimics KLKB1 to prevent the formation of angiotensin II by inhibiting renin activity. 241 P38: The 6-cysteine protein/merozoite surface protein P38 (P38, PF3D7_0508000) functions in red-
242
blood cell invasion and binds to human glycophorin-A (GLPA, P02724) (Paul et al., 2017). We
243
found that P38 was structurally similar to human titin (TTN, Q8WZ42) and fibroblast growth factor
244
receptor 1 (FGFR1, P11362). The region from FGFR1 similar to P38 contained the two Ig-like
245
protein domains ‘Immunoglobulin I-set domain (PF07679)’ and the ‘Immunoglobulin domain
246
(PF00047)’. The regions similar to P38 from TTN also contained immunoglobulin-like domains. The
247
‘Immunoglobulin I-set domain’ are present in several adhesion proteins in human, where Ig-like
248
domains play an important role in homophilic cell adhesion (Leshchyns’ka & Sytnyk, 2016). This
249
suggests that P38, in addition to binding to GLPA, functions in adhesion owing to its similarity to the
250
Ig-like domains. 251 P38: The 6-cysteine protein/merozoite surface protein P38 (P38, PF3D7_0508000) functions in red-
242
blood cell invasion and binds to human glycophorin-A (GLPA, P02724) (Paul et al., 2017). We
243
found that P38 was structurally similar to human titin (TTN, Q8WZ42) and fibroblast growth factor
244
receptor 1 (FGFR1, P11362). The region from FGFR1 similar to P38 contained the two Ig-like
245
protein domains ‘Immunoglobulin I-set domain (PF07679)’ and the ‘Immunoglobulin domain
246
(PF00047)’. The regions similar to P38 from TTN also contained immunoglobulin-like domains. The
247
‘Immunoglobulin I-set domain’ are present in several adhesion proteins in human, where Ig-like
248
domains play an important role in homophilic cell adhesion (Leshchyns’ka & Sytnyk, 2016). This
249
suggests that P38, in addition to binding to GLPA, functions in adhesion owing to its similarity to the
250
Ig-like domains. 3.3. Structural analysis of parasite proteins experimentally known to interact with human
197
proteins
198 We performed a literature review and identified 74 P. falciparum proteins that interact with human
199
molecules at various stages in its life-cycle (File S3). We also included three large P. falciparum
200
gene families which are thought to play a role in parasite virulence—PfEMP1 (61 proteins), RIFIN
201
(158 proteins), and STEVOR (32 proteins) (File S3). Overall, 206 of these proteins were represented
202
by at least one structure in our database of PDB and high-quality AlphaFold structures. To
203
understand whether molecular mimicry plays a role in how these proteins interact with the host, we
204
asked the question: do these 206 P. falciparum proteins share sequence and/or tertiary structural
205
similarity with human proteins? 206 We found that 31 proteins (15%) shared structural similarity with at least one human protein. Of
207
these, three proteins aligned to human proteins which were restricted in vertebrates at the sequence-
208
level (orthologs in only mouse, rat, and/or zebrafish). They were the parasite circumsporozoite
209
protein (CSP, PF3D7_0304600) and two PfEMP1 proteins (PF3D7_0800100 and PF3D7_0617400). 210
CSP was aligned to human thrombospondin (TSP1, P07996). Interestingly, as per previous sequence-
211
based approaches, CSP mimics a cytoadhesive region in mammalian thrombospondin (Cerami et al.,
212
1992; Robson et al., 1988). 213 5 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 9, 2023. ;
https://doi.org/10.1101/2023.02.08.527763
doi:
bioRxiv preprint Next, from these 31 proteins, we removed all parasite proteins which shared sequence similarity with
214
human proteins – 15, 16, and 15 proteins aligned to human proteins by BLASTP, DIAMOND, and
215
SSEARCH36 respectively (Figure 2A). Only 11 of these 31 P. falciparum proteins shared structure
216
similarity but not sequence similarity to human proteins (Figure 2A). For these 11 proteins, we
217
visually inspected their structural alignments with human proteins (File S4) and here we present the
218
biological relevance of their interactions for seven proteins. 219
Three PfEMP1 proteins: P. falciparum erythrocyte membrane protein 1 (PfEMP1) proteins primarily
220
function in adhesion of infected erythrocytes to the vasculature. Here, our results suggest potential
221
novel functions for three members of this large gene-family. 3.3. Structural analysis of parasite proteins experimentally known to interact with human
197
proteins
198 Of the 61 PfEMP1 family proteins
222
analyzed in this study, six shared structural similarity, but not sequence similarity, to human proteins. 223
Two PfEMP1 proteins (PF3D7_0100300 and PF3D7_0600400) were similar to the human tyrosine-
224
protein phosphatase non-receptor type 23 (PTPN23). Additionally, PTPN23 was the second-best
225
Foldseek alignment for one more PfEMP1 protein (PF3D7_0937600). Interestingly, PTPN23
226
interacts with six human proteins that function in cytokine signaling (PTPN4, PTPN9, PTPN13,
227
PTPN14, and GRAP2), suggesting that the three PfEMP1 proteins could be involved in immune
228
modulation of the host by interfering in cytokine signaling (Figure S2). 229
AMA1: The apical membrane antigen (AMA1, PF3D7_1133400) interacts with the host erythrocyte
230
membrane transport protein Kx protein (Kato et al., 2005) and is considered a potential vaccine target
231
(Remarque et al., 2008). AMA1 has multiple crystallised structures, in addition to its AlphaFold
232
prediction. AMA1 was aligned to human plasma kallikrein protein (KLKB1), a member of the
233
plasma kallikrein–kinin system (KKS) in human. KKS proteins are known to interact with immune
234
and complement systems (Wu, 2015). KLKB1 has also been implicated in the renin-angiotensin
235
system (RAS), where it converts prorenin to renin (Schmaier, 2003). Some studies have reported an
236
anti-malarial effect for RAS components. For instance, angiotensin II (which is formed by the
237
cleavage of angiotensin to angiotensin I by renin and conversion to angiotensin II by angiotensin-
238
converting enzyme) has been shown to inhibit different stages of multiple Plasmodium species and
239
also reduce cerebral malaria pathogenesis (reviewed in (De et al., 2022)). We propose that AMA1
240
mimics KLKB1 to prevent the formation of angiotensin II by inhibiting renin activity. 241
P38: The 6-cysteine protein/merozoite surface protein P38 (P38, PF3D7_0508000) functions in red-
242
blood cell invasion and binds to human glycophorin-A (GLPA, P02724) (Paul et al., 2017). We
243
found that P38 was structurally similar to human titin (TTN, Q8WZ42) and fibroblast growth factor
244
receptor 1 (FGFR1, P11362). The region from FGFR1 similar to P38 contained the two Ig-like
245
protein domains ‘Immunoglobulin I-set domain (PF07679)’ and the ‘Immunoglobulin domain
246
(PF00047)’. The regions similar to P38 from TTN also contained immunoglobulin-like domains. The
247
‘Immunoglobulin I-set domain’ are present in several adhesion proteins in human, where Ig-like
248
domains play an important role in homophilic cell adhesion (Leshchyns’ka & Sytnyk, 2016). 3.3. Structural analysis of parasite proteins experimentally known to interact with human
197
proteins
198 284
The perforin-like protein 3 (PLP3, PF3D7_0923300) was aligned to human macrophage-expressed
285
gene 1 (MPEG1, Q2M385), which functions in the host innate immune response. While PLP is
286
expressed in both sexual and asexual stages of the parasite, it primarily functions in the mosquito-
287
stage of the parasite (S. Garg et al., 2020). Thus, it is possible that PLP3 has an additional function in
288
the human host to avoid the immune response. 289
Nearly one-fifth of the P. falciparum proteins that shared structural similarity to human proteins
290
(386/2,120) had no detectable sequence similarity to a human protein (File S5). On average, 352 P. 291
falciparum proteins had structural similarity but no sequence similarity to the control proteins. 292
Overall, such P. falciparum proteins with structure similarity but not sequence similarity were not the
293
same for each of these 16 species (Figure S4). 294
To prioritise the proteins, we categorized them with available annotations from PlasmoDB. Category
295
1 was predicted function, where the top scoring alignment was with a human protein with a GO term
296
of interest
‘immune system process’ ‘cell adhesion’ ‘cytoskeleton’ or ‘signalling’ Category 2
297 in ‘MAPK family signaling cascades’. The next best alignment was mitogen-activated protein kinase
259
kinase kinase kinase 4 (MAP4K4, O95819), which also functions in the MAPK signaling. Thus, in
260
addition to its key role in red blood cell invasion, structural similarity analysis suggests a possible
261
novel role for CyPRA in modulating the host MAPK signaling pathway. 262 in ‘MAPK family signaling cascades’. The next best alignment was mitogen-activated protein kinase
259
kinase kinase kinase 4 (MAP4K4, O95819), which also functions in the MAPK signaling. Thus, in
260
addition to its key role in red blood cell invasion, structural similarity analysis suggests a possible
261
novel role for CyPRA in modulating the host MAPK signaling pathway. 262 SHLP2: In at least one instance, our approach identified structural similarity in protein domains
263
between the parasite and human proteins, which was missed by sequence similarity searches. 264
Foldseek aligned the region containing the ‘Metallophos’ protein domain between the parasite
265
protein ‘Shewanella-like protein phosphatase 2’ (SHLP2, PF3D7_1206000) and the human
266
phosphatase 'Serine/threonine-protein phosphatase with EF-hands 1’ (PPEF1, O14829). 267 3.3. Structural analysis of parasite proteins experimentally known to interact with human
197
proteins
198 Category 3 was the gene’s expression, where we selected proteins whose genes was
299
expressed in the 90th percentile in at least one of the human life-cycle stages of the parasite—
300
sporozoite and ring—or one of the intraerythrocytic stages (Wichers et al., 2019; Zanghì et al., 2018). 301 in ‘MAPK family signaling cascades’. The next best alignment was mitogen-activated protein kinase
259
kinase kinase kinase 4 (MAP4K4, O95819), which also functions in the MAPK signaling. Thus, in
260
addition to its key role in red blood cell invasion, structural similarity analysis suggests a possible
261
novel role for CyPRA in modulating the host MAPK signaling pathway. 262
SHLP2: In at least one instance, our approach identified structural similarity in protein domains
263
between the parasite and human proteins, which was missed by sequence similarity searches. 264
Foldseek aligned the region containing the ‘Metallophos’ protein domain between the parasite
265
protein ‘Shewanella-like protein phosphatase 2’ (SHLP2, PF3D7_1206000) and the human
266
phosphatase 'Serine/threonine-protein phosphatase with EF-hands 1’ (PPEF1, O14829). 267
3.4. Large-scale analysis of the structural similarity between Plasmodium falciparum and
268
human proteins
269
The previous section shows that our approach can identify instances of molecular mimicry which
270
mediate host-Plasmodium interactions. This motivated us to extend the approach to search all tertiary
271
structures from P. falciparum against a database of 415,164 structures from human and the 15
272
negative control species. Of the parasite’s 4,326 tertiary structures, 3,649 (84%) were aligned to a
273
structure from human and/or negative control species, and 3,350 (77%) could be aligned to a human
274
structure. 275
Of these 3,350 structures, 59 structures were similar to only vertebrate proteins, and 27 aligned to
276
only mammalian structures. Only eight structures aligned exclusively to a human structure. We
277
further examined the structural alignments of these eight structures and identified potential novel
278
biological functions for some of them. As mentioned above (section 3.3), the parasite protein CSP
279
was aligned to human thrombospondin-1. The parasite protein 40S ribosomal protein S30 (RPS30,
280
PF3D7_0219200) was shared tertiary structural similarity to human FAU ubiquitin-like and
281
ribosomal protein S30 (FAU, P62861), which functions in apoptosis (Pickard et al., 2011) and is
282
mapped to the GO term ‘innate immune response in mucosa’ in UniProt. This suggests that RPS30
283
could provide a biological advantage to P. falciparum by modulating the human immune response. 3.3. Structural analysis of parasite proteins experimentally known to interact with human
197
proteins
198 As mentioned above
279
was aligned to human thrombospondin-1. The parasite prot
280
PF3D7_0219200) was shared tertiary structural similarity t
281
ribosomal protein S30 (FAU, P62861), which functions in
282
mapped to the GO term ‘innate immune response in mucos
283
could provide a biological advantage to P. falciparum by m
284
The perforin-like protein 3 (PLP3, PF3D7_0923300) was a
285
gene 1 (MPEG1, Q2M385), which functions in the host inn
286
expressed in both sexual and asexual stages of the parasite,
287
stage of the parasite (S. Garg et al., 2020). Thus, it is possib
288
the human host to avoid the immune response. 289
Nearly one-fifth of the P. falciparum proteins that shared st
290
(386/2,120) had no detectable sequence similarity to a hum
291
falciparum proteins had structural similarity but no sequenc
292
Overall, such P. falciparum proteins with structure similari
293
same for each of these 16 species (Figure S4). 294
To prioritise the proteins, we categorized them with availab
295
1 was predicted function, where the top scoring alignment w
296
of interest—‘immune system process’, ‘cell adhesion’, ‘cyt
297
was likely export from the cell, where the P. falciparum pro
298
peptide. Category 3 was the gene’s expression, where we s
299 in ‘MAPK family signaling cascades’. The next best alignment was mitogen-activated protein kinase
259
kinase kinase kinase 4 (MAP4K4, O95819), which also functions in the MAPK signaling. Thus, in
260
addition to its key role in red blood cell invasion, structural similarity analysis suggests a possible
261
novel role for CyPRA in modulating the host MAPK signaling pathway. 262
SHLP2: In at least one instance, our approach identified structural similarity in protein domains
263
between the parasite and human proteins, which was missed by sequence similarity searches. 264
Foldseek aligned the region containing the ‘Metallophos’ protein domain between the parasite
265
protein ‘Shewanella-like protein phosphatase 2’ (SHLP2, PF3D7_1206000) and the human
266
phosphatase 'Serine/threonine-protein phosphatase with EF-hands 1’ (PPEF1, O14829). 267
3.4. Large-scale analysis of the structural similarity between Plasmodium falciparum and
268
human proteins
269
The previous section shows that our approach can identify instances of molecular mimicry which
270
mediate host-Plasmodium interactions. This motivated us to extend the approach to search all tertiary
271
structures from P. falciparum against a database of 415,164 structures from human and the 15
272
negative control species. 3.3. Structural analysis of parasite proteins experimentally known to interact with human
197
proteins
198 Of the parasite’s 4,326 tertiary structures, 3,649 (84%) were aligned to a
273
structure from human and/or negative control species, and 3,350 (77%) could be aligned to a human
274
structure. 275
Of these 3,350 structures, 59 structures were similar to only vertebrate proteins, and 27 aligned to
276
only mammalian structures. Only eight structures aligned exclusively to a human structure. We
277
further examined the structural alignments of these eight structures and identified potential novel
278
biological functions for some of them. As mentioned above (section 3.3), the parasite protein CSP
279
was aligned to human thrombospondin-1. The parasite protein 40S ribosomal protein S30 (RPS30,
280
PF3D7_0219200) was shared tertiary structural similarity to human FAU ubiquitin-like and
281
ribosomal protein S30 (FAU, P62861), which functions in apoptosis (Pickard et al., 2011) and is
282
mapped to the GO term ‘innate immune response in mucosa’ in UniProt. This suggests that RPS30
283
could provide a biological advantage to P. falciparum by modulating the human immune response. 284
The perforin-like protein 3 (PLP3, PF3D7_0923300) was aligned to human macrophage-expressed
285
gene 1 (MPEG1, Q2M385), which functions in the host innate immune response. While PLP is
286
expressed in both sexual and asexual stages of the parasite, it primarily functions in the mosquito-
287
stage of the parasite (S. Garg et al., 2020). Thus, it is possible that PLP3 has an additional function in
288
the human host to avoid the immune response. 289
Nearly one-fifth of the P. falciparum proteins that shared structural similarity to human proteins
290
(386/2,120) had no detectable sequence similarity to a human protein (File S5). On average, 352 P. 291
falciparum proteins had structural similarity but no sequence similarity to the control proteins. 292
Overall, such P. falciparum proteins with structure similarity but not sequence similarity were not the
293
same for each of these 16 species (Figure S4). 294
To prioritise the proteins, we categorized them with available annotations from PlasmoDB. Category
295
1 was predicted function, where the top scoring alignment was with a human protein with a GO term
296
of interest—‘immune system process’, ‘cell adhesion’, ‘cytoskeleton’, or ‘signalling’. Category 2
297
was likely export from the cell, where the P. falciparum protein was predicted to contain a signal
298
peptide. 3.4. Large-scale analysis of the structural similarity between Plasmodium falciparum and
268
human proteins
269 The previous section shows that our approach can identify instances of molecular mimicry which
270
mediate host-Plasmodium interactions. This motivated us to extend the approach to search all tertiary
271
structures from P. falciparum against a database of 415,164 structures from human and the 15
272
negative control species. Of the parasite’s 4,326 tertiary structures, 3,649 (84%) were aligned to a
273
structure from human and/or negative control species, and 3,350 (77%) could be aligned to a human
274
structure. 275 Of these 3,350 structures, 59 structures were similar to only vertebrate proteins, and 27 aligned to
276
only mammalian structures. Only eight structures aligned exclusively to a human structure. We
277
further examined the structural alignments of these eight structures and identified potential novel
278
biological functions for some of them. As mentioned above (section 3.3), the parasite protein CSP
279
was aligned to human thrombospondin-1. The parasite protein 40S ribosomal protein S30 (RPS30,
280
PF3D7_0219200) was shared tertiary structural similarity to human FAU ubiquitin-like and
281
ribosomal protein S30 (FAU, P62861), which functions in apoptosis (Pickard et al., 2011) and is
282
mapped to the GO term ‘innate immune response in mucosa’ in UniProt. This suggests that RPS30
283
could provide a biological advantage to P. falciparum by modulating the human immune response. 284
The perforin-like protein 3 (PLP3, PF3D7_0923300) was aligned to human macrophage-expressed
285
gene 1 (MPEG1, Q2M385), which functions in the host innate immune response. While PLP is
286
expressed in both sexual and asexual stages of the parasite, it primarily functions in the mosquito-
287
stage of the parasite (S. Garg et al., 2020). Thus, it is possible that PLP3 has an additional function in
288
the human host to avoid the immune response. 289 Nearly one-fifth of the P. falciparum proteins that shared structural similarity to human proteins
290
(386/2,120) had no detectable sequence similarity to a human protein (File S5). On average, 352 P. 291
falciparum proteins had structural similarity but no sequence similarity to the control proteins. 292
Overall, such P. falciparum proteins with structure similarity but not sequence similarity were not the
293
same for each of these 16 species (Figure S4). 294 Nearly one-fifth of the P. falciparum proteins that shared structural similarity to human proteins
290
(386/2,120) had no detectable sequence similarity to a human protein (File S5). On average, 352 P. 3.3. Structural analysis of parasite proteins experimentally known to interact with human
197
proteins
198 251 CyPRA: The cysteine-rich protective antigen (CyPRA, PF3D7_0423800) interacts with two other
252
parasite proteins (PfRH5 and PfRipr) to form a complex on the surface of an invading merozoite and
253
is considered a potential vaccine target (Ragotte et al., 2020). CyPRA was represented by multiple
254
structures from both the PDB and AFdb. The only human protein which shared tertiary structural
255
similarity to both PDB and AlphaFold structures was the V(D)J recombination-activating protein 2
256
(RAG2, P55895) (Figure 2B). We identified the human interacting partners of RAG2 using StringDB
257
(Figure S3). Seven interactors functioned in ‘signaling by Interleukins’, of which four were involved
258 6 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 9, 2023. ;
https://doi.org/10.1101/2023.02.08.527763
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 9, 2023. ;
https://doi.org/10.1101/2023.02.08.527763
doi:
bioRxiv preprint in ‘MAPK family signaling cascades’. The next best alignm
259
kinase kinase kinase 4 (MAP4K4, O95819), which also fun
260
addition to its key role in red blood cell invasion, structural
261
novel role for CyPRA in modulating the host MAPK signa
262
SHLP2: In at least one instance, our approach identified str
263
between the parasite and human proteins, which was misse
264
Foldseek aligned the region containing the ‘Metallophos’ p
265
protein ‘Shewanella-like protein phosphatase 2’ (SHLP2, P
266
phosphatase 'Serine/threonine-protein phosphatase with EF
267
3.4. Large-scale analysis of the structural similarity betw
268
human proteins
269
The previous section shows that our approach can identify
270
mediate host-Plasmodium interactions. This motivated us t
271
structures from P. falciparum against a database of 415,164
272
negative control species. Of the parasite’s 4,326 tertiary str
273
structure from human and/or negative control species, and
274
structure. 275
Of these 3,350 structures, 59 structures were similar to only
276
only mammalian structures. Only eight structures aligned e
277
further examined the structural alignments of these eight st
278
biological functions for some of them. 3.4. Large-scale analysis of the structural similarity between Plasmodium falciparum and
268
human proteins
269 The fourth example is sporozoite invasion-
327
associated protein 1 (SIAP1, PF3D7_0408600), which was aligned to human collagen alpha-1(VII)
328
chain (COL7A1, Q02388). StringDB identified seven proteins that interact with COL7A1 and are
329
mapped to the GO term ‘cell adhesion’ (GO:0007155) (Figure S7). Additionally, six proteins were
330
mapped to the KEGG pathway ‘ECM-receptor interaction’ (hsa04512). Thus, we posit that SIAP1
331
could play a role in adhesion of the parasite to the host extracellular matrix. 332
An additional 35 parasite proteins were present in two of the three categories listed above. All
333
warrant further investigation, but two are noteworthy. First, the sporozoite surface protein P36
334
(PF3D7_0404500) was similar to human PIGR, like two other members of the 6-cysteine protein
335
family - P12 and P41. In fact, P36 was aligned to a similar region in PIGR compared to P12 and P41
336
This suggests that P12 potentially binds to IgM and helps the parasite evade the host immune
337
response. When we analyzed all Foldseek alignments (not just the best hits), we found that an
338
additional four 6-cysteine proteins were also similar to PIGR - 6-cysteine protein P12p (P12p,
339
PF3D7_0612800), 6-cysteine protein P52 (P52, PF3D7_0404500), 6-cysteine protein (P48/45,
340
PF3D7_1346700), and 6-cysteine protein P47 (P47, PF3D7_1346800). Second, the LamG domain-
341
containing protein (PF3D7_0723200), which possesses a signal peptide, shared tertiary structure
342
similarity to the human adhesion G-protein coupled receptor V1 (ADGRV1, Q8WXG9), a cell-
343
surface protein involved in cell adhesion. StringDB found that five of its interacting partners function
344
in ‘cell-cell adhesion’ (GO:0098609), suggesting that the P. falciparum protein assists parasite
345
infection and survival by functioning in cell adhesion. 346 A total of 169 P. falciparum proteins could be placed in at least one of the categories, with 97
302
proteins in category 1, 43 in category 2, 95 in category 3, 38 in two categories, and 13 proteins in all
303
three categories (Figure S5, Table 2). Seven of the nine top human proteins aligned to these P. 304
falciparum 13 proteins were taxonomically restricted to vertebrates. 305 The 51 P. falciparum proteins present in more than category represent instances of mimicry (File
306
S6). Here, we present the biological relevance of a subset of these proteins. The four 6-cysteine
307
proteins present in these 13 proteins are one of the most abundant surface antigens in the P. 3.4. Large-scale analysis of the structural similarity between Plasmodium falciparum and
268
human proteins
269 291
falciparum proteins had structural similarity but no sequence similarity to the control proteins. 292
Overall, such P. falciparum proteins with structure similarity but not sequence similarity were not the
293
same for each of these 16 species (Figure S4). 294 To prioritise the proteins, we categorized them with available annotations from PlasmoDB. Category
295
1 was predicted function, where the top scoring alignment was with a human protein with a GO term
296
of interest—‘immune system process’, ‘cell adhesion’, ‘cytoskeleton’, or ‘signalling’. Category 2
297
was likely export from the cell, where the P. falciparum protein was predicted to contain a signal
298
peptide. Category 3 was the gene’s expression, where we selected proteins whose genes was
299
expressed in the 90th percentile in at least one of the human life-cycle stages of the parasite—
300
sporozoite and ring—or one of the intraerythrocytic stages (Wichers et al., 2019; Zanghì et al., 2018). 301 To prioritise the proteins, we categorized them with available annotations from PlasmoDB. Category
295
1 was predicted function, where the top scoring alignment was with a human protein with a GO term
296
of interest—‘immune system process’, ‘cell adhesion’, ‘cytoskeleton’, or ‘signalling’. Category 2
297
was likely export from the cell, where the P. falciparum protein was predicted to contain a signal
298
peptide. Category 3 was the gene’s expression, where we selected proteins whose genes was
299
expressed in the 90th percentile in at least one of the human life-cycle stages of the parasite—
300
sporozoite and ring—or one of the intraerythrocytic stages (Wichers et al., 2019; Zanghì et al., 2018). 301 7 7 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 9, 2023. ;
https://doi.org/10.1101/2023.02.08.527763
doi:
bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 9, 2023. ;
https://doi.org/10.1101/2023.02.08.527763
doi:
bioRxiv preprint A total of 169 P. 3.4. Large-scale analysis of the structural similarity between Plasmodium falciparum and
268
human proteins
269 falciparum proteins could be placed in at least one of the categories, with 97
302
proteins in category 1, 43 in category 2, 95 in category 3, 38 in two categories, and 13 proteins in all
303
three categories (Figure S5, Table 2). Seven of the nine top human proteins aligned to these P. 304
falciparum 13 proteins were taxonomically restricted to vertebrates. 305
The 51 P. falciparum proteins present in more than category represent instances of mimicry (File
306
S6). Here, we present the biological relevance of a subset of these proteins. The four 6-cysteine
307
proteins present in these 13 proteins are one of the most abundant surface antigens in the P. 308
falciparum (Lyons et al., 2022) and have been attributed to virulence in related parasites (Wasmuth e
309
al., 2012). These are promising candidates for mediators of host-parasite interactions. The first
310
example was the P92/merozoite surface protein (P92, PF3D7_1364100), which interacts with human
311
Factor H to downregulate the alternative complement pathway (Kennedy et al., 2016). We found that
312
P92 shared structural similarity to human T-cell surface protein tactile (CD96, P40200), a
313
transmembrane protein that is expressed by both T and NK cells (Georgiev et al., 2018). 314
Interestingly, multiple roles for NK cells have been proposed at different stages of P. falciparum
315
infection, including cytotoxic activity against the erythrocyte and hepatocyte stages (reviewed in
316
(Wolf et al., 2017)). Additionally, six of the proteins CD96 interacts with function in immune
317
response (GO:0006955) (Figure S6). Therefore, it is possible that P92 assists parasite infection by
318
modulating the human immune response. 319
The second and third examples are the 6-cysteine protein P12/merozoite surface protein P12 (P12,
320
PF3D7_0612700) and merozoite surface protein 41 (P41, PF3D7_0404900), which were similar to
321
human polymeric immunoglobulin receptor PIGR (PIGR, P01833). PIGR is a known receptor for
322
immunoglobulin M (IgM) (Pleass et al., 2016) and IgM is known to block the invasion of red blood
323
cells by Plasmodium merozoites (Oyong et al., 2019). In fact, four other P. falciparum proteins -
324
VAR2CSA, TM284VAR1, DBLMSP, and DBLMSP2, bind to IgM and affect IgM-mediated
325
complement activation for evading the human immune response (Ji et al., 2022). Our structure-based
326
approach suggests a similar role for P12 and P41. 3.4. Large-scale analysis of the structural similarity between Plasmodium falciparum and
268
human proteins
269 308
falciparum (Lyons et al., 2022) and have been attributed to virulence in related parasites (Wasmuth et
309
al., 2012). These are promising candidates for mediators of host-parasite interactions. The first
310
example was the P92/merozoite surface protein (P92, PF3D7_1364100), which interacts with human
311
Factor H to downregulate the alternative complement pathway (Kennedy et al., 2016). We found that
312
P92 shared structural similarity to human T-cell surface protein tactile (CD96, P40200), a
313
transmembrane protein that is expressed by both T and NK cells (Georgiev et al., 2018). 314
Interestingly, multiple roles for NK cells have been proposed at different stages of P. falciparum
315
infection, including cytotoxic activity against the erythrocyte and hepatocyte stages (reviewed in
316
(Wolf et al., 2017)). Additionally, six of the proteins CD96 interacts with function in immune
317
response (GO:0006955) (Figure S6). Therefore, it is possible that P92 assists parasite infection by
318
modulating the human immune response. 319 The second and third examples are the 6-cysteine protein P12/merozoite surface protein P12 (P12,
320
PF3D7_0612700) and merozoite surface protein 41 (P41, PF3D7_0404900), which were similar to
321
human polymeric immunoglobulin receptor PIGR (PIGR, P01833). PIGR is a known receptor for
322
immunoglobulin M (IgM) (Pleass et al., 2016) and IgM is known to block the invasion of red blood
323
cells by Plasmodium merozoites (Oyong et al., 2019). In fact, four other P. falciparum proteins -
324
VAR2CSA, TM284VAR1, DBLMSP, and DBLMSP2, bind to IgM and affect IgM-mediated
325
complement activation for evading the human immune response (Ji et al., 2022). Our structure-based
326
approach suggests a similar role for P12 and P41. The fourth example is sporozoite invasion-
327
associated protein 1 (SIAP1, PF3D7_0408600), which was aligned to human collagen alpha-1(VII)
328
chain (COL7A1, Q02388). StringDB identified seven proteins that interact with COL7A1 and are
329
mapped to the GO term ‘cell adhesion’ (GO:0007155) (Figure S7). Additionally, six proteins were
330
mapped to the KEGG pathway ‘ECM-receptor interaction’ (hsa04512). Thus, we posit that SIAP1
331
could play a role in adhesion of the parasite to the host extracellular matrix. 332 An additional 35 parasite proteins were present in two of the three categories listed above. All
333
warrant further investigation, but two are noteworthy. First, the sporozoite surface protein P36
334
(PF3D7_0404500) was similar to human PIGR, like two other members of the 6-cysteine protein
335
family - P12 and P41. Discussion
347 In this study, we present, to the best of our knowledge, the first genome-scale search of tertiary
348
structural similarity between P. falciparum proteins and human proteins. We demonstrate the
349
usefulness of our approach by showing that approximately 7% of the P. falciparum proteins had
350
similarity to human proteins that could be detected only at the level of tertiary structure, and not
351
primary sequence. Using available molecular and -omics datasets from PlasmoDB, we shortlisted a
352
set of 51 instances of mimicry that are candidate mediators of host-parasite interactions. 353 We compared whether the source of the structural model made a difference. It made a considerable
354
difference (<50% agreement) for 10% of proteins. We emphasize that this is for AlphaFold structures
355
that were filtered for high confidence; we anticipate that for lower confidence AlphaFold structures,
356
an agreement with a PDB structure would be worse. Improvement in the AlphaFold software and its
357
evolutionary models will lead to better structural models, but users must remain vigilant of accuracy
358
metrics when using this promising resource. If a particular gene of interest has a protein structural
359
model available in the PDB, we recommend incorporating it in the analysis. A recent notable study
360
attempted to overcome this challenge by improving AlphaFold predictions for two parasites -
361
Trypanosoma cruzi and Leishmania infantum (Wheeler, 2021). They attributed the poor accuracy of
362
tertiary structure prediction for several parasite proteins to the low number of representative parasite
363
sequences used to predict them and demonstrated that they could improve the AlphaFold predictions
364
by increasing the number of parasite sequences used to model these structures. 365 We validated our approach on known mediators of host-parasite interactions and we identified known
366
similarity between CSP and thrombospondin-1. We also identified potential novel functions of
367
multiple parasite proteins, which improves our understanding of existing P. falciparum-human
368
interactions. One promising example involves multiple members of the 6-cysteine proteins found in
369
P. falciparum. Members of this gene-family are expressed in multiple stages of the P. falciparum
370
life-cycle in both the hosts (Lyons et al., 2022). Interestingly, while several members of this family
371
have been implicated in critical functions like immune-evasion and host cell invasion, the precise
372
roles for several of these proteins, like P12, P12p, P38, and P41, remain unknown (Lyons et al.,
373
2022). 3.4. Large-scale analysis of the structural similarity between Plasmodium falciparum and
268
human proteins
269 In fact, P36 was aligned to a similar region in PIGR compared to P12 and P41. 336
This suggests that P12 potentially binds to IgM and helps the parasite evade the host immune
337
response. When we analyzed all Foldseek alignments (not just the best hits), we found that an
338
additional four 6-cysteine proteins were also similar to PIGR - 6-cysteine protein P12p (P12p,
339
PF3D7_0612800), 6-cysteine protein P52 (P52, PF3D7_0404500), 6-cysteine protein (P48/45,
340
PF3D7_1346700), and 6-cysteine protein P47 (P47, PF3D7_1346800). Second, the LamG domain-
341
containing protein (PF3D7_0723200), which possesses a signal peptide, shared tertiary structure
342
similarity to the human adhesion G-protein coupled receptor V1 (ADGRV1, Q8WXG9), a cell-
343
surface protein involved in cell adhesion. StringDB found that five of its interacting partners function
344
in ‘cell-cell adhesion’ (GO:0098609), suggesting that the P. falciparum protein assists parasite
345
infection and survival by functioning in cell adhesion. 346 8 8 . CC-BY 4.0 International license
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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bioRxiv preprint Discussion
347 This is unfortunate as instances of
393
mimicry have been identified in such disordered regions. For example, it has been proposed that
394
viruses modulate host cellular processes by mimicking regions in disordered regions (Dolan et al.,
395
2015; A. Garg et al., 2022; Xue et al., 2014). Such mimicking regions in disordered regions can be
396
potentially identified by supplementing our structure-based approach with a k-mer based approach. 397 Second, since we discarded AlphaFold predictions with low prediction accuracy, we ended up
392
removing structures with a high level of intrinsic protein disorder. This is unfortunate as instances of
393
mimicry have been identified in such disordered regions. For example, it has been proposed that
394
viruses modulate host cellular processes by mimicking regions in disordered regions (Dolan et al.,
395
2015; A. Garg et al., 2022; Xue et al., 2014). Such mimicking regions in disordered regions can be
396
potentially identified by supplementing our structure-based approach with a k-mer based approach. 397
Third, the influence of the structure aligner must be considered. Foldseek is orders of magnitude
398
faster than other currently available structural aligners and the only software that allows, in a
399
reasonable time, the type of comparison presented here. However, the trade-off in terms of accuracy
400
is a matter of debate (Holm, 2022; van Kempen et al., 2022). The alignment speed of DALI makes it
401
unsuitable for large-scale structural similarity searches. 402
In conclusion, we present, to the best of our knowledge, the first genome-level search of tertiary
403
structure similarity between P. falciparum and human proteins and use the results of the search to
404
identify instances of molecular mimicry between the parasite and the host. The extensive
405
experimental data for P. falciparum on PlasmoDB guided our efforts to unearth the biological
406
relevance of the identified similarities. The list of P. falciparum mimics catalogued in our study
407
represent excellent candidates for experimental validation. Our results help further our insights into
408
the molecular nature of Plasmodium-human interactions and will be important to inform efforts on
409
developing vaccines and therapeutics against malaria. 410 Third, the influence of the structure aligner must be considered. Foldseek is orders of magnitude
398
faster than other currently available structural aligners and the only software that allows, in a
399
reasonable time, the type of comparison presented here. Discussion
347 Therefore, our structure-similarity-based approach improved our knowledge of how some of
374
these proteins contribute to immune evasion, by identifying a potential binding-partner (IgM) and
375
function for these proteins. 376 There are four important points to consider when interpreting the results of this analysis. First, we
377
noticed that the number of parasite proteins with structural similarity, but not sequence similarity, to
378
human and negative control proteins was similar for the majority of species studied. Some these
379
would be distant homologs which are not detected by sequence-based tools employed in this study. 380
Indeed, the failure of homology detection has been shown to be one of the important reasons for the
381
presence of lineage-specific genes (Weisman et al., 2020). In fact, multiple studies have
382
demonstrated the effectiveness of using structure data to identify distant homologs that are missed by
383
sequence-based approaches (Andorf et al., 2022; Monzon et al., 2022). Other proteins, however,
384
would represent false-positives of our approach based on our definition of ‘molecular mimics’, in
385
which we define that a mimic confers benefit to the parasite. To this end, we used experimental data
386
on P. falciparum from PDB to shortlist 46 proteins in P. falciparum that are structurally similar to
387
human proteins and have a high probability of benefiting the parasite via its mimicry of a human
388
protein. It is important to note that the majority of parasites lack similar resources, making such an
389
analysis for them problematic. This highlights the need to develop resources, like PlasmoDB, for
390
other parasites of global health concern. 391 9 . CC-BY 4.0 International license
available under a
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The copyright holder for this preprint
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bioRxiv preprint . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 9, 2023. ;
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bioRxiv preprint Second, since we discarded AlphaFold predictions with low prediction accuracy, we ended up
392
removing structures with a high level of intrinsic protein disorder. Discussion
347 However, the trade-off in terms of accuracy
400
is a matter of debate (Holm, 2022; van Kempen et al., 2022). The alignment speed of DALI makes it
401
unsuitable for large-scale structural similarity searches. 402 In conclusion, we present, to the best of our knowledge, the first genome-level search of tertiary
403
structure similarity between P. falciparum and human proteins and use the results of the search to
404
identify instances of molecular mimicry between the parasite and the host. The extensive
405
experimental data for P. falciparum on PlasmoDB guided our efforts to unearth the biological
406
relevance of the identified similarities. The list of P. falciparum mimics catalogued in our study
407
represent excellent candidates for experimental validation. Our results help further our insights into
408
the molecular nature of Plasmodium-human interactions and will be important to inform efforts on
409
developing vaccines and therapeutics against malaria. 410 Figures
411 412
Figure 1 A) The 7F9N protein tertiary structure from the RCSB Protein Data Bank. The chains from
413
the P. falciparum RIFIN protein (RIF, PF3D7_1000500) are colored in green and the chains from the
414
human LAIR protein (LAIR1, Q6GTX8) are colored in purple. B) Box-plot representing the overall
415
prediction accuracy for each protein in the AFdb. Each point in this distribution represents the
416
proportion of all residues with a pLDDT score above 70 for one AlphaFold structure. The median
417
value is the parallel bar within the box. The limits of the box are the 25th and 75th percentiles, the
418
whiskers extend 1.5 times the interquartile range, and the dots are the outliers. 419 412 Figure 1 A) The 7F9N protein tertiary structure from the RCSB Protein Data Bank. The chains from
413
the P. falciparum RIFIN protein (RIF, PF3D7_1000500) are colored in green and the chains from the
414
human LAIR protein (LAIR1, Q6GTX8) are colored in purple. B) Box-plot representing the overall
415
prediction accuracy for each protein in the AFdb. Each point in this distribution represents the
416
proportion of all residues with a pLDDT score above 70 for one AlphaFold structure. The median
417
value is the parallel bar within the box. The limits of the box are the 25th and 75th percentiles, the
418
whiskers extend 1.5 times the interquartile range, and the dots are the outliers. 419 10 . CC-BY 4.0 International license
available under a
which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
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bioRxiv preprint Figure 2 A) An UpSet plot of the 206 parasite proteins that interact with human molecules and are
represented by at least one structure in our database. This plot displays the number of these
interacting parasite proteins aligned to human proteins by DIAMOND, BLAST, Foldseek, and/or
SSEARCH36. B) Tertiary structure alignments of the parasite structures (gray) and human structure
(red). Figures
411 These alignments were generated and visualized using Foldseek. 420 Figure 2 A) An UpSet plot of the 206 parasite proteins that interact with human molecules and are
421
represented by at least one structure in our database. This plot displays the number of these
422
interacting parasite proteins aligned to human proteins by DIAMOND, BLAST, Foldseek, and/or
423
SSEARCH36. B) Tertiary structure alignments of the parasite structures (gray) and human structures
424
(red). These alignments were generated and visualized using Foldseek. 425 Figure 2 A) An UpSet plot of the 206 parasite proteins that interact with human molecules and are
421
represented by at least one structure in our database. This plot displays the number of these
422
interacting parasite proteins aligned to human proteins by DIAMOND, BLAST, Foldseek, and/or
423
SSEARCH36. B) Tertiary structure alignments of the parasite structures (gray) and human structures
424
(red). These alignments were generated and visualized using Foldseek. 425 11 . CC-BY 4.0 International license
available under a
which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
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bioRxiv preprint 426
Figure 3 An UpSet plot of all Plasmodium falciparum proteins which were aligned to human
427
proteins by Foldseek, DIAMOND, BLASTP, and/or SSEARCH36. 428 426
Figure 3 An UpSet plot of all Plasmodium falciparum proteins which were aligned to human
427
proteins by Foldseek, DIAMOND, BLASTP, and/or SSEARCH36. 428 426 Figure 3 An UpSet plot of all Plasmodium falciparum proteins which were aligned to human
427
proteins by Foldseek, DIAMOND, BLASTP, and/or SSEARCH36. 428 Tables
429 Table 1 The number of tertiary protein structures for each species used in this study. Structures were
430
downloaded from two sources - the AlphaFold Protein structure Database (AFdb) and the RCSB Protein Data
431
Bank (PDB). These structures were filtered according to the steps outlined in Section 2.1. The number protein-
432
coding genes were taken from UniProt. 433 Categor
y
Species
Protein-
coding
genes in
UniProt
AlphaFold
structures
(that
passed
filtering)
PDB
chains
Uniprot
proteins
mapping
to PDB
structures
Total
tertiary
structures
Uniprot
proteins
with
tertiary
structures
Control
Arabidopsis
thaliana
27,498
20,460
4,560
899
25,020
20,612
Control
Caenorhabditis
elegans
19,838
14,683
1,036
231
14,968
14,736 12 . CC-BY 4.0 International license
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bioRxiv preprint Control
Candida albicans
SC5314 / ATCC
MYA-2876
6,759
4,777
285
62
5,813
4,790
Control
Danio rerio
26,355
19,541
1,026
157
20,567
19,627
Control
Dictyostelium
discoideum
12,727
8,259
345
55
8,604
8,266
Control
Drosophila
melanogaster
13,821
9,685
2,624
505
12,309
9,877
Control
Escherichia coli
strain K12
4,402
4,220
11,242
1,087
15,462
4,332
Control
Glycine max
55,855
39,168
314
42
39,482
39,180
Host
Homo sapiens
20,594
17,226
125,073
8,215
142,299
17,568
Control
Methanocaldococ
cus jannaschii
strain DSM 2661
1,787
1,708
511
99
2,219
1,709
Control
Mus musculus
21,968
16,911
13,419
2,227
30,330
17,562
Control
Oryza sativa
subsp. japonica
43,673
22,852
410
77
23,262
22,873
Parasite
Plasmodium
falciparum isolate
3D7
5,372
2,909
1,417
207
4,326
2,949
Control
Rattus norvegicus
22,816
16,555
7,512
668
24,067
16,705
Control
Saccharomyces
cerevisiae strain
ATCC 204508 /
S288c
6,060
4,745
15,228
1,215
19,973
4,878
Control
Schizosaccharomy
ces pombe strain
972 / ATCC
24843
5,122
4,224
1,388
295
5,612
4,260
Control
Zea mays
39,208
24,721
456
94
25,177
24,757
. CC-BY 4.0 International license
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y p
)
g
p y
p
p
p
p
y 13 . CC-BY 4.0 International license
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bioRxiv preprint . Tables
429 ;
https://doi.org/10.1101/2023.02.08.527763
doi:
bioRxiv preprint Q8ID66/
PF3D7_136410
0
6-cysteine
protein P92
P40200
T-cell surface
protein tactile
AF-Q8ID66-
F1-
model_v2.pdb.g
z
AF-P40200-F1-
model_v2.pdb.g
z
2.01E-06
71
Q8IFM8/
PF3D7_042380
0
cysteine-rich
protective
antigen
P55895
V(D)J
recombination-
activating
protein 2
AF-Q8IFM8-
F1-
model_v2.pdb.g
z
AF-P55895-F1-
model_v2.pdb.g
z
7.19E-08
184
Q8IIU7/
PF3D7_110560
0
translocon
component
PTEX88
Q16531
DNA damage-
binding protein
1
AF-Q8IIU7-F1-
model_v2.pdb.g
z
5V3O_A.pdb.g
z
1.42E-10
156
Q8IM47/
PF3D7_140470
0
cysteine-rich
small secreted
protein CSS
O75161
Nephrocystin-4
AF-Q8IM47-
F1-
model_v2.pdb.g
z
AF-O75161-F1-
model_v2.pdb.g
z
3.57E-05
68
Q9U0K0/
PF3D7_040860
0
sporozoite
invasion-
associated
protein 1
Q02388
Collagen
alpha-1(VII)
chain
AF-Q9U0K0-
F1-
model_v2.pdb.g
z
AF-Q02388-F2-
model_v2.pdb.g
z
7.23E-03
38
437
Conflict of Interest
438
The authors declare that the research was conducted in the absence of any commercial or financial
439
relationships that could be construed as a potential conflict of interest. 440
Author Contributions
441
Conceived and designed the analyses: VM & JW. Performed the analyses: VM. Wrote the
442
manuscript: VM & JW. 443
Funding
444
This work was supported by Natural Sciences and Engineering Research Council of Canada
445
(NSERC) Discovery Grant (#04589-2020) to JDW and an Eyes High Postdoctoral recruitment
446
scholarship to VM. The funders had no role in study design, data collection and analysis, decision to
447
publish, or preparation of the manuscript. 448
Acknowledgments
449
We thank Kaylee Rich and Dr. Constance Finney for their discussions on various aspects of the
450
manuscript. We acknowledge the high-performance computing resources made available by the
451
Faculty of Veterinary Medicine and Research Computing at the University of Calgary. 452
. CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 9, 2023. Tables
429 CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 9, 2023. ;
https://doi.org/10.1101/2023.02.08.527763
doi:
bioRxiv preprint 434 Table 2 Top Foldseek alignments for the 13 P. falciparum proteins that were present in all three categories
435
outlined in section 3.4. 436 seek alignments for the 13 P. falciparum proteins that were present in all three categories
n 3.4. Table 2 Top Foldseek alignments for the 13 P. falciparum proteins that were present in all three categories
outlined in section 3.4. P. falciparum
protein
(UniProt ID /
PlasmoDB ID)
Protein name
Human
protein
Protein name
P. falciparum
structure
Human
structure
e-value
Foldsee
k score
A0A144A2G5/
PF3D7_111670
0
dipeptidyl
aminopeptidas
e 1
P53634
Dipeptidyl
peptidase 1
AF-
A0A144A2G5-
F1-
model_v2.pdb.g
z
AF-P53634-F1-
model_v2.pdb.g
z
3.91E-28
761
C0H473/
PF3D7_031400
0
HSP20-like
chaperone
Q9P035
Very-long-
chain (3R)-3-
hydroxyacyl-
CoA
dehydratase 3
AF-C0H473-
F1-
model_v2.pdb.g
z
AF-Q9P035-
F1-
model_v2.pdb.g
z
1.71E-09
314
C6KSX0/
PF3D7_061270
0
6-cysteine
protein P12
P01833
Polymeric
immunoglobuli
n receptor
AF-C6KSX0-
F1-
model_v2.pdb.g
z
6UE7_C.pdb.gz
3.44E-07
84
Q8I1Y0/
PF3D7_040490
0
6-cysteine
protein P41
P01833
Polymeric
immunoglobuli
n receptor
4YS4_A.pdb.gz
5D4K_B.pdb.gz
1.11E-04
67
Q8I395/
PF3D7_090540
0
high
molecular
weight rhoptry
protein 3
Q5TBA9
Protein furry
homolog
AF-Q8I395-F1-
model_v2.pdb.g
z
AF-Q5TBA9-
F10-
model_v2.pdb.g
z
5.06E-03
48
Q8I423/
PF3D7_050800
0
6-cysteine
protein P38
Q8WZ42
Titin
AF-Q8I423-F1-
model_v2.pdb.g
z
AF-Q8WZ42-
F19-
model_v2.pdb.g
z
8.90E-06
79
Q8I4R4/
PF3D7_125220
0
chitinase
P36222
Chitinase-3-
like protein 1
AF-Q8I4R4-F1-
model_v2.pdb.g
z
1HJW_B.pdb.g
z
1.15E-06
169
Q8IAV9/
PF3D7_081230
0
sporozoite
surface
protein 3
O75161
Nephrocystin-4
AF-Q8IAV9-
F1-
model_v2.pdb.g
z
AF-O75161-F1-
model_v2.pdb.g
z
2.43E-05
62 14 14 . CC-BY 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted February 9, 2023. Tables
429 ;
https://doi.org/10.1101/2023.02.08.527763
doi:
bioRxiv preprint Q8ID66/
PF3D7_136410
0
6-cysteine
protein P92
P40200
T-cell surface
protein tactile
AF-Q8ID66-
F1-
model_v2.pdb.g
z
AF-P40200-F1-
model_v2.pdb.g
z
2.01E-06
71
Q8IFM8/
PF3D7_042380
0
cysteine-rich
protective
antigen
P55895
V(D)J
recombination-
activating
protein 2
AF-Q8IFM8-
F1-
model_v2.pdb.g
z
AF-P55895-F1-
model_v2.pdb.g
z
7.19E-08
184
Q8IIU7/
PF3D7_110560
0
translocon
component
PTEX88
Q16531
DNA damage-
binding protein
1
AF-Q8IIU7-F1-
model_v2.pdb.g
z
5V3O_A.pdb.g
z
1.42E-10
156
Q8IM47/
PF3D7_140470
0
cysteine-rich
small secreted
protein CSS
O75161
Nephrocystin-4
AF-Q8IM47-
F1-
model_v2.pdb.g
z
AF-O75161-F1-
model_v2.pdb.g
z
3.57E-05
68
Q9U0K0/
PF3D7_040860
0
sporozoite
invasion-
associated
protein 1
Q02388
Collagen
alpha-1(VII)
chain
AF-Q9U0K0-
F1-
model_v2.pdb.g
z
AF-Q02388-F2-
model_v2.pdb.g
z
7.23E-03
38 437 The authors declare that the research was conducted in the absence of any commercial or financial
439
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Interrelation of individual-personal qualities and dynamics of functional states participants of the sea Arctic expedition
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Acta biomedica scientifica
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A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Vol. 7, N 5-1 A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Vol. 7, N 5-1 ВЗАИМОСВЯЗЬ ИНДИВИДУАЛЬНО-ЛИЧНОСТНЫХ КАЧЕСТВ
И ДИНАМИКИ ФУНКЦИОНАЛЬНЫХ СОСТОЯНИЙ УЧАСТНИКОВ МОРСКОЙ
АРКТИЧЕСКОЙ эксп ед и ц и и ВЗАИМОСВЯЗЬ ИНДИВИДУАЛЬНО-ЛИЧНОСТНЫХ КАЧЕСТВ
И ДИНАМИКИ ФУНКЦИОНАЛЬНЫХ СОСТОЯНИЙ УЧАСТНИКОВ МОРСКОЙ
АРКТИЧЕСКОЙ эксп ед и ц и и Автор, ответственный за переписку:
Тункина Мария Александровна,
e-mail: masha.tynkina@yandex.ru Статья поступила: 05.06.2022
Статья принята: 10.10.2022
Статья опубликована: 08.12.2022 ABSTRACT Tunkina M.A. 1,
Korneeva Ya.A. 1,
Simonova N.N. 2,
Trofimova A.A. 1 The issues of psychological adaptation of a person to the extreme conditions
of the Arctic continue to be relevant at the present time. Dynamic monitoring of hu
man functional states is an effective technology that allows timely identification
of the level of human adaptation to the conditions of the North and implementation
of preventive measures to maintain health in extreme working conditions. The aim of the study is to identify and describe the relationship of individual-
personal qualities and the dynamics of the functional states of the participants
of the marine Arctic expedition. 1 Northern (Arctic) Federal University
named after M.V. Lomonosov
(Severnoy Dvini Embankment 17,
Arkhangelsk 163002, Russian Federation)
2 Lomonosov Moscow State University
(Leninskiye Gory 1, Moscow 119991,
Russian Federation) Methods. Psychophysiological testing using the device UPFT-1/30-«Psychophysiolog»
(variational cardiointervalometry, complex visual-motor reaction); color preference
test (M. Luscher, adapted by L.N. Sobchik) with calculation of interpretation coeffi
cients by G.A. Aminev; Five-factor personality questionnaire (R. McCrae and P. Costa,
adapted by A.B. Khromov); statistical methods: descriptive statistics, frequency analy
sis, correlation analysis using Spearman's rank correlation coefficient. The study was
conducted daily in the morning and evening for20 days on the research vessel Mikhail
Somov in June 2021. The study involved 39people, including participants in the sci
entific and educational expeditionary project "Arctic Floating University - 2021". Results. The dynamics of psychophysiological and projective psychological param
eters of the functional states of the expedition members as a whole is character
ized by favorable levels, which indicates their adaptation to the conditions of high
latitudes. Corresponding author:
Maria A. tunkina,
e-mail: masha.tynkina@yandex.ru Corresponding author:
Maria A. tunkina,
e-mail: masha.tynkina@yandex.ru p
yp
j
g
y
Results. The dynamics of psychophysiological and projective psychological param
eters of the functional states of the expedition members as a whole is character
ized by favorable levels, which indicates their adaptation to the conditions of high
latitudes. According to the results of the correlation analysis, statistically significant relation
ships were revealed between the dynamics of functional states and such individual-
personal qualities as: extraversion - introversion and attachment - separation. Conclusion. The results of the study are consistent with the data of previous expe
ditions of the "Arctic floating university" and other polar expeditions. РЕЗЮМЕ Тункина М.А. 1,
Корнеева Я.А. 1,
Симонова Н.Н. 2,
Трофимова А.А. 1 Обоснование. Вопросы психологической адаптации человека к экстремаль
ным условиям Арктики продолжают быть актуальными и в настоящее
время. Динамический мониторинг функциональных состояний человека -
эффективная технология, позволяющая своевременно выявлять уровень
адаптированности человека к условиям Севера и осуществлять профи
лактические мероприятия для поддержания здоровья в экстремальных
условиях труда. 1 ФГАОУ ВО «Северный (Арктический)
федеральный университет имени
М.В. Ломоносова» (163002, г. Архангельск,
наб. Северной Двины, 17, Россия)
2 ФГБОУ ВО «Московский
государственный университет
имени М.В. Ломоносова» (119991,
г. Москва, Ленинские горы, 1, Россия) Цель. Выявить и описать взаимосвязь индивидуально-личностных качеств
и динамики функциональных состояний участников морской арктической
экспедиции. Методы. Психофизиологическое тестирование с помощью прибора УПФТ-
1/30 «Психофизиолог» (вариационная кардиоинтервалометрия, сложная
зрительно-моторная реакция); тест цветовых предпочтений (М. Люшер,
адаптированный Л.Н. Собчик) с расчётом коэффициентов интерпретации
Г.А. Аминева; пятифакторный личностный опросник (Р. Маккрей и П. Коста,
адаптированный А.Б. Хромовым); статистические методы: описатель
ные статистики, анализ частот, корреляционный анализ с применением
коэффициента ранговой корреляции Спирмена. Исследование проводилось
ежедневно в утреннее и вечернее время в течение 20 дней на научно-экспеди
ционном судне «Михаил Сомов» в июне2021 г. В исследовании приняли участие
39 человек, среди нихучастники научно-образовательного экспедиционного
проекта «Арктический плавучий университет - 2021». Автор, ответственный за переписку:
Тункина Мария Александровна,
e-mail: masha.tynkina@yandex.ru Результаты. Динамика психофизиологических и проективных психоло
гических параметров функциональных состояний участников экспедиции
в целом характеризуется благоприятными уровнями, что свидетельству
ет об их адаптированности к условиям высоких широт. По результатам
корреляционного анализа выявлены статистически значимые связи между
динамикой функциональных состояний и такими индивидуально-личност
ными качествами как экстраверсия - интроверсия и привязанность - отде-
лённость. Обсуждение. Результаты исследования согласуются с данными предыду
щих экспедиций «Арктического плавучего университета» и других полярных
экспедиций. Выявленные взаимосвязи функциональных состояний с разны
ми характеристиками личности участников свидетельствуют в пользу
комплексного характера исследования с применением разных групп диа
гностических методов. Ключевые слова: функциональное состояние человека, работоспособность,
стресс, вариационная кардиоинтервалометрия, динамический мониторинг,
Арктический плавучий университет, экспедиционный метод, Арктика для цитирования: Тункина М.А., Корнеева Я.А., Симонова Н.Н., Трофимова А.А. Взаимосвязь индивидуально-личностных качеств и динамики функциональных
состояний участников морской арктической экспедиции. Acta biomedica scientifica. 2022; 7(5-1): 228-241. doi: 10.29413/ABS.2022-7.5-1.24 228 Психология и психиатрия A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Том 7, № 5-1 Received: 05.06.2022
Accepted: 10.10.2022
Published: 08.12.2022 Corresponding author:
Maria A. tunkina,
e-mail: masha.tynkina@yandex.ru ОБОСНОВАНИЕ ко на физиологическом, но и на психологическом уров
не. На сегодняшний день вопрос об объёме и выборе
методик исследования функциональных состояний яв
ляется дискуссионным. В ходе анализа отечественных
и зарубежных публикаций, посвящённых разработке
технологии динамического мониторинга функциональ
ных состояний в экстремальных условиях, и исследова
ний, посвящённых изучению адаптации у участников ар
ктических и антарктических морских экспедиций, было
выявлено выделение как минимум двух основных групп
методов [15, 23-25]. Арктика относится к региону, который обладает при
родно-ресурсным, экономическим, транспортно-логи
стическим потенциалом. Сегодня к арктическим терри
ториям также проявляется научный интерес [1]. В многочисленных исследованиях [2-8] отмечается
неблагоприятное влияние климатогеографических и ге
офизических условий Арктического региона на здоро
вье и деятельность человека. В первую очередь, влия
ние низких температур с высокой скоростью ветра, пе
репады атмосферного давления, резкая смена светового
режима («полярная ночь» и «полярный день») и др. Со
гласно исследованиям [9-11], под воздействием факто
ров «высоких широт» может развиваться метеочувстви
тельность, быстрее наступает истощение адаптивных ре
зервов организма с последующим развитием «синдрома
полярного напряжения» и различных хронических забо
леваний, прежде всего сердечно-сосудистых, онкологи
ческих, респираторных. К другой группе факторов, так
же оказывающих влияние на жизнеобеспечение чело
века в Арктике, относятся социально-бытовые факторы. Экспедиционная и вахтовая формы трудовой деятель
ности, которые часто встречаются на Севере, сопряже
ны с групповой изоляцией, ограничением личного про
странства, однообразностью обстановки и др. [12-14]. 1) методы аппаратурной психофизиологической ди
агностики, которые считаются наиболее надёжными, по
скольку регистрируют изменения на уровне физиологи
ческих и психофизиологических систем, в частности ана
лиз вариабельности сердечного ритма, анализ показате
лей нервной системы, оценка работоспособности на ос
нове сенсомоторной реакции и др.; 1) методы аппаратурной психофизиологической ди
агностики, которые считаются наиболее надёжными, по
скольку регистрируют изменения на уровне физиологи
ческих и психофизиологических систем, в частности ана
лиз вариабельности сердечного ритма, анализ показате
лей нервной системы, оценка работоспособности на ос
нове сенсомоторной реакции и др.; 2) психологические проективные и субъективно
оценочные (опросные) методы, позволяющие качествен
но изучить состояния и настроения человека на уровне
его субъективных ощущений и переживаний; Комплексный подход к оценке динамики функци
ональных состояний человека, предполагающий ис
следование характеристик в динамике разными мето
дами, помогает исследовать компенсаторный харак
тер адаптации участников экспедиций. В рамках риск
ориентированного подхода внимание также уделяется
личностному потенциалу работников. Поэтому в по
добных исследованиях часто данные методы исполь
зуются вместе. Таким образом, важным при организации и выпол
нении деятельности под воздействием неблагоприят
ных условий Арктики является вопрос об адаптации
человека. ОБОСНОВАНИЕ С каждым годом возрастает актуальность ис
следований, посвящённых оценке функционального со
стояния человека и изучению психологических особен
ностей вахтовиков и участников полярных экспедиций
[15-19]. А рост количества публикаций по данной про
блематике обусловливает необходимость систематиза
ции уже имеющихся научных результатов [20]. Определяя индивидуально-личностные особенно
сти работников, осуществляющих свою деятельность
в Арктике, при соотнесении с их динамикой различных
параметров функциональных состояний возможно вы
явить качества личности, способствующие более успеш
ной психологической адаптированности к условиям Се
вера. В качестве одного из инструментов комплексной
оценки индивидуально-личностных характеристик яв
ляется Пятифакторный личностный опросник FFI (Боль
шая пятёрка) Р. Маккрея и П. Коста [26], который актив
но применяется в аналогичных исследованиях и макси
мально близких выборках: полярников, вахтовых работ
ников, космонавтов [13, 27-30]. у
у
р
у
[
]
Динамический мониторинг функциональных состо
яний человека является эффективной технологией, по
зволяющей своевременно диагностировать и профи-
лактировать ухудшение состояния здоровья работни
ков и возникновение нежелательных ситуаций в экстре
мальных условиях труда. По динамике изменений инте
гральных показателей сердечно-сосудистой и нервной
систем, работоспособности, стресса можно сделать вы
вод об адаптированности человека к условиям Севера. В связи с существующими сложностями организации по
левых исследований в Арктике наиболее часто прово
дятся двухкратные (в начале и конце выезда сотрудни
ков) или трёхкратные замеры состояний специалистов
(в начале, середине и конце вахтовых заездов/морских
рейсов), что может не позволять определить наиболее
сложные с точки зрения адаптации дни, а также факто
ры, их обуславливающие [21, 22]. Так, в пользу необхо
димости проведения ежедневного мониторинга состоя
ния участников экспедиции указывает факт недостаточ
ности исследований со схожим дизайном. Таким образом, в настоящем исследовании будет
определена адаптированность участников морской ар
ктической экспедиции посредством динамического на
блюдения объективных психофизиологических и проек
тивных психологических параметров их функциональ
ных состояний, а также выявлены индивидуально-пси
хологические качества, способствующие более успеш
ной адаптации в условиях «высоких широт». ABSTRACT The revealed
interrelations of functional states with different personality characteristics of the par
ticipants testify in favor of the complex nature of the study using different groups
of diagnostic methods. Key words: human functional state, working capacity, stress, variational cardioint
ervalometry, dynamic monitoring, Arctic floating university, expeditionary method,
Arctic For citation: Tunkina M.A., Korneeva Ya.A., Simonova N.N., Trofimova A.A. Interrelation
of individual-personal qualities and dynamics of functional states participants of the sea
Arctic expedition. Acta biomedica scientifica. 2022; 7(5-1): 228-241. doi: 10.29413/
ABS.2022-7.5-1.24 229 Psychology and psychiatry Psychology and psychiatry Психология и психиатрия A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Vol. 7, N 5-1 М АТЕРИАЛЫ И МЕТОДЫ Исследование проводилось ежедневно в утреннее и
вечернее время в течение 20 дней на научно-экспедици
онном судне «Михаил Сомов» в июне 2021 г. Начало ис
следования - второй день экспедиции, конец исследо
вания - последний день экспедиционного рейса. Марш
рут экспедиции: г. Архангельск - мыс Желания (о. Новая
Земля) - о. Хейса (Земля Франца-Иосифа (ЗФИ)) - о. Гуке
ра (ЗФИ) - о. Греэм-Белл (ЗФИ) - г. Архангельск. Марш
рут в Баренцевом море включал вертолётные высадки
на острова для сбора научного материала и проведения
научно-исследовательских работ. Для оценки операторской работоспособности при
менялась методика сложной зрительно-моторной реак
ции («СЗМР-35», прибор УПФТ-1/30 «Психофизиолог»). Анализ проводился на основе следующих параметров:
«оценка уровня сенсомоторных реакций» (ОУСР, отн. ед.)
и «уровень операторской работоспособности» (баллы). Время сложной зрительно-моторной реакции (реакции
с переключением) характеризует скорость проведения
возбуждения по рефлекторной дуге. Методика позволя
ет определить скорость и качество выполнения заданий;
на основе сочетания этих двух параметров делается вы
вод об уровне операторской работоспособности (высо
кий, средний или низкий) по параметрам двухальтерна
тивной сложной зрительно-моторной реакции (35 сти
мулов). Сущность методики «СЗМР-35» - в определении
времени и стабильности зрительно-моторной реакции
на световые стимулы (зелёный и красный). Участие в исследовании было добровольным. Все участники исследования подписали информацион
ное согласие и согласие на обработку персональных дан
ных. Программа и методы исследования рассмотрены
на этическом комитете ФГБОУ ВО «Северный государ
ственный медицинский университет» Минздрава Рос
сии (г. Архангельск, Россия) и рекомендованы к приме
нению (протокол № 04-06-21 от 09.06.2021). В исследовании приняли участие 39 человек (19 муж
чин и 20 женщин в возрасте от 20 до 72 лет, средний воз
раст - 33,6 ± 2 года), среди них участники научно-обра
зовательного экспедиционного проекта «Арктический
плавучий университет - 2021», сотрудники Националь
ного парка «Русская Арктика», участники проекта «Хо
зяин Арктики» и члены экипажа. Среди участников ис
следования: у
(
р
)
2. На психологическом уровне изучались неосоз
наваемые компоненты психофизиологических состоя
ний с применением метода цветовых выборов (8-цвето-
вого теста М. Люшера в русской адаптации Л.Н. Собчик)
[32, 33]. Для анализа вычислялись интерпретационные
коэффициенты «работоспособность», «стресс» и «веге
тативный баланс», разработанные Г.А. Аминевым [34]. Значения от 9,1 до 16,0 баллов указывают на снижен
ную работоспособность, от 16,1 до 20,9 балла - доста
точная и высокая работоспособность. Значения по по
казателю стресса могут быть в диапазоне от 0 до 41,8;
значения равные и выше 20 указывают на наличие про
явлений стрессового состояния, значения до 20 баллов
свидетельствуют о тенденции к образованию стресса. ГИПОТЕЗЫ ИССЛЕДОВАНИЯ 1. На психофизиологическом уровне анализиро
вались параметры сердечно-сосудистой и вегетатив
ной нервной систем (методика вариационной кардио-
интервалометрии «ВКМ», прибор УПФТ-1/30 «Психофи
зиолог»): средняя длительность интервалов R-R между
синусовыми сокращениями (RRNN, мс), оценка функци
онального состояния (VSR, отн. ед.), уровень функцио
нального состояния (УФС, баллы). Согласно данным по
казателя RRNN можно оценить уровень регуляторных
возможностей человека, соотношение симпатическо
го и парасимпатического отделов вегетативной нерв
ной системы и сделать вывод о сердечном ритме (нор-
мокардия, тахикардия или брадикардия). Интегральный
количественный показатель VSR отражает общее функ
циональное состояние организма человека по параме
трам его сердечной деятельности. Ожидается увидеть взаимосвязь динамики объек
тивных показателей функциональных состояний с экс-
тернальностью/интернальностью участников экспеди
ции; а динамики проективных показателей функциональ
ных состояний - с эмоциональностью/эмоциональной
сдержанностью участников. ЦЕЛЬ РАБОТЫ Выявить и описать взаимосвязь индивидуально-лич
ностных качеств и динамики функциональных состояний
участников морской арктической экспедиции. В настоящее время перспективным направлением
является многофакторный и комплексный подход, учи
тывающий разнообразие проявлений адаптации не толь В настоящее время перспективным направлением
является многофакторный и комплексный подход, учи
тывающий разнообразие проявлений адаптации не толь 230 Psychology and psychiatry Психология и психиатрия Этапы исследования: 1. Описание динамики объективных психофизиоло
гических и проективных психологических параметров
функциональных состояний участников экспедиции. A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Vol. 7, N 5-1 A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Vol. 7, N 5-1 о
ской арктической экспедиции (2 раза в сутки ежеднев
но: утром и вечером). Для каждого участника экспеди
ции по каждому параметру было построено уравнение
регрессии с вычислением коэффициента, отражающе
го средний прирост показателя за единицу времени. Графически этот коэффициент демонстрирует наклон
линии тренда. Таким образом, чем больше коэффици
ент, тем выше рост (при положительных значениях) или
спад (при отрицательных величинах) показателя в ди
намике наблюдения. В качестве примера представлена
динамика показателя оценки функционального состо
яния (VSR, отн. ед.) методики «ВКМ» одного из участни
ков экспедиции в течение 20-дневного периода в ве
чернее время (рис. 1). [35]. Опросник состоит из 75 противоположных по зна
чению высказываний, каждое из которых выявляет важ
ные характеристики личности и особенности поведения
человека в некоторых жизненных ситуациях. Результаты
предоставляются в виде первичных и вторичных факто
ров (индивидуально-личностных качеств). [35]. Опросник состоит из 75 противоположных по зна
чению высказываний, каждое из которых выявляет важ
ные характеристики личности и особенности поведения
человека в некоторых жизненных ситуациях. Результаты
предоставляются в виде первичных и вторичных факто
ров (индивидуально-личностных качеств). 4. Статистический анализ данных проводился с
мощью пакета программ «IBM SPSS Statistics 23.00» с ис
пользованием следующих методов: описательные стати
стики, анализ частот, корреляционный анализ с приме
нением коэффициента ранговой корреляции Спирмена. При проверке связи в корреляционном анализе учиты
вались результаты с силой связи не ниже 0,3. М АТЕРИАЛЫ И МЕТОДЫ Оценка вегетативного баланса производилась в соот
ветствии со следующими нормами: от 0 до +9,8 балла -
преобладание тонуса симпатической нервной системы,
т. е. мобилизация всех функций, подготовка к активным
действиям, а от 0 до -9,8 - преобладание тонуса пара
симпатической нервной системы, т. е. работа организ
ма направлена на отдых, восстановление сил, сбереже
ние ресурсов. • 24 человека ранее имели экспедиционный опыт
от двух недель до нескольких месяцев, 15 человек уча
ствовали в экспедиции впервые; • 24 человека ранее имели экспедиционный опыт
от двух недель до нескольких месяцев, 15 человек уча
ствовали в экспедиции впервые; • 20 участников являются жителями приарктических
территорий (территорий, приравненных к Крайнему Се
веру) и 1 участник проживает в Арктическом регионе; • 32 человека имеют оконченное высшее образова
ние (бакалавриат, специалитет), 7 участников на момент
исследования имеют неоконченное высшее (студенты). В данном исследовании, вслед за В.И. Медведевым
и А.Б. Леоновой, функциональное состояние понимает
ся как «интегральный комплекс наличных характеристик
тех функций и качеств человека, которые прямо или кос
венно обуславливают выполнение деятельности» [31]. Оценка функционального состояния участников
производилась с применением психофизиологических
(аппаратурных) и психологических (проективных) мето
дов в течение 20-дневного экспедиционного периода. Исследование индивидуально-личностных качеств про
изводилось однократно в начале экспедиции. 3. Оценка индивидуально-личностных качеств
участников экспедиции производилась с применени
ем Пятифакторного личностного опросника (FFI) Р. Мак-
крей и П. Коста [26], в русской адаптации А.Б. Хромова 3. Оценка индивидуально-личностных качеств
участников экспедиции производилась с применени
ем Пятифакторного личностного опросника (FFI) Р. Мак-
крей и П. Коста [26], в русской адаптации А.Б. Хромова 231 Психология и психиатрия Psychology and psychiatry A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Том 7, № 5-1 A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Том 7, № 5-1 ми сокращениями (RRNN, мс) в утренних и вечерних за
мерах. Среди участников только три человека имеют
тенденцию к учащению сердечного ритма (тахикардии). В динамике проективного показателя работоспособ
ности (интерпретационный коэффициент Г.А. Аминева,
разработанный для 8-цветового теста М. Люшера) так
же выявлена тенденция к средним значениям. Значимое
снижение данного показателя наблюдается в утреннее
время 11-го дня исследования (перед высадкой на о. Гу
кера). Мы предполагаем, что подобное снижение рабо
тоспособности (в сочетании с усилением парасимпати
ческой нервной системы по проективному показателю)
может быть связано с недостаточным восстановлением
участников между высадками, которые осуществлялись
друг за другом в течение двух дней. За день до высадки
о. Гукера, часть участников исследования высаживались
на другой остров архипелага ЗФИ - о. Хейса. В начале экспедиции наиболее ярко изменения
в уровне функционального состояния проявились во
второй день исследования (третий день экспедиции). Данные изменения характеризовались усилением на
пряжения регуляторных систем и снижением психофи
зиологических резервов организма участников в утрен
нее время (с последующим улучшением показателей ве
чером того же дня). В конце экспедиции, согласно ча
стотному анализу показателя «уровень функциональ
ного состояния» (УФС), увеличилось число участников
с оптимальным, близким к оптимальному и допустимым
уровням функционального состояния. Согласно средним значениям проективного по
казателя стресса (интерпретационный коэффициент
Г.А. Аминева, разработанный для 8-цветового теста
М. Люшера), в динамике арктического рейса у участни
ков не выявлено выраженного стрессового состояния. Тенденция к увеличению стресса (в пределах нормы) на
блюдается вечером 6-го дня исследования, но уже ве
чером 7-го дня исследования (после высадки на о. Но
вая Земля) наблюдается снижение данного показателя. В середине экспедиционного периода (в утреннее и ве
чернее время 11-го дня исследования) также фиксиру
ется тенденция к образованию стресса. В этот день чле
ны экспедиции пересекли самую северную для марш
рута экспедиции точку (80-ю параллель северной ши
роты) и участвовали в высадке на о. Гукера. При этом
важно отметить, что данные изменения происходили
в пределах нормы. Динамика состояния вегетативной нервной систе
мы (ВНС) в течение экспедиционного периода оцени
валась согласно объективным данным (с использова
нием аппаратурной методики «ВКМ») и проективным
данным (проективный показатель «вегетативный ба
ланс» цветовой тест М. Люшера). Полученные данные
не противоречат друг другу. Состояние вегетативной
нервной системы в утреннее и вечернее время соглас
но средним значениям проективного показателя преи
мущественно характеризуется тенденцией к усилению
симпатической системы. РЕЗУЛЬТАТЫ 2. Вычислить и описать коэффициенты среднего
прироста значений объективных психофизиологиче
ских и проективных психологических параметров функ
циональных состояний участников в течение морской
арктической экспедиции. Динамика функционального состояния участни
ков в течение 20-дневного экспедиционного пери
ода. Согласно объективным данным методики «ВКМ»
(табл. 1), в целом динамика интегрального показате
ля «оценка функционального состояния» (VSR, отн. ед.)
участников экспедиции в течение всего рейса характе
ризуется тенденцией к преобладанию допустимых зна
чений. Наблюдается преимущественно умеренное на
пряжение регуляторных систем при достаточном объ
ёме и адекватном расходовании психофизиологических
резервов сердечно-сосудистой и вегетативной нервной
систем. Сердечный ритм большинства участников ха
рактеризуется как нормальный (согласно средним зна
чениям индивидуальных динамик участников по пока
зателю длительности интервалов R-R между синусовы- 3. Выявить и описать взаимосвязь индивидуально
личностных качеств участников экспедиции и коэффи
циентов среднего прироста значений объективных пси
хофизиологических и проективных психологических па
раметров функциональных состояний участников в те
чение арктического рейса. Технология расчёта коэффициента, отражающе
го средний прирост показателя. Индивидуальные объ
ективные и проективные показатели функциональных
состояний в течение экспедиции оценивались у каж
дого участника в течение всего периода научной мор VSR,
0|Тн.ед
Оценка функционального состояния (VSR), вечерние замеры
Коэффициент
наклона линии
линейной
регрессии/линии
индивидуального
тренда
по показателю
оценки
функционального
состояния (VSR)
4
5
9
10
11
12
Дни исследования
13
14
15
16
17
18
19
20
1
2
3
6
7
8
РИС. 1. FIG. 2. Динамика показателя оценки функционального состояния
Dynamics of the general functional state (VSR) o f the study
(VSR) участ ника исследования в течение экспедиционного
participant during the expedition period in the evening
периода в вечернее время
P
h l
d
hi
232
П Коэффициент
наклона линии
линейной
регрессии/линии
индивидуального
тренда
по показателю
оценки
функционального
состояния (VSR) Коэффициент
наклона линии
линейной
регрессии/линии
индивидуального
тренда
по показателю
оценки
функционального
состояния (VSR) Дни исследования FIG. 2. Dynamics of the general functional state (VSR) o f the study
participant during the expedition period in the evening Динамика показателя оценки функционального состояния
(VSR) участ ника исследования в течение экспедиционного
периода в вечернее время 232 Psychology and psychiatry Психология и психиатрия A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Том 7, № 5-1 Преобладание же парасимпа
тической нервной системы, активизация которой свя
зана с ориентацией организма на отдых, восстановле
ние сил и сбережение ресурсов, проявляется в нача
ле экспедиции (утром 1-го дня исследования), в сере
дине (утром 11-го и 14-го дней исследования) и в конце
экспедиции (утром 16-го дня исследования и вечером
17-го и 18-го дней исследования). Далее были вычислены и проанализированы ко
эффициенты среднего прироста основных параметров
функциональных состояний участников, измеренных
за 20-дневной период экспедиции. В таблице 2 представ
лены показатели прироста общего функционального со
стояния организма (по объективным данным), работо
способности (по объективным и проективным данным)
и стресса (по проективным данным). Согласно объективным и проективным данным,
смещение показателя вегетативного баланса в сторо
ну преобладания симпатического звена вегетативной
нервной регуляции указывает на более интенсивный ха
рактер функционирования адаптационных процессов. В конце экспедиции функциональное состояние мно
гих участников характеризовалось усилением парасим
патического отдела и сбалансированным влиянием от
делов вегетативной нервной системы, что было связа
но с периодом отдыха и восстановлением гомеостати
ческого равновесия. Согласно данным таблицы 2, мы видим незначи
тельные положительные и отрицательные приросты,
при этом информативная ценность их заключается
в оценке математического знака. Несмотря на тенден
цию к снижению в утренние и вечерние часы показа
теля «оценка функционального состояния» (VSR), кото
рый представляет собой интегральную характеристику
общего функционального состояния организма участ
ников, итоговый прирост по данному показателю по
ложительный. Это объясняется тем, что ряд участников
имеют в индивидуальной динамике показателя функци
онального состояния в утреннее время отрицательный
прирост, а в вечерние часы - положительный прирост,
что может в целом указывать на улучшение состояния
в течение экспедиции. Динамика показателя операторской работоспособ
ности в течение экспедиционного периода (методика
«СЗМР-35») характеризовалась тенденцией к средним
(достаточным) значениям (табл. 1). Согласно средним
значениям участников (табл. 1), по параметру «оценка
уровня сенсомоторных реакций» (ОУСР) в середине пе
риода наблюдалась тенденция к сниженной работоспо
собности в утреннее время 8-го дня (после первой вы
садки участников на сушу на о. Новая Земля). А в утрен
нее время 15-го и 16-го дней исследования, после за
вершения активных научно-исследовательских работ,
связанных с высадками на острова, наблюдалось увели
чение числа участников с повышенной работоспособно
стью (среднее значение ОУСР равно 0,67 и 0,65 отн. ед. соответственно). В течение рейса работоспособность большинства
участников имеет положительный прирост в утреннее и
вечернее время. Данный результат можно считать поло
жительным объективным маркером адаптации к аркти
ческой морской экспедиции. A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Том 7, № 5-1 При этом в динамике про- 233 Psychology and psychiatry Психология и психиатрия 234
Psychology and psychiatry
Психология и психиатрия
ТА Б Л И Ц А 1
T A B LE 1
ОПИСАТЕЛЬНЫЕ СТАТИСТИКИ ОБЪЕКТИВНЫХ И ПРОЕКТИВНЫХ ПАРАМЕТРОВ
DESCRIPTIVE STATISTICS OF OBJECTIVE AND PROJECTIVE PARAMETERS
ФУНКЦИОНАЛЬНЫХ СОСТОЯНИЙ УЧАСТНИКОВ В ТЕЧЕНИЕ МОРСКОЙ
OF THE FUNCTIONAL STATES OF PARTICIPANTS DURING THE MARINE ARCTIC
АРКТИЧЕСКОЙ ЭКСПЕДИЦИИ (M ± SE)
EXPEDITION (M ± SE)
Методики
Дни исследования /
Показатели
Пересечение
70-й параллели с.ш. Незначительная
бортовая качка
Высадка
на о. Новая Земля
Пересечение
80-й параллели с.ш. Высадка на о. Хейса
Высадка на о. Гукера
Высадка
на о. Греэм-Белл
Пересечение
75-й параллели с.ш. 1
2
3
4
5
б
7
8
9
10
11
12
13
14
15
16
17
18
19
20
VSR (утро), М ± SE
0,5 ±
0,08
0,4 ±
0,07
0,5 ±
0,07
0,5 ±
0,06
0,5 ±
0,05
0,5 ±
0,06
0,6 ±
0,05
0,5 ±
0,06
0,5 ±
0,05
0,6 ±
0,07
0,6 ±
0,09
0,5 ±
0,06
0,5 ±
0,06
0,5 ±
0,06
0,5 ±
0,05
0,5 ±
0,05
0,5 ±
0,06
0,5 ±
0,06
0,6 ±
0,06
0,6 ±
0,06
вкм
VSR (вечер), М ± SE
0,5 ±
0,07
0,7 ±
0,05
0,6 ±
0,06
0,6 ±
0,05
0,6 ±
0,05
0,5 ±
0,06
0,5 ±
0,06
0,6 ±
0,05
0,5 ±
0,05
0,5 ±
0,05
0,5 ±
0,05
0,6 ±
0,05
0,5 ±
0,08
0,6 ±
0,05
0,6 ±
0,05
0,5 ±
0,06
0,6 ±
0,05
0,5 ±
0,05
0,5 ±
0,05
0,6 ±
0,05
УФС (утро). Me
3
2
3
3
3
3
4
4
3
3
4
4
3
3
3
4
3
4
3
4
УФС (вечер). A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Том 7, № 5-1 Me
3
4
4
4
4
3
3
4
3
3
3
4
3
4
4
3
4
3
3
4
ОУСР (утро), М ± SE
0,4 ±
0,06
0,5 ±
0,07
0,6 ±
0,05
0,5 ±
0,06
0,5 ±
0,05
0,6 ±
0,05
0,5 ±
0,06
0,4 ±
0,05
0,5 ±
0,06
0,6 ±
0,05
0,5 ±
0,09
0,5 ±
0,05
0,6 ±
0,05
0,5 ±
0,05
0,6 ±
0,05
0,7 ±
0,04
0,6 ±
0,06
0,6 ±
0,06
0,6 ±
0,06
0,6 ±
0,06
C3MP-35
ОУСР (вечер), М ± SE
0,4 ±
0,05
0,5 ±
0,05
0,5 ±
0,05
0,5 ±
0,05
0,6 ±
0,04
0,5 ±
0,05
0,6 ±
0,05
0,6 ±
0,05
0,6 ±
0,05
0,5 ±
0,05
0,5 ±
0,05
0,5 ±
0,05
0,5 ±
0,07
0,5 ±
0,05
0,6 ±
0,05
0,6 ±
0,05
0,5 ±
0,05
0,6 ±
0,04
0,5 ±
0,05
0,6 ±
0,05
УОП (утро). Me
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
УОП (вечер). Me
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
Работоспособность
17,1 ±
17,1 ±
16,6±
1 7,6 ±
16,9 ±
17,2 ±
17,6 ±
17,4 ±
15,9 ±
17,8 ±
17,2 ±
16,8 ±
16,8 ±
16,8 ±
17,0 ±
16,6±
1 7,4 ±
16,9 ±
17,1 ±
17,1 ±
(утро), М ± SE
0,57
0,59
0,45
0,51
0,54
0,47
0,52
0,50
0,42
0,47
0,63
0,40
0,46
0,48
0,46
0,45
0,47
0,54
0,51
0,71
Работоспособность
16,2 ±
16,9 ±
1 7,6 ±
16,9 ±
16,8 ±
16,7 ±
17,8 ±
17,5 ±
17,4 ±
17,3 ±
16,8 ±
16,8 ±
17,1 ±
16,9 ±
17,1 ±
16,8 ±
16,6±
16,4 ±
17,2 ±
17,7 ±
(вечер), М ± SE
0,42
0,51
0,44
0,49
0,54
0,52
0,42
0,41
0,40
0,43
0,55
0,47
0,57
0,43
0,41
0,44
0,50
0,50
0,51
0,40
Цветовой
Стресс (утро), М ± SE
7,1 ±
1,37
8,4 ±
1,53
6,8 ±
1,21
8,0 ±
1,43
8,0 ±
1,30
8,0 ±
1,55
8,7 ±
1,49
7,9 ±
1,16
6,7 ±
1,28
8,3 ±
1,73
9,4 ±
2,55
6,8 ±
1,16
6,2 ±
1,43
8,0 ±
1,31
7,4 ±
1,23
8,0 ±
1,48
8,9 ±
1,46
9,2 ±
1,60
6,9 ±
1,44
6,6 ±
1,16
тест
М.Люшера
Стресс (вечер). A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Том 7, № 5-1 8,9 ±
8,8 ±
6,7 ±
8,0 ±
7,9 ±
10,0±
5,0 ±
6,7 ±
7,2 ±
7,6 ±
9,9 ±
7,8 ±
7,2 ±
8,4 ±
6,8 ±
7,7 ±
9,4 ±
8,9 ±
7,6 ±
6,2 ±
М ± SE
1,29
1,56
1,05
1,44
1,27
1,49
1,14
1,07
1,24
1,02
1,45
1,24
1,24
1,29
1,35
1,53
1,59
1,62
1,47
1,13
Вегетативный баланс
-0,2 ±
1,5 ±
1,1 ±
1,4 ±
1,8 ±
2,2 ±
0,5 ±
0,7 ±
1,6 ±
1,9 ±
-0,9 ±
1,6 ±
0,8 ±
-0,3 ±
0,2 ±
-0,3 ±
0,9 ±
0,2 ±
1,2 ±
1,3 ±
(утро), М ± SE
1,08
0,92
0,87
0,95
0,82
0,90
0,96
0,91
0,85
0,95
1,34
0,80
0,80
0,84
0,84
0,81
0,89
1,00
0,80
0,81
Вегетативный баланс
0,1 ±
1,6 ±
2,0 ±
1,1 ±
1,6 ±
1,5 ±
2,2 ±
1,6 ±
1,5 ±
1,0 ±
0,3 ±
0,3 ±
0,8 ±
0,3 ±
0,9 ±
0,7 ±
-0,3 ±
-0,4 ±
1,0 ±
2,1 ±
(вечер), М ± SE
0,71
0,88
0,87
0,94
0,80
0,94
0,87
0,83
0,76
0,83
0,99
0,83
0,84
0,77
0,77
0,79
0,84
0,86
0,89
0,81
Примечание. VSR - оценка функционального состояния; УФС -уровень функционального состояния; ОУСР - оценка уровня сенсомоторных реакций; УОП - уровень операторской работоспособности. T A B LE 1
DESCRIPTIVE STATISTICS OF OBJECTIVE AND PROJECTIVE PARAMETERS
OF THE FUNCTIONAL STATES OF PARTICIPANTS DURING THE MARINE ARCTIC
EXPEDITION (M ± SE) ACTA BIOMEDICA SCIENTIFICA, 2022, Vol. 7, N 5-1 A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Том 7, № 5-1
ТА Б Л И Ц А 2
T A B LE 2
ОПИСАТЕЛЬНЫЕ СТАТИСТИКИ КОЭФФИЦИЕНТОВ
DESCRIPTIVE STATISTICS OF THE COEFFICIENTS
с р е д н е г о п р и р о с т а з н а ч е н и й о б ъ е к т и в н ы х
o f t h e a v e r a g e in c r e a s e in t h e v a l u e s
п с и х о ф и з и о л о г и ч е с к и х и п р о е к т и в н ы х
o f o b je c t iv e p s y c h o p h y s io l o g ic a l a n d p r o je c t iv e
п с и х о л о г и ч е с к и х п а р а м е т р о в ф у н к ц и о н а л ь н ы х
p s y c h o l o g ic a l p a r a m e t e r s o f t h e f u n c t io n a l
с о с т о я н и й у ч а с т н и к о в в ТЕЧЕНИЕ МоРСкоЙ
STATES oF THE PARTICIPANTS DuRING THE MARINE
а р к т и ч е с к о й э к с п е д и ц и и
a r c t ic e x p e d it io n
Параметр
M ± SE
Средний прирост показателя VSR (утренние замеры)
-0,001 ± 0,007
Средний прирост показателя VSR (вечерние замеры)
-0,001 ± 0,003
Средний прирост показателя VSR (за весь период)
0,0003 ± 0,001
Средний прирост показателя ОУСР (утренние замеры)
0,009 ± 0,004
Средний прирост показателя ОУСР (вечерние замеры)
0,001 ± 0,002
Средний прирост показателя ОУСР (за весь период)
0,002 ± 0,001
Средний прирост проективного показателя работоспособности (утренние замеры)
-0,017 ± 0,020
Средний прирост проективного показателя работоспособности (вечерние замеры)
0,003 ± 0,017
Средний прирост проективного показателя работоспособности (за весь период)
-0,001 ± 0,009
Средний прирост проективного показателя стресса (утренние замеры)
0,012 ± 0,064
Средний прирост проективного показателя стресса (вечерние замеры)
-0,022 ± 0,066
Средний прирост проективного показателя стресса (за весь период)
-0,004 ± 0,032 A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Том 7, № 5-1 ективного показателя работоспособности наблюдается
тенденция к снижению, как в течение утреннего перио
да, так и за весь период пребывания на судне. ACTA BIOMEDICA SCIENTIFICA, 2022, Vol. 7, N 5-1 В вечер
ние часы участники на субъективном уровне чувству
ют себя более работоспособными, что объясняет поло
жительный прирост проективного показателя в вечер
них замерах. вергались параметры основных и дополнительных шкал
Пятифакторного личностного опросника (FFI) Р. Маккрей
и П. Коста и коэффициенты среднего прироста значе
ний объективных психофизиологических и проектив
ных психологических параметров функциональных со
стояний участников в течение морской арктической экс
педиции (перечень которых представлен в таблице 2). На рисунке 2 отражены основные взаимосвязи между
индивидуально-личностными качествами участников
и их динамикой функциональных состояний (по объек
тивным и проективным данным). Кроме этого, интересна динамика проективного по
казателя стресса. Несмотря на то, что результаты в целом
отражают норму, анализ прироста показателя в утрен
ние часы указывает на тенденцию к образованию стрес
са, которая может быть связана с биоритмами участни
ков, поскольку несколько научных групп осуществля
ли научно-исследовательскую деятельность на судне
в сменном режиме. И возможно, что к утреннему заме
ру их организм не успевал восстановиться. Кроме это
го, большинство вертолётных высадок на острова осу
ществлялись именно в утреннее время, что могло вызы
вать волнение и напряжение у участников, которое сни
жалось в вечернее время, уже после высадок. По результатам корреляционного анализа выявлены
статистически значимые связи между динамикой функ
циональных состояний и такими индивидуально-лич
ностными качествами, как экстраверсия - интроверсия
и привязанность - отделённость. Согласно данным рисунка 2, чем сильнее выраже
но у участника самоуважение, тем более благоприят
ным был уровень его общего функционального состо
яния организма в утреннее время. Также утром более
успешно адаптировались к экспедиции люди, более из
бирательные в оказании помощи другим и склонные вы
держивать оптимальную дистанцию в общении. Кроме
этого, значение имеет выраженность экстравертиро-
ванных и интровертированных черт. Согласно резуль
татам корреляционного анализа, у экстравертов с выра
женной потребностью в поиске впечатлений в услови
ях экспедиции скорее расходовались психофизиологи
ческие резервы организма. В то время как интроверты,
способные переносить уединение и монотонию легче, Взаимосвязь индивидуально-личностных качеств
и динамики функциональных состояний участников мор
ской арктической экспедиции. Для установления связи
между индивидуально-личностными качествами участ
ников и показателями их функциональных состояний
в динамике морской арктической экспедиции был про
ведён корреляционный анализ с применением коэффи
циента ранговой корреляции Спирмена. Данный коэф
фициент выбран в связи с тем, что часть данных распре
делены ненормально. Корреляционному анализу под 235 Psychology and psychiatry Психология и психиатрия A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Vol. 7, N 5-1
ИС. 2. ACTA BIOMEDICA SCIENTIFICA, 2022, Vol. 7, N 5-1 орреляционная плеяда взаимосвязи индивидуально-лич
остных качеств и коэффициентов среднего прирост а зна
й
б
ф
FIG. 2. Correlation pleiad o f the relationship o f individual-personal
qualities and coefficients o f average increase in the values o f objec
i
h
h
i l
i
l
d
j
i
h l
i
l A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Vol. 7, N 5-1 A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Vol. 7, N 5-1 A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Vol. 7, N 5-1 ОБСУЖ ДЕНИЕ ные и активные люди, способные проявлять инициати
ву, первыми идти на контакт с окружающими и как след
ствие объединяющие вокруг себя группу, так и более
спокойные, сохраняющие дистанцию в общении люди,
лучше справляющиеся с монотонной работой и прора
батывающие детально поставленные задачи. Кроме это
го, нельзя исключать профессиональную принадлеж
ность участников, которая оказывает влияние на лич
ностные характеристики. Так, например, в сравнитель
ном исследовании личностных характеристик жителей
высоких широт с применением диагностической мето
дики «Большой пятёрки» авторами (G.D. Steel, P. Suedfeld,
A. Peri и L.A. Palinkas) [29] было выявлено, что учёные,
как профессиональная группа, имели более низкие
баллы по шкале экстраверсии, чем военнослужащие,
и по шкале доброжелательности, чем технический и об
служивающий персонал. ОБСУЖ ДЕНИЕ Говоря о результатах нашего исследования, можно
отметить, что в целом они согласуются с данными пре
дыдущих экспедиций «Арктического плавучего универ
ситета» в 2013 г. [21] и 2015 г. [22]. Так, в 2013 г. была так
же выявлена тенденция к преобладанию симпатической
нервной системы у участников на протяжении всего рей
са. Согласно результатам исследования, в 2015 г. было
установлено, что у участников в возрасте до 35 лет на
блюдается повышение активности парасимпатических
влияний к окончанию рейса, и, наоборот, активность
симпатоадреналовой системы с вкладом в гумораль
ный компонент регуляции увеличивается у лиц старше
35 лет. В целом данные, полученные в ходе экспедиции
в 2015 г., свидетельствуют об отсутствии значимого не
гативного воздействия краткосрочного морского рейса
в «высоких широтах» на функциональное состояние ор
ганизма участников. Что также согласуется с результата
ми, полученными в настоящем исследовании. Одним из ограничений настоящего исследования яв
ляется относительно небольшой объём выборки. Кро
ме этого, сезонность проведения исследования влияет
на психоэмоциональное и функциональное состояние
организма. Проведение более продолжительного ис
следования с привлечением большего числа участников
помогло бы нам глубже изучить тему. А также расшире
ние спектра методов оценки функциональных состоя
ний, включающих также выявление признаков утомле
ния, дезадаптации и др., позволило бы расширить вари
ативность получаемых связей с индивидуально-личност
ными качествами. Полученные нормальные значения по проективному
показателю стресса у участников согласуются с резуль
татами исследования C.A. Alfano, J. Bower, C. Connaboy
и коллег (2021) [36], посвящённого психическому здо
ровью, физическим симптомам и биомаркерам стресса
у участников экспедиции во время девятимесячного пе
риода пребывания в экстремальных условиях Антаркти
ды. При анализе изменений, наблюдаемых по разным ме
сячным показателям и маркерам стресса, исследователям
не удалось выявить какой-либо один период как одно
значно сложный. ОБСУЖ ДЕНИЕ Мы предполагаем, что на выраженность
стресса оказывает влияние продолжительность пребы
вания в экстремальных условиях экспедиции. Важно от
метить, что при отсутствии ярко выраженного стрессо
вого состояния у участников, был выявлен незначитель
ный положительный прирост стресса в утренние часы,
что свидетельствует о тенденции к накоплению стресса. Согласно данным исследования С.В. Пряничникова (2020)
[16], у людей, пребывающих более продолжительное вре
мя в посёлке Баренцбург (архипелаг Шпицберген), более
6 месяцев, показатели психоэмоционального состояния
характеризуются увеличением уровня личностной тре
вожности, проявляющейся в снижении эмоционального
фона и увеличении напряжённости. Также удалось уста
новить, что повышение уровня личностной тревожности
связано с показателями кардиогемодинамики. В статье представлена часть результатов морской
научно-исследовательской экспедиции в Арктический
регион. Планируется дальнейшее выявление и описа
ние статистически значимых взаимосвязей индивиду
ально-личностных качеств и динамики функциональ
ных состояний участников экспедиции. По результатам проведённого исследования можно
сформулировать следующие рекомендации и направле
ния оптимизации деятельности участников экспедиции: • предварительно не допускать истощения психо
физиологических резервов перед началом экспедиции; • в первые дни экспедиции обращать внимание
на функциональное состояние, соблюдать баланс меж
ду активной деятельностью и отдыхом; • при возможности организовывать работу в соот
ветствии с собственными биоритмами; • в период высадок на острова обеспечивать пол
ноценный отдых для сохранения достаточной работо
способности; Полученные результаты исследования, отражающие
взаимосвязь функциональных состояний с разными ха
рактеристиками личности участников, свидетельствуют
в пользу комплексного характера исследования с приме
нением разных групп диагностических методов. Соотно
шение характеристик функциональных состояний в дина
мике экспедиционного рейса и индивидуально-личност
ных качеств открывает перспективы для выявления и про
гноза развития процесса адаптации, а также более диф
ференцированной системы сопровождения сотрудников. • участникам с более выраженными экстравертиро-
ванными чертами с вниманием относиться к реализации
своей активности, связанной с расходованием психофи
зиологических резервов. РИС. 2. Correlation pleiad o f the relationship o f individual-personal
qualities and coefficients o f average increase in the values o f objec
tive psycho-physiological and projective psychological parameters
o f the functional states of participants during the marine Arctic
expedition Корреляционная плеяда взаимосвязи индивидуально-лич
ностных качеств и коэффициентов среднего прирост а зна
чений объективных психофизиологических и проективных
психологических параметров функциональных состояний
участников в течение морской арктической экспедиции чем экстраверты, к вечеру, наоборот, имели более бла
гоприятные показатели функционального состояния. Участники, которые брали на себя меньше ответствен
ности в групповой работе, нестремясь заниматьлидер
ское положение, также имели в вечернее время более
оптимальное состояние. сохраняли энергию в вечернее время и способность ра
ботать, не замечая усталости. Общительные участники склонны чаще испытывать
стресс. При этом в условиях экспедиции стрессу более
подвержены активные люди, предпочитающие зани
маться какой-то деятельностью. Более работоспособными в вечернее время были
активные, открытые для общения участники, способ
ные к доверительным отношениям, в том числе к обсуж
дению своих переживаний и трудностей. У участников
с более выраженной подозрительностью, которая мо
жет проявляться в переживаниях по поводу недооцен
ки себя в коллективе, быстрее развивалось психофизи
ологическое напряжение. Более активные люди лучше Таким образом, выявленные взаимосвязи свидетель
ствуют о том, что преобладание экстравертированных
или интровертированных черт, наряду со склонностью
к привязанности, основанной на потребности в тёплых
и доверительных отношениях, или отделённости, пред
полагающей развитое самоуважение, выступают инди
видуально-личностными маркерами в оценке функци
ональных состояний. 236 Psychology and psychiatry Психология и психиатрия A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Том 7, № 5-1 A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Том 7, № 5-1 ЛИТЕРАТУРА 1 1. Иванов И.С. (ред.). Аркт ический регион: Проблемы
международного сотрудничества. Хрестоматия в 3 томах. М.: Аспект Пресс; 2013. 1. Иванов И.С. (ред.). Аркт ический регион: Проблемы
международного сотрудничества. Хрестоматия в 3 томах. М.: Аспект Пресс; 2013. 2. Хаснулин В.И. Здоровье человека и космогеофизиче
ские факторы Севера. Экология человека. 2013; 12: 3-13. 3. Чащин В.П., Гудков А.Б., Попова О.Н., Одланд Ю.О.,
Ковшов А.А. Характеристика основных факторов риска нару
шений здоровья населения, проживающего на территориях
активного природопользования в Арктике. Экология человека. 2014; 1: 3-12. 4. Popova T, Kulganov V, Lobanov A, Andronov S. Neuro-psy
chological adaptation of patients with chronic bronchitis in Arctic
conditions. Eur Respir J. 2018; 52: PA801. doi: 10.1183/13993003. congress-2018.PA801 4. Popova T, Kulganov V, Lobanov A, Andronov S. Neuro-psy
chological adaptation of patients with chronic bronchitis in Arctic
conditions. Eur Respir J. 2018; 52: PA801. doi: 10.1183/13993003. congress-2018.PA801 Выдвинутая нами гипотеза нашла частичное под
тверждение. Установлено, что общий уровень функци
онального состояния организма, измеренный объектив
ным методом, взаимосвязан с интроверсией и избегани
ем новых впечатлений, а также равнодушием и самоува
жением. Участники экспедиции с умеренной выражен
ностью данных характеристик характеризовались более
благоприятным уровнем функционального состояния. 5. Атьков О.Ю., Горохова С.Г., Сериков В.В., Алчинова И.Б.,
Полякова М.В., Панкова Н.Б., и др. Результаты медицинских
и психофизиологических исследований во время кругосветно
го арктического перелёта. Вестник РАМН. 2019; 74(4): 261-271. doi: 10.15690/vramn1110 5. Атьков О.Ю., Горохова С.Г., Сериков В.В., Алчинова И.Б.,
Полякова М.В., Панкова Н.Б., и др. Результаты медицинских
и психофизиологических исследований во время кругосветно
го арктического перелёта. Вестник РАМН. 2019; 74(4): 261-271. doi: 10.15690/vramn1110 6. Фролков В.К., Нагорнев С.Н., Бобровницкий И.П., Жер
нов В.А., Зубаркина М.М. Патофизиологические механизмы
неблагоприятного влияния климатогеографических факторов
Арктики на здоровье человека и технологии восстановитель
ной медицины. Физиотерапевт. 2020; 1: 57-63. doi: 10.33920/
med-14-2002-10 6. Фролков В.К., Нагорнев С.Н., Бобровницкий И.П., Жер
нов В.А., Зубаркина М.М. Патофизиологические механизмы
неблагоприятного влияния климатогеографических факторов
Арктики на здоровье человека и технологии восстановитель
ной медицины. Физиотерапевт. 2020; 1: 57-63. doi: 10.33920/
med-14-2002-10 Уровень операторской работоспособности, изме
ренный объективным методом, взаимосвязан с доверчи
востью и открытостью участников экспедиции. У участ
ников экспедиции с более высоким уровнем активности
наблюдаются более высокая работоспособность и более
сниженный стресс, согласно проективным параметрам. С большей выраженностью стресса также связана общи
тельность как черта личности. 7. Корчин В.И., Корчина Т.Я., Терникова Е.М., Бикбулато
ва Л.Н., Лапенко В.В. Влияние климатогеографических факторов
Ямало-Ненецкого автономного округа на здоровье населения
(обзор). Журнал медико-биологических исследований. 2021; 9(1):
77-88. doi: 10.37482/2687-1491-Z046 7. Конфликт интересов Наиболее значимыми с точки зрения изменений ста
ли 2-й, 11-й и 14-й дни исследования. В начале экспеди
ции, в утренние часы 2-го дня исследования увеличилось
число участников с неблагоприятным функциональным
состоянием. Далее в утренние часы 11-го дня исследова
ния, на субъективном уровне наблюдался спад работо
способности, произошло усиление парасимпатического
отдела вегетативной нервной системы. И после заверше
ния этапа высадок на острова, с 14-го дня исследования
на несколько дней увеличились объективные показате
ли операторской работоспособности и произошли из
менения в динамике вегетативной нервной системы: по
проективному показателю вегетативного баланса сни
зились значения, отражающие тенденцию к преоблада
нию симпатического тонуса, в динамике объективного
показателя функционального состояния стала преобла
дать тенденция к сбалансированному влиянию отделов
вегетативной нервной системы, а по проективному по
казателю стала преобладать тенденция к усилению па
расимпатического влияния. Авторы данной статьи заявляют об отсутствии кон
фликта интересов. Благодарность Авторы выражают благодарность участникам ис
следования и организаторам научно-образовательно
го проекта «Арктический плавучий университет - 2021»
на научно-экспедиционном судне «Михаил Сомов». Финансирование Исследование выполнено в рамках реализации го
сударственного задания на проведение фундаменталь
ных научных исследований по теме «Оценка психологи
ческих рисков в профессиональной деятельности спе
циалистов экстремального профиля» (проект FSRU-2020-
006, 2020-2022 гг.). A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Vol. 7, N 5-1 A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Vol. 7, N 5-1 обходима умеренная выраженность интровертиро-
ванных качеств личности, при этом для сохранения
требуемого уровня работоспособности важно разви
тие открытости и активности как индивидуально-лич
ностных качеств. свидетельствуют об отсутствии значимого негативного
влияния «высоких широт» на функциональное состояние
организма участников 20-дневной морской арктической
экспедиции. Согласно объективным данным, функцио
нальное состояние характеризуется тенденцией к пре
обладанию допустимых значений с умеренным напря
жением регуляторных систем. Благодаря достаточным
адаптационным возможностям сердечно-сосудистой
и вегетативной нервной систем участники сохраняют
нормальные показатели самочувствия и работоспособ
ности. Динамика работоспособности в течение рейса ха
рактеризовалась тенденцией к средним (достаточным)
значениям согласно объективным и проективным дан
ным. Динамика проективного показателя стресса в це
лом отражает норму, при этом фиксируется незначитель
ный положительный прирост стресса в утренние часы,
что свидетельствует о тенденции к накоплению стрес
са. В целом, полученные объективные и проективные
данные не носят противоречивый характер, а скорее
дополняют друг друга. ЗАКЛЮ ЧЕНИЕ Анализ динамики объективных психофизиологиче
ских и проективных психологических параметров функ
циональных состояний участников экспедиции в целом Безусловно, что для более успешного взаимодей
ствия в экспедиционной команде необходимы участни
ки с различными характеристиками: как жизнерадост 237 Психология и психиатрия Psychology and psychiatry A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Том 7, № 5-1 doi: 10.1016/S0140-
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ный университет имени М.В. Ломоносова», e-mail: masha.tynkina@yandex.ru, https://orcid.org/0000-0002-1403-1639 Корнеева Яна Александровна - кандидат психологических наук, доцент, доцент кафедры психологии, ФГ АОУ ВО «Северный (Арктический) федеральный университет имени М.В. Ло
моносова», e-mail: ya.korneeva@narfu.ru, https://orcid.org/0000-0001-9118-9539 колаевна - кандидат биологических наук, доктор психологических наук, профессор, ведущий научный сотрудник лаборатории психологии труда факуль
О «Московский государственный университет имени М.В. Ломоносова», e-mail: n23117@mail.ru, https://orcid.org/0000-0001-5658-6811 Симонова Наталья Николаевна - кандидат биологических наук, доктор психологических наук, профессор, ведущий научный сотрудник лаборатории психологии труда факуль
тета психологии, ФГБОУ ВО «Московский государственный университет имени М.В. Ломоносова», e-mail: n23117@mail.ru, https://orcid.org/0000-0001-5658-6811
Трофимова Анна Алексеевна - магистрант 2-го курса, ФГАОУ ВО «Северный (Арктический) федеральный университет имени М.В. Ломоносова», e-mail: annatrofimova100@gmail.com,
https://orcid.org/0000-0002-1751-7773 ,
уд р
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,
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Трофимова Анна Алексеевна - магистрант 2-го курса, ФГАОУ ВО «Северный (Арктический) федеральный университет имени М.В. Ломоносова», e-mail: annatrofimova100@gmail.com,
https://orcid.org/0000-0002-1751-7773 A C T A B IO M E D IC A S C IE N T IF IC A , 2022, Том 7, № 5-1 34. Aminev GA. Instruction and interpretation of the eight-
color test based on mathematical processing. Mathematical meth
ods in engineering psychology. Ufa; 1982: 19-24. (In Russ.). 30. Palinkas LA, Gunderson EK, Holland AW, Miller C, John
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son JC. Predictors of behavior and performance in extreme en
vironments: The Antarctic space analogue program. Aviat Space
Environ Med. 2000; 71(6): 619-625. 35. Khromov AB. Five-factor personality questionnaire. Kurgan;
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2000. (In Russ.). 31. Leonova AB. Psychodiagnostics o f human functional states. Moscow; 1984. (In Russ.). 36. Alfano CA, Bower J, Connaboy C, Agha NH, Baker FL,
Smith KA, et al. Mental health, physical symptoms and biomark
ers of stress during prolonged exposure to Antarctica>s extreme
environment. Acta Astronautica. 2021; 181(4). doi: 10.1016/
j.actaastro.2021.01.051 36. Alfano CA, Bower J, Connaboy C, Agha NH, Baker FL,
Smith KA, et al. Mental health, physical symptoms and biomark
ers of stress during prolonged exposure to Antarctica>s extreme
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color test based on mathematical processing. Mathematical meth
ods in engineering psychology. Ufa; 1982: 19-24. (In Russ.). Information about the authors Maria A. Tunkina - Expert at the Department of Targeted Scientific Programs and Projects of the Research Department, Northern (Arctic) Federal University named after M.V. Lomonosov
University, e-mail: masha.tynkina@yandex.ru, https://orcid.org/0000-0002-1403-1639
Yana A. Korneeva - Cand. Sc. (Psychol.), Docent, Associate Professor at the Department of Psychology, Northern (Arctic) Federal University named after M.V. Lomonosov University,
e-mail: ya.korneeva@narfu.ru, https://orcid.org/0000-0001-9118-9539
Natalia N. Simonova - Cand. Sc. (Biol.), Dr. Sc. (Psychol.), Professor, Leading Research Officer at the Laboratory of Occupational Psychology, Faculty of Psychology, Lomonosov Moscow State
University, e-mail: n23117@mail.ru, https://orcid.org/0000-0001-5658-6811
Anna A. Trofimova - 2nd year Master's Degree Student, Northern (Arctic) Federal University named after M.V. Lomonosov, e-mail: annatrofimova100@gmail.com, https://orcid.org/0000-
0002-1751-7773 Natalia N. Simonova - Cand. Sc. (Biol.), Dr. Sc. (Psychol.), Professor, Leading Research Officer at the Laboratory of Occupational Psychology, Faculty of Psychology, Lomonosov Moscow State
University, e-mail: n23117@mail.ru, https://orcid.org/0000-0001-5658-6811 Anna A. Trofimova - 2nd year Master's Degree Student, Northern (Arctic) Federal University named after M.V. Lomonosov, e-mail: annatrofimova100@gmail.com, https://orcid.org/0000-
0002-1751-7773 Статья опубликована в рамках V Всероссийской научно-практической конференции молодых учёных с международным участием «Фундаментальные и прикладные аспекты в ме
дицине и биологии». 241 Psychology and psychiatry Психология и психиатрия
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